Genetically modified filamentous fungi for the production of exogenous proteins with low or no N-linked glycosylation.

Abstract

Provided is an Ascomycota filamentous fungus that contains a deletion or disruption of the stt3 and / or cwh8 genes and that has been genetically engineered to produce proteins with reduced or no mammalian protein N-glycans.

Description

Technical Field

[0001] The present invention relates to a genetically modified Ascomycota filamentous fungus in which the expression and / or activity of STT3 and / or CWH8 protein is reduced. Genetically modified filamentous fungi are used for the strong production of recombinant proteins that have partially or no N-linked glycosylation.

Background Art

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

[0003] As eukaryotes, yeasts and fungi can perform post-translational modifications including N- and O-glycosylation, but protein glycosylation in yeasts and fungi is different from that in mammalian cells. To overcome these problems, the possibility of redesigning the N-glycosylation pathway has been investigated, especially in species most frequently used for the production of heterologous proteins (e.g., Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, and species of the genus Aspergillus and the genus Trichoderma).

[0004] Parsaie Nasab et al. (2013, Appl Environ Microbiol., 79(3):997-1007) describe a synthetic N-glycosylation pathway for producing recombinant proteins that hold human N-glycans in S. cerevisiae. Their research, also described in U.S. Patent Application Publication No. 2011 / 0207214, discloses cells modified to express lipid-binding oligosaccharide (LLO) flippase activity in the ER membrane. The flippase allows for the flipping of LLOs containing one, two, or three mannose molecules from the cytoplasmic side of the ER to the luminal side. The research is further reviewed along with other related studies by De Wachter et al. (2018, Engineering of Yeast Glycoprotein Expression. In: Advances in Biochemical Engineering / Biotechnology. Springer, Berlin, Heidelberg).

[0005] U.S. Patents 7,029,872, 7,326,681, 7,629,163, and 7,981,660 disclose cell lines having a recombinant glycosylation pathway that enables the execution of a series of enzymatic reactions that mimic the processing of glycoproteins in humans. These involve modifying eukaryotes, such as unicellular and multicellular fungi, which typically produce high-mannose-containing N-glycans, to produce N-glycans such as Man5GlcNAc2 or other structures along the human glycosylation pathway.

[0006] U.S. Patent No. 9,359,628 discloses genetically modified strains of the genus Pichia that can produce proteins with smaller glycans. In particular, the genetically modified strains can express either or both α-1,2-mannosidase and glucosidase II. The genetically modified strains can be further modified so that the OCH1 gene is disrupted. Methods for producing glycoproteins with smaller glycans using such genetically modified strains of the genus Pichia are also provided.

[0007] U.S. Patents No. 8,268,585 and No. 8,871,493, filed with the applicants of the present invention, disclose transformation systems in the field of filamentous fungal hosts for expressing and secreting heterologous proteins or polypeptides. Processes for producing large quantities of polypeptides or proteins in an economical manner are also disclosed. The systems include transformed or transfected fungal strains of the genus Chrysosporium, more specifically Chrysosporium lucknowense and its mutants or derivatives. Transformants containing Chrysosporium coding sequences, as well as regulatory sequences for Chrysosporium genes, are also disclosed.

[0008] Thermothelomyces heterothallica (Th. heterothallica) strain C1 (recently renamed from Myceliophthora thermophila, which was previously named Chrysosporium lucknowense) is a thermostable ascomycete filamentous fungus that produces high levels of cellulase, making it attractive for the commercial production of these and other enzymes.

[0009] Wild-type C1 was deposited in accordance with the Budapest Convention on the deposit date of August 29, 1996, under the designation VKM F-3500 D. High-cellulose (HC) and low-cellulose (LC) strains have also been deposited, as described, for example, in U.S. Patent No. 8,268,585.

[0010] U.S. Patent No. 9,695,454 discloses a composition comprising filamentous fungal cells, such as Trichoderma cells, in which protease activity is reduced and the fucosylation pathway is expressed. Further disclosed is a method for producing glycoproteins having fucosylated N-glycans using genetically modified filamentous fungal cells, such as Trichoderma cells, as an expression system.

[0011] U.S. Patents 7,449,308 and 7,935,513 disclose eukaryotic host cells having modified oligosaccharides, which may be further modified by heterologous expression of a series of glycosyltransferases, sugar transporters, and mannosidases to become host cells for the production of therapeutic glycoproteins in mammals, such as humans. The N-glycans produced in the modified host cells have a Man5GlcNAc2 core structure and may subsequently be further modified by heterologous expression of one or more enzymes, such as glycosyltransferases, sugar transporters, and mannosidases, to obtain human-like glycoproteins.

[0012] U.S. Patent Nos. 8,268,585 and 8,871,493 disclose transformation systems in the field of filamentous fungal hosts for expressing and secreting heterologous proteins or polypeptides. Processes for producing large quantities of polypeptides or proteins in an economical manner are also disclosed. The systems include fungal strains of the genus Chrysosporium, more specifically Chrysosporium lucknowense and its mutants or derivatives. Transformants containing Chrysosporium coding sequences, as well as regulatory sequences for Chrysosporium gene expression, are also disclosed.

[0013] U.S. Patent No. 9,175,296 discloses a fungal host strain of Chrysosporium lucknowense. Also disclosed are methods for the homogeneous and / or heterogeneous production of pure protein with a purity of 75% or higher, methods for the production of artificial proteinmixes, and methods for simplified screening of strains that functionally express a desired enzyme. U.S. Patent No. 9,175,296 further discloses isolated promoter sequences suitable for transcriptional regulation of gene expression in Chrysosporium lucknowense (recently renamed Thermothelomyces heterothallica) and a method for isolating a fungal host strain of Chrysosporium lucknowense in which protease secretion is less than 20% of the protease secretion of Chrysosporium lucknowense strain UV18-25.

[0014] There is a need for expression systems to produce recombinant proteins that can produce proteins with or without partial N-linked glycosylation in high yield, so that the proteins are suitable for a wide variety of industrial and pharmaceutical applications. [Overview of the project]

[0015] The present invention provides genetically modified Ascomycetes filamentous fungi that are genetically modified to produce proteins with little or no N-glycan modification of mammalian proteins. In particular, the present invention provides Thermothelomyces heterothallica strain C1 as a representative Ascomycetes filamentous fungus that is genetically modified to produce recombinant proteins with little or no N-glycan modification. In some embodiments, the fungi disclosed herein are modified to delete the stt3 and / or cwh8 genes.

[0016] This invention is partly based on the finding that genetically modified Th. heterothallica, as disclosed herein, produces proteins with fewer or no glycans compared to unmodified strains. This is in contrast to conventionally described expression systems, which produce proteins with greater variability in the resulting N-glycans.

[0017] Conveniently, the recombinant Ascomycete filamentous fungal cells of the present invention enable the production of heterologous proteins with partial post-translational modifications. These proteins may be used in a wide variety of applications where fully glycosylated proteins are unsuitable. For example, proteins can be designed for desired solubility and / or bioactivity. Proteins with lower N-glycan content may exhibit reduced immunogenicity compared to fully glycosylated proteins. The partially N-glycosylated proteins of the present invention may be used as key substances for addition or other protein modifications. Furthermore, non-N-glycosylated proteins may be used in pharmacokinetic / pharmacodynamic studies as potential regulatory proteins for various glycosylation forms and mixtures. Moreover, since therapeutic effects often depend on N-glycosylation, the partially glycosylated proteins described herein may have different therapeutic effects compared to naturally glycosylated proteins.

[0018] Conveniently, the recombinant Ascomycete filamentous fungal cells of the present invention produce proteins with high yield and stability. Protein levels obtained using the Th. heterothallica cells of the present invention are significantly higher than those obtained using mammalian cells such as CHO cells or yeast.

[0019] Therefore, the present invention provides an effective system for producing eukaryotic recombinant proteins that are low in or completely free of N-glycans, suitable for a wide variety of applications in the pharmaceutical and non-pharmaceutical industries.

[0020] According to one embodiment, the present invention provides a genetically modified Ascomycete filamentous fungus capable of producing a target protein with low or no N-linked glycosylation, wherein the genetically modified filamentous fungus comprises at least one cell in which the expression and / or activity of STT3 and / or CWH8 are reduced.

[0021] According to some embodiments, at least one cell comprises at least one exogenous polynucleotide encoding a protein of interest.

[0022] According to some embodiments, the expression and / or activity of STT3 is reduced in at least one cell.

[0023] According to some embodiments, STT3 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 acids of Thermothelomyces heterothallica STT3. According to certain embodiments, Thermothelomyces heterothallica STT3 comprises the amino acids of SEQ ID NO: 27.

[0024] According to some embodiments, the expression and / or activity of CWH8 is reduced in at least one cell.

[0025] According to some embodiments, CWH8 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 acids of Thermothelomyces heterothallica CWH8. According to certain embodiments, Thermothelomyces heterothallica CWH8 comprises the amino acids of SEQ ID NO: 28.

[0026] According to some embodiments, the recombinant filamentous fungus comprises at least one cell in which the expression and / or activity of STT3 and CWH8 proteins is reduced.

[0027] According to some embodiments, the genetic recombination includes deletion or disruption of the stt3 gene. According to some embodiments, the genetic recombination includes deletion or disruption of the stt3 gene such that the recombinant filamentous fungus produces less amount of the catalytic subunit of the oligosaccharyltransferase (OST) complex. According to some embodiments, the genetic recombination includes deletion or disruption of the stt3 gene such that the recombinant filamentous fungus fails to produce the catalytic subunit of the oligosaccharyltransferase (OST) complex.

[0028] According to some embodiments, the genetic recombination includes deletion or disruption of the cwh8 gene. According to some embodiments, the genetic recombination includes deletion or disruption of the cwh8 gene such that the recombinant filamentous fungus produces less amount of the functional dolichol pyrophosphate phosphatase. According to some embodiments, the genetic recombination includes deletion or disruption of the cwh8 gene such that the recombinant filamentous fungus fails to produce the functional dolichol pyrophosphate phosphatase.

[0029] According to some embodiments, the recombinant filamentous fungus expresses a protein with less amount of N-linked glycosylation. According to certain embodiments, the recombinant filamentous fungus expresses a protein with N-linked glycosylation less than 20% compared to the non-recombinant fungus. According to additional embodiments, the recombinant filamentous fungus expresses a protein with no N-linked glycosylation.

[0030] According to some embodiments, the ascomycete filamentous fungus is of a genus within the family of the Pezizomycotina.

[0031] In some embodiments, the Ascomycetes phylum filamentous fungi are genera selected from the group consisting of Thermothelomyces, Myceliophthora, Trichoderma, Aspergillus, Penicillium, Rasamsonia, Chrysosporium, Corynascus, Fusarium, Neurospora, and Talaromyces.

[0032] According to some embodiments, the Ascomycetes phylum filamentous fungi include Thermothelomyces heterothallica (also known as Myceliophthora thermophila), Myceliophthora lutea, Aspergillus nidulans, Aspergillus funiculosus, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Trichoderma harzianum, Trichoderma longibrachiatum, and Trichoderma viride. It is a species 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.

[0033] 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. 29.

[0034] In some embodiments, the Ascomycete filamentous fungus is Thermothelomyces heterothallica. In some embodiments, the Ascomycete filamentous fungus is Thermothelomyces heterothallica C1.

[0035] In some embodiments, C1 is a strain selected from the group consisting of W1L#100I(prt-Δalp1Δchi1Δalp2Δpyr5) deposit number CBS141153, UV18-100f(prt-Δalp1,Δpyr5) deposit number CBS141147, W1L#100I(prt-Δalp1Δchi1Δpyr5) deposit number CBS141149, and UV18-100f(prt-Δalp1Δpep4Δalp2Δprt1Δpyr5) deposit number CBS141143 and their derivatives. Each possibility represents a distinct embodiment of the present invention.

[0036] In some embodiments, the protein of interest is selected from the group consisting of enzymes, structural proteins, vaccine antigens, and their components.

[0037] In some embodiments, the protein of interest is a secreted protein. In certain embodiments, the protein of interest is a leader peptide. In other embodiments, the protein of interest is an intracellular protein. In certain embodiments, the intracellular protein is a membrane or vesicle-associated protein.

[0038] 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 antibody or a multispecific antibody.

[0039] In some embodiments, the protein of interest is a therapeutic protein.

[0040] In some embodiments, the protein of interest is a vaccine protein antigen.

[0041] The polynucleotide encoding the target protein may form part of a DNA construct or expression vector.

[0042] According to some embodiments, at least one exogenous polynucleotide is a DNA construct or expression vector further comprising at least one regulatory element executable by the Ascomycete filamentous fungus. According to certain embodiments, the regulatory element is selected from the group consisting of regulatory elements endemic to the fungus and regulatory elements heterologous to the fungus.

[0043] In some embodiments, genetically modified Ascomycetes filamentous fungi are designed to produce secretory proteins.

[0044] In some embodiments, the Ascomycetes filamentous fungi according to the present invention are genetically modified to express antibodies.

[0045] In some embodiments, the Ascomycetes filamentous fungi are strains further recombinant in which one or more genes encoding endogenous proteases are deleted.

[0046] According to some embodiments, the recombinant Ascomycetes filamentous fungi include 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 distinct embodiment of the present invention. According to certain embodiments, the recombinant filamentous fungi include at least one cell in which the expression and / or activity of at least 9, 10, 11, 12, 13, or 14 proteases is reduced. Each possibility represents a distinct embodiment of the present invention.

[0047] In another aspect, the present invention: (a) A step of reducing the expression and / or activity of the STT3 protein of Ascomycetes filamentous fungi; and / or (b) A step of reducing the expression and / or activity of the CHW8 protein of Ascomycetes filamentous fungi. This invention provides a method for producing filamentous fungi of the Ascomycetes phylum that can produce proteins containing low or no N-linked glycosylation.

[0048] According to some embodiments, the method is: (a) deleting or disrupting the stt3 gene of an Ascomycete filamentous fungus to reduce the production of a functional catalytic subunit of the oligosaccharide transferase (OST) complex; and / or (b) The step of deleting or disrupting the chw8 gene of an Ascomycete filamentous fungus in order to reduce the production of functional dolichol pyrophosphate phosphatase. Includes.

[0049] In some embodiments, fungi fail to produce the functional catalytic subunit of the oligosaccharide transferase (OST) complex.

[0050] According to some embodiments, fungi fail to produce functional dolichol pyrophosphate phosphatase.

[0051] In some embodiments, fungi fail to produce the functional catalytic subunit and functional dolichol pyrophosphate phosphatase of the oligosaccharide transferase (OST) complex.

[0052] In some embodiments, the method further includes the step of introducing an exogenous polynucleotide encoding a heterologous protein of interest into an Ascomycete filamentous fungus, thereby expressing a heterologous protein of interest that is low in or completely lacking in N-glycans in the fungus.

[0053] In another aspect, the present invention provides a method for producing heterologous proteins with little or no N-glycan modification, the method being: (i) Providing a genetically modified Ascomycete filamentous fungus comprising at least one cell having reduced expression and / or activity of STT3 and / or CWH8 as described herein, and at least one exogenous polynucleotide encoding the protein of interest according to the present invention; (ii) the step of culturing Ascomycetes filamentous fungi under conditions suitable for expressing the protein; and (iii) Step of recovering the protein Includes.

[0054] In some embodiments, the protein is a heterologous mammalian protein recombinantly expressed in Ascomycetes. In some specific embodiments, the protein is a human protein recombinantly expressed in Ascomycetes. In other embodiments, the protein is a companion animal and / or livestock protein recombinantly expressed in Ascomycetes.

[0055] In a further embodiment, the present invention provides recombinant proteins produced by genetically modified Ascomycetes filamentous fungi according to the present invention.

[0056] In some embodiments, the recombinant proteins produced by the genetically modified Ascomycetes filamentous fungi according to the present invention are pharmaceutical-grade proteins.

[0057] In a further embodiment, the present invention provides a method for producing at least one protein of interest, the method comprising the steps of culturing a genetically modified filamentous fungus in a suitable medium as described herein; and recovering at least one protein product.

[0058] According to some embodiments, the recovery step includes recovering proteins from the growth medium, such as a fungal mass or broth.

[0059] 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.

[0060] It should be understood that any combination of each aspect and embodiment disclosed herein is expressly included within the scope of the disclosure of the present invention.

[0061] These and further aspects and features of the present invention will become apparent from the following detailed description, examples and claims. [Brief explanation of the drawing]

[0062] [Figure 1] Lipid-binding oligosaccharide biosynthesis pathway and oligosaccharide transfer to nascent polypeptides in the endoplasmic reticulum (ER) membrane of eukaryotic cells. [Figure 2] N-glycan patterns and abundance of different glycan forms on native fungal proteins produced in bioreactors in non-glycodenatured C1 strain (2A) and stt3 deletion strain M3210 (2B). [Figure 3] N-glycan patterns and abundance of different glycan forms on native fungal proteins produced in bioreactors in non-glycodenatured C1 strain (3A) and cwh8 deletion strain M3211 (3B). [Figure 4]N-glycan patterns and abundance of different glycan forms on monoclonal antibodies produced in non-glycolytic C1 strain (4A) and stt3 deletion strain M3480 (4B). [Figure 5] N-glycan patterns and abundance of different glycan forms on monoclonal antibodies produced in non-glycolytic C1 strain (5A) and cwh8 deletion strain M3481 (5B). [Modes for carrying out the invention]

[0063] The present invention provides an alternative, highly efficient system for producing proteins with low or no N-linked glycosylation. The system of the present invention is partly based on the filamentous fungus Thermothelomyces heterothallica C1 and its specific strains, which have been previously developed as natural biofactories and secondary metabolites of proteins. In some embodiments, the present invention provides genetically modified fungi in which the expression and / or activity of STT3 and / or CWH8 proteins is reduced. In some embodiments, the genetically modified fungi have reduced or inactivated expression and / or activity of multiple proteases.

[0064] Proteins produced by genetically modified fungi, as described herein, are suitable for a wide variety of pharmaceutical and non-pharmaceutical applications.

[0065] According to one embodiment, the present invention provides a genetically modified filamentous fungus that produces a target protein, the genetically modified filamentous fungus comprising at least one cell in which the expression and / or activity of the STT3 and / or CWH8 proteins are reduced.

[0066] In an additional embodiment, the present invention provides a genetically modified filamentous fungus capable of producing recombinant proteins with low or no N-linked glycosylation, wherein the genetic modification is: (i) Deletion or disruption of the stt3 gene in which the genetically modified filamentous fungus fails to produce the catalytic subunit of the oligosaccharide transferase (OST) complex; (ii) Deletion or disruption of the cwh8 gene such that the genetically modified filamentous fungus fails to produce functional dolichol pyrophosphate phosphatase; or (iii) Deletion or disruption of both the stt3 and cwh8 genes. Includes.

[0067] The term “disruption” means that a gene may be structurally disrupted, involving at least one mutation or structural change, to the extent that the disrupted gene is unable to direct the efficient expression of a full-length, fully functional gene product. The term “disruption” also encompasses the possibility that the disrupted gene or one of its products may be functionally inhibited or inactivated to the extent that the gene does not express a full-length and / or fully functional gene product or is unable to express it efficiently. Functional inhibition or inactivation may result from structural disruption and / or interruption of expression at either the transcriptional or translational level. The term “disruption” also encompasses attenuation or knockdown of gene expression.

[0068] Protein glycosylation, that is, the covalent bonding of oligosaccharides to the side chains of newly synthesized polypeptide chains within cells, is an ordered process in eukaryotic cells involving a series of enzymes that sequentially add and remove sugar moieties. N-glycosylation is the process by which oligosaccharides bind to asparagine residues in the side chains, particularly asparagine in the sequence Asn-Xaa-Ser / Thr (where Xaa represents any amino acid except Pro).

[0069] N-glycosylation begins within the endoplasmic reticulum (ER), where the oligosaccharide Glc3Man9GlcNAc2 is assembled on the lipid carrier, dolichol pyrophosphate, and subsequently transferred to a selected asparagine residue of the polypeptide entering the ER lumen. Figure 1 illustrates the biosynthetic pathway of lipid-bound oligosaccharides and the transfer of oligosaccharides to nascent polypeptides across the ER membrane in eukaryotic cells. The biosynthesis of lipid-bound oligosaccharides requires the activity of several specific glycosyltransferases. It begins on the cytoplasmic side of the ER membrane and ends in the lumen where oligosaccharide transferases (OSTs) select the NXS / T sequence of the nascent polypeptide, generating an N-glycosidic bond between the asparagine side-chain amide and the oligosaccharide. Flipping of lipid-bound oligosaccharides from the outside to the inside of the ER is carried out by flippers located in the ER membrane. Following the transfer to the nascent polypeptide, the oligosaccharide is typically trimmed by glycosidase and mannosidase, and the nascent glycoprotein is then transferred to the Golgi apparatus for further processing.

[0070] The synthesis of dolichol pyrophosphate-linked oligosaccharides is essentially converted in known eukaryotes. However, further processing of oligosaccharides as glycoproteins involves transport along vastly different secretory pathways between lower eukaryotes such as fungi or yeasts and higher eukaryotes such as animals and plants. Therefore, the final composition of the sugar side chains varies among different organisms and is host-dependent.

[0071] In microorganisms such as yeast, additional mannose and / or mannose phosphate sugars are typically added, resulting in a "hypermannosylated" N-glycan that may contain 30 to 50 or fewer mannose residues.

[0072] In animal cells, including human, companion animal, and other mammalian cells, newly synthesized glycoproteins are transported to the Golgi apparatus where mannose residues are removed by Golgi-specific 1,2-mannosidase. Processing continues as the protein passes through the Golgi apparatus by numerous modifying enzymes, including N-acetylglucosamine transferases (GnT I, GnT II, ​​GnT III, GnT IV, GnT V, GnT VI), mannosidase II, and fucosyltransferase, which add and remove specific sugar residues. Finally, the N-glycan is acted upon by galactosyltransferase (GalT) and sialyltransferase (ST), and the completed glycoprotein is released from the Golgi apparatus. Animal glycoprotein N-glycans have bi-, tri-, or tetraanthentory structures and typically contain galactose, mannose, fucose, and N-acetylglucosamine. Generally, the terminal residues of N-glycans consist of sialic acid.

[0073] Unlike yeast, Th. heterothallica does not possess hypermannosylated N-glycans, but rather oligomannose glycans - Man3~Man 8~9 -It also possesses hybrid glycans (Man3HexNac-Man8HexNac) containing both Man and HexNAc residues. The exact structures of these hybrid glycans are not fully known. The hybrid glycans have typical mannose residues but also unknown HexNAc attached via bonds that have not yet been characterized.

[0074] This invention is directed towards genetic modification of the N-glycosylation pathway to produce a smaller amount of N-glycan.

[0075] As used herein, “glycan” refers to an oligosaccharide chain that can be bound to a carrier such as an amino acid, peptide, polypeptide, lipid, or reducing end conjugate. The present invention particularly refers to N-linked glycans ("N-glycans") conjugated to a polypeptide N-glycosylation site such as -Asn-Xaa-Ser / Thr- (wherein Xaa is any amino acid residue except Pro) by an N-linking to the side-chain amide nitrogen of an asparagine residue (Asn). The present invention may further relate to glycans as part of a dolichol-phosphorus-oligosaccharide (Dol-PP-OS) precursor lipid structure, which is a precursor of N-linked glycans in the endoplasmic reticulum of eukaryotic cells. The precursor oligosaccharide is bound to two phosphate residues on the dolichol lipid by their reducing ends.

[0076] The term "stt3 gene" refers to the gene encoding the dolicol diphosphooligosaccharide-protein glycosyltransferase subunit. It is the catalytic subunit of the oligosaccharide transferase (OST) complex that catalyzes the first step in protein N-glycosylation: the initial transfer of a defined glycan (Glc3Man9GlcNAc2 in eukaryotes) from the lipid carrier dolicol pyrophosphate to an asparagine residue within the Asn-X-Ser / Thr consensus motif of a nascent polypeptide chain. The STT3 protein catalyzes this reaction:

[0077] (Math 1) Dolicol diphosphooligosaccharide-(1→4)-N-acetyl-β-D-glucosaminyl-(1→4)-N-acetyl-β-D-glucosaminyl) + L-asparaginyl-[protein]→ Dolicol diphosphate + H+ + N4-(oligosaccharide-(1→4)-N-acetyl-β-D-glucosaminyl-(1→4)-N-acetyl-β-D-glucosaminyl)-L-asparaginyl-[protein]

[0078] The genetically modified Ascomycetes filamentous fungi of the present invention are genetically modified by deletion or disruption of the stt3 gene so that the fungi fail to produce the functional catalytic subunit of the oligosaccharide transferase (OST) complex. The genetically modified Ascomycetes filamentous fungi of the present invention do not exhibit detectable oligosaccharide transferase (OST) activity.

[0079] The term "cwh8 gene" refers to the gene that encodes dolicol diphosphatase. CWH8 catalyzes the reaction:

[0080] (Math 2) Dolicol diphosphate + H2O = Dolicol phosphate + H+ + phosphate

[0081] The genetically modified Ascomycetes filamentous fungi of the present invention are genetically modified by deletion or disruption of the cwh8 gene so that the fungi fail to produce functional dolichol diphosphatase. The genetically modified Ascomycetes filamentous fungi of the present invention do not exhibit detectable dolichol diphosphatase activity.

[0082] As defined herein, Ascomycetes refer to any fungal strain belonging to the tribe of Pezizomycotina. The Pezizomycotina subphylum includes the following groups: Genus: Thermothelomyces (species: including heterothallica and thermophila), Myceliophthora (including species lutea and unnamed species), Corynascus (including the species fumimontanus), Neurospora crassa (including the species Neurospora) This includes the order Sordariales; Genus: Fusarium (including the species graminearum and venenatum), Trichoderma (including species reesei, harzianum, longibrachiatum, and viride) This includes the order Hypocreales; Genus: Chrysosporium (including the species *Chrysosporium lucknowense*) The order Onygenales, which includes this order; Genus: Rasamsonia (including the species emersonii), Penicillium (including the species verrucosum), Aspergillus (including species funiculosus, nidulans, niger, and oryzae), Talaromyces (including the species piniphilus (formerly Penicillium funiculosum)) Eurotiales, which includes This includes, but is not limited to, these.

[0083] It should be understood that the above list is not definitive and provides an incomplete list of industrially relevant Ascomycete filamentous fungal species.

[0084] It may be a filamentous fungal species of the Ascomycota other than the subphylum Pezizomycotina, but the tribe does not include the subphylum Saccharomycotina, but includes the most commonly known industrially related genera of non-filamentous fungi such as Saccharomyces, Komagataella (including the former Pichia pastoris), Kluyveromyces, or the subphylum Taphrinomycotina, as well as some other industrially related genera of commonly known non-filamentous fungi such as Schizosaccharomyces.

[0085] All of the above classification categories are defined according to the NCBI Taxonomy Browser (ncbi.nlm.nih.gov / taxonomy) as of the patent application date.

[0086] It must be acknowledged 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 can clearly determine if a particular fungal strain belongs to one of the groups defined above.

[0087] According to a particular embodiment, the filamentous fungus genus is selected from the group consisting of Myceliophthora, Thermothelomyces, Aspergillus, Penicillium, Trichoderma, Rasamsonia, Chrysosporium, Corynascus, 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 following: graminearum, Fusarium venenatum, Neurospora crassa, and Talaromyces piniphilus.

[0088] In particular, the present invention provides Thermothelomyces heterothallica strain C1 as a model for Ascomycetes filamentous fungi capable of producing large amounts of stable protein.

[0089] The terms “Thermothelomyces” and its species “Thermothelomyces heterothallica” and “thermophila” are used herein in the broadest sense as 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.

[0090] It is noteworthy that the authors mentioned above (Marin-Felix et al., 2015) proposed dividing 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 the newly established genus Thermothelomyces, which includes previously heat-tolerant Myceliophthora species, rather than to the genus Myceliophthora, which still includes non-heat-tolerant species. Since C1 can form ascospores with some other Thermothelomyces (formerly Myceliophthora) strains with opposite mating types, C1 is best classified as Th. heterothallica strain C1 rather than Th. thermophila C1.

[0091] Furthermore, it should be acknowledged that fungal taxonomy has constantly changed over time, and therefore, the current names listed above may sometimes be preceded by a variety of older names other than Myceliophthora thermophila, which are now considered synonymous (van Oorschot, 1977. Persoonia 9(3):403). For example, Thermothelomyces heterothallica (Marin-Felix et al., 2015. Mycologica, 3:619-63) is synonymous with Corynascus heterotchallicus, Thielavia heterothallica, Chrysosporium lucknowense, thermophile, and Sporotrichium thermophile (Alpinis 1963. Nova Hedwigia 5:74).

[0092] It should be further made clear that the present invention encompasses any strain containing a ribosomal DNA (rDNA) sequence exhibiting 99% or higher homology to Sequence ID No. 29, and all such strains are considered to be congenerally related to Thermothelomyces heterothallica.

[0093] In particular, the term Th. Heterothallica strain C1 encompasses genetically modified subordinate strains derived from wild-type strains that have been mutated using random or targeted approaches, for example, by using 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 and their derivatives.

[0094] It should be clearly understood 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, when genetically modified according to the teachings of the present invention, are capable of producing at least one protein product according to the teachings of the present invention. As used herein, the term “offspring” refers to offspring that are not or partially recombined from the parent fungal strain, e.g., cells from cells. The term “parent strain” refers to the corresponding fungal strain in which the expression or activity of the specific protease according to the present invention is not reduced.

[0095] Several 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 other Ascomycete filamentous fungal hosts. As a result, they can tolerate higher rates of carbon supply, leading to high yield production by this fungus.

[0096] According to some embodiments, the fungal growth medium includes a carbon source selected from the group consisting of glucose, sucrose, xylose, arabinose, galactose, fructose, lactose, cellobiose, glycerol, and any combination thereof.

[0097] This invention is particularly directed toward designing with little to no N-glycosylation modification. Note that O-glycans may be present, removed, or altered by further genetic recombination of fungi.

[0098] The terms “low expression” or “inhibited expression” of a protein as used herein are interchangeable and include, but are not limited to, deletion or disruption of the gene encoding that protein.

[0099] The terms “reduced activity” or “inhibited activity” of a protein as used herein are interchangeable and include, but are not limited to, post-translational modifications that result in reduced or lost activity of that protein.

[0100] It should be understood that the genetic modification according to the present invention is such that the genetically modified fungus can grow at a rate sufficient to be suitable for its intended use.

[0101] The above terms also encompass genetically modified subordinate strains derived from wild-type strains that have been mutated using random or targeted approaches, for example, by using UV mutagenesis or by deleting one or more endogenous genes.

[0102] Generally, Th. heterothallica fungi, and strain C1 in particular, exhibit higher biomass production compared to yeast strains when grown under appropriate conditions. Th. heterothallica fungi can be grown in large quantities of three-dimensional (3D) liquid cultures and solid media. Several strains developed by the applicants of this invention are less sensitive to feedback inhibition by glucose and other fermentable sugars present in the fungal growth medium as a carbon source compared to conventional yeasts and other fungi, and can tolerate higher rates of carbon source supply, leading to higher yields. Furthermore, some of these strains yield significantly lower medium viscosity when grown in commercial fermenters compared to the high viscosity obtained with non-glucose-suppressed wild-type Th. heterothallica fungi or other filamentous fungi known to be used for protein production. The lower viscosity may be due to morphological changes in the strains, from having long, highly contangled hyphae in the parent strain to short, less contangled hyphae in the developed strains. Low culture medium viscosity is a significant advantage in large-scale industrial production in fermenters. For example, the Th. Heterothallica C1 strain UV18-25, deposit number VKM F-3631 D, which exhibits low sensitivity to glucose inhibition, is industrially propagated to produce recombinant enzymes in volumes exceeding 100,000 liters.

[0103] The term “heterogeneous” is used herein to describe genes, enzymes, proteins, or peptide sequences that are not found or expressed in nature in Ascomycetes.

[0104] The term "endogenous" refers to genes, enzymes, proteins, or peptide sequences that are naturally present in Ascomycetes, such as those used for intracellular localization signals.

[0105] The term “exogenous” is used herein to describe synthetic polynucleotides that are exogenously introduced into Ascomycetes via transformation, when referring to polynucleotides. Exogenous polynucleotides may be introduced into Ascomycetes in a stable or transient manner to produce ribonucleic acid (RNA) molecules and subsequent polypeptide molecules.

[0106] Expression vector According to some embodiments, the genetically modified Ascomycetes filamentous fungi described herein contain at least one exogenous polynucleotide encoding the protein of interest.

[0107] The polynucleotide encoding the target protein may form part of a DNA construct or expression vector.

[0108] The terms “expression construct,” “DNA construct,” or “expression cassette” are interchangeable in this specification and refer to artificially constructed or isolated nucleic acid molecules that contain a nucleic acid sequence encoding a protein of interest and are assembled to express the protein of interest in target host cells. An expression construct typically contains a suitable regulatory sequence that is viably bound to the nucleic acid sequence encoding the protein of interest. An expression construct may further contain a nucleic acid sequence encoding a marker of choice.

[0109] The terms “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” are used herein to refer to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and their modified forms, either as distinct fragments of a larger construct or as components. A nucleic acid sequence may be a coding sequence, i.e., a sequence that codes for the final product in a cell, such as a protein. A nucleic acid sequence may also be a regulatory sequence, such as a promoter.

[0110] The terms “peptide,” “polypeptide,” and “protein” are used herein to refer to polymers of amino acid residues. The term “peptide” typically refers to an amino acid sequence consisting of 2 to 50 amino acids, while “protein” refers to an amino acid sequence consisting of more than 50 amino acid residues.

[0111] Sequences that are “homologous” to a reference sequence (such as nucleic acid sequences and amino acid sequences) refer, as herein, to percentage identity between sequences, where percentage identity is at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. Each possibility represents a distinct embodiment of the invention. Sequence homology described herein is encompassed by the invention. Protein homology is encompassed insofar as they maintain the activity of the original protein. Homologous nucleic acid sequences include variations related to codon use and genetic code denaturation. Sequence identity may be determined using nucleotide / amino acid sequence comparison algorithms known in the art.

[0112] Nucleic acid sequences encoding target proteins may be optimized for expression. Examples of such sequence modifications include, but are not limited to, altered G / C content, commonly known as codon optimization, which is closer to that typically found in Ascomycetes, and the removal of codons typically found in fungi.

[0113] The phrase "codon optimization" refers to the selection of DNA nucleotides suitable for use within a structural gene or fragment that approaches codon use within the organism of interest, and / or the modification of a nucleic acid sequence to enhance expression in the host cell of interest by replacing at least one codon of the native sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50) with a codon that is more frequently or most frequently used in the host cell's gene while maintaining the native amino acid sequence. Different species exhibit a particular bias towards certain codons of a particular amino acid. Codon bias (differences in codon use between organisms) often correlates with messenger RNA (mRNA) translation efficiency and, similarly, appears to depend, among other things, 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 is generally a reflection of the codons most frequently used in protein synthesis. Therefore, based on codon optimization, genes can be regulated for optimal gene expression within a given organism. Thus, an optimized gene or nucleic acid sequence refers to a gene whose nucleotide sequence, originally natural or spontaneously occurring, has been modified to utilize codons that are statistically preferred or statistically favored within the organism.

[0114] The term "regulatory sequence" refers to DNA sequences that control the expression (transcription) of codon sequences, such as promoters and terminators.

[0115] The term “promoter” refers to a regulatory DNA sequence that controls or directs the transcription of another DNA sequence in vivo or in vitro. Generally, promoters are located within the 5' region of the transcribed sequence (i.e., preceding, upstream). Promoters may be delivered whole from natural sources, or they may consist of different elements derived from different naturally occurring promoters, or they may 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). In most cases, the precise boundaries of regulatory sequences are not fully defined, and often cannot be fully defined, and for this reason, some variant DNA sequences may have the same promoter activity.

[0116] The term "terminator" refers to another regulatory DNA sequence that controls transcription termination. The terminator sequence is viablely bound to the 3' end of the nucleic acid sequence being transcribed.

[0117] According to some embodiments, the filamentous fungus is Th. heterothallica, and the protein of interest is expressed in a construct having regulatory elements of Th. heterothallica. According to certain embodiments, the construct expressing the protein of interest comprises a Th. heterothallica promoter and / or a Th. heterothallica terminator.

[0118] The terms "Th. Heterothallica promoter" and "Th. Heterothallica terminator" refer to promoter and terminator sequences suitable for use in Th. Heterothallica, i.e., capable of directing gene expression in Th. Heterothallica. In some specific embodiments, C1 promoters and C1 terminators are used, which refer to promoter and terminator sequences capable of directing gene expression in C1.

[0119] According to some embodiments, the Th. Heterothallica promoter / terminator is derived from an endogenous gene in Th. Heterothallica. According to other embodiments, the Th. Heterothallica promoter / terminator is derived from an exogenous gene in Th. Heterothallica.

[0120] Appropriate constitutive promoters and terminators include, for example, the phosphoglycerate kinase gene (PGK) (Uniprot:G2QLD8, NCBI reference sequence:XM_003665967), glyceraldehyde 3-phosphate dehydrogenase (GPD) (Uniprot:G2QPQ8, NCBI reference sequence:XM_003666768), and phosphofructokinase (PFK) (Uniprot:G2Q605, NCBI reference sequence:XM_003666768). This includes C1 glycosphagocytes such as the reference sequence: XM_003659879; or the β-glucosidase 1 gene bgl1 (acceptance number: XM_003662656); or triose phosphate isomerase (TPI) (Uniprot: G2QBR0, NCBI reference sequence: XM_003663200); or actin (ACT) (Uniprot: G2Q7Q5, NCBI reference sequence: XM_003662111); or the C1 cbh1 promoter (Genbank AX284115) or the C1 chi1 promoter (Genbank HI550986). Additional promoters that can be used are the Aspergillus nidulans gpdA promoter; and the synthetic promoter described by Rantasalo et al. (2018 NAR 46(18):e111). Typical terminators that can be used include the C1 chitinase 1 gene chi1 (Genbank HI550986), the cellobiohydrolase 1 cbh1 (Genbank AX284115), or the yeast adh1 terminator.

[0121] The term "viable binding" means that the selected nucleic acid sequence is in close proximity to a regulatory element (promoter or terminator) that allows the regulatory element to regulate the expression of the selected nucleic acid sequence.

[0122] Expression constructs according to some embodiments of the present invention include a Th. heterothallica promoter sequence and a Th. heterothallica terminator sequence viably conjugated to a protein-coding nucleic acid sequence. In some specific embodiments of the present invention, expression constructs include a C1 promoter sequence and a C1 terminator sequence viably conjugated to an enzyme-coding nucleic acid sequence.

[0123] Specific expression constructs can be assembled by a wide variety of methods, including conventional molecular biological techniques such as polymerase chain reaction (PCR), restriction endonuclease digestion, in vitro and in vivo assembly methods, and gene synthesis methods, or combinations thereof. Representative expression constructs and methods for their construction are given in the following Examples section.

[0124] Deletion of the stt3 and / or cwh8 genes Gene deletion techniques allow for the partial or complete removal of a gene, thereby eliminating its expression. In such a method, gene deletion can be achieved by homologous recombination using plasmids constructed to contain adjacent 5' and 3' regions of the gene in close proximity.

[0125] Gene deletion may also be performed by inserting a disruption nucleic acid construct, also referred to herein as a deletion construct, into the gene. The disruption construct may simply be a selectable marker gene containing homologous 5' and 3' regions to the gene. The selectable marker allows for the identification of transformants containing the disrupted gene. Alternatively, or further, the disruption nucleic acid construct may contain one or more polynucleotides encoding a heterologous protein expressed in the host cell.

[0126] The following Examples section provides representative deletion constructs and procedures for performing the deletions for stt3 and cwh8. As described herein, the stt3 and cwh8 genes are deleted using disruption constructs containing selectable markers, as shown in Example 1 below.

[0127] Deletions can be confirmed using PCR with an appropriate primer adjacent to the disrupted construct.

[0128] Genetically modified Th. heterothallica The genetically modified Th. heterothallica cells according to the present invention, which produce proteins with few or no N-linked glycans, are generated by modifying the cells, such as by deleting at least one of the two endogenous Th. heterothallica genes, stt3 and cwh8, so that the gene fails to produce functional proteins.

[0129] Endogenous gene deletions have been described above and are also shown in the Examples section below.

[0130] In yet another aspect, the present invention provides a method for producing an exogenous protein, comprising the steps of culturing a genetically modified fungus, particularly the Th. heterothallica C1 fungus of the present invention, in a suitable medium; and recovering the protein product thereof.

[0131] 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 lignocellulosic biomass containing polymeric carbohydrates such as molasses containing fermentable sugars, starch, cellulose, and hemicellulose, which are waste products obtained from ethanol production or other biological production from starch, sugar beets, and sugarcane.

[0132] According to some embodiments, exogenous proteins are purified from fungal growth media.

[0133] According to other embodiments, exogenous proteins are extracted from fungal masses. Any method known in the art for extracting and purifying proteins from plant tissues can be used.

[0134] In a further embodiment, the present invention provides exogenous proteins produced by genetically modified fungi, particularly the genetically modified Th. heterothallica C1 of the present invention.

[0135] Exogenous polynucleotide expression is carried out by introducing an expression construct containing nucleic acids encoding proteins expressed in fungi into fungal cells, particularly the nucleus. In particular, the genetic recombination according to the present invention means the integration of the expression construct into the host genome.

[0136] The introduction of expression constructs into fungal cells, i.e., fungal transformation, can be carried out by methods known in the art, for example, using the protoplast transformation method described in the Examples section below.

[0137] To facilitate the easy selection of transformed cells, selection markers may be transformed into fungal cells. “Selection markers” refer to polynucleotides encoding gene products that give a specific phenotype not present in non-transformed cells, such as antibiotic resistance (resistance markers), the ability to utilize specific resources (utilization / nutrient requirement markers), or the expression of a reporter protein that can be detected, for example, by spectral measurement. Nutritional requirement markers are typically preferred as a means of selection in the food or pharmaceutical industries. Selection markers can reside on separate nucleotides co-transformed with an expression construct, or on the same polynucleotide of the expression construct. Following transformation, positive transformants are selected, for example, by culturing the cells on a selective medium with the selected selection marker. In some cases, a split marker system is used, where the selection marker is split into two plasmids, and the functional selection marker is formed only when the two plasmids are simultaneously transformed and join together via homologous recombination.

[0138] [Table 1] TIFF0007873500000002.tif71160

[0139] [Table 2]

[0140] The following embodiments are provided to more fully illustrate certain 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.

[0141] Examples Example 1-C1 Deletion of stt3 and cwh8 genes To reduce or eliminate N-glycosylation of secreted proteins in C1 cells, the genes encoding the dolichol diphosphooligosaccharide protein glycosyltransfer catalytic subunit STT3 and the dolichol diphosphatase CWH8 were deleted. STT3 is a subunit of the multimeric oligosaccharide transferase (OST) complex and is essential for the complex's catalytic activity (Zufferey et al. 1995, EMBO Journal 14, 4949-4960). The OST complex catalyzes the transfer of oligosaccharides from the lipid carrier dolichol pyrophosphate to selected asparagine residues on polypeptide chains. Dolichol pyrophosphate phosphatase CWH8 is presumed to be involved in the recycling of the lipid carrier dolichol pyrophosphate. Although CWH8 is not essential for cell survival, deletion of the corresponding gene results in a loss of N-glycosylation (van Berkel et al. 1999, Glycobiology 9, 243-253).

[0142] The deletions were performed by individually transforming C1 with stt3 and cwh8 deletion constructs (one deletion / strain). DNA constructs for deleting stt3 or cwh8 were constructed in two separate plasmids. The 5' sequence plasmid contained the stt3 / cwh8 5' facile region fragment and the first half of the pyr4 marker gene for integration. The 3' group plasmid contained the remaining half of the pyr4 marker and the stt3 / cwh8 3' facile region fragment for integration. The pyr4 marker fragments in these two plasmids overlap each other. During the transformation of C1 with the two plasmids, the overlapping region undergoes homologous recombination between the plasmids, while the 5' and 3' facile region fragments are recombined with genomic DNA on both sides of the gene to be deleted. Recombination between selective marker fragments requires that the marker gene be functional, thus allowing for selective growth of the transformant. Approximately 500 bp from the end of the 5' adjacent region was added after the remaining half of the PYR4 marker to allow for loop-out of the marker gene if necessary. Different fragments of the stt3 and cwh8 5' and 3' sequences of the vector were amplified from C1 genomic DNA and cloned into a backbone vector (pRS426) using the marker gene by yeast recombinant cloning (Colot et al. 2006, PNAS 103, 10352-10357).

[0143] The 5' sequence of the stt3 deletion construct is described in SEQ ID NO: 1. The 5' adjacency sequence corresponds to positions 1-930 of SEQ ID NO: 1, and the first half of the pyr4 marker gene corresponds to positions 938-2,717 of SEQ ID NO: 1. The 3' sequence of the stt3 deletion construct is described in SEQ ID NO: 2. The remaining half of the pyr4 marker gene corresponds to positions 1-1,257 of SEQ ID NO: 2. The direct repeat sequence corresponds to positions 1,266-1,777 of SEQ ID NO: 2. The 3' adjacency sequence corresponds to positions 1,785-2,715 of SEQ ID NO: 2.

[0144] The 5' sequence of the cwh8 deletion construct is described in SEQ ID NO: 3. The 5' adjacency sequence corresponds to positions 1-1,000 of SEQ ID NO: 3, and the first half of the pyr4 marker gene corresponds to positions 1,008-2,787 of SEQ ID NO: 3. The 3' sequence of the cwh8 deletion construct is described in SEQ ID NO: 4. The remaining half of the pyr4 marker gene corresponds to positions 1-1,257 of SEQ ID NO: 4. The direct repeat sequence corresponds to positions 1,266-1,765 of SEQ ID NO: 4. The 3' adjacency sequence corresponds to positions 1,774-2,973 of SEQ ID NO: 4.

[0145] Both sequences of the stt3 / cwh8 deletion construct were excised from the plasmid backbone and simultaneously transformed into the C1 strain DNL132, which has nine deletions in the protease gene. A pair of one 5' sequence vector and one 3' sequence vector was used for each transformation.

[0146] Transformant colonies grown on selective medium plates were cultured as striae on selective medium. Identification of transformants with accurate integration of the deletion construct was performed by PCR. Mycelium from transformant striae was dissolved in 20 mM NaOH and incubated at 100°C to lyse the cells. 1-2 μl of this lysate was used as a template for PCR using the Phire Plant PCR Kit (Thermo Fisher). The oligonucleotide primers used are shown in Table 1.

[0147] Integration of the deletion construct into the stt3 locus was demonstrated by two PCR reactions. Integration at the 5' end of the gene was verified using the primers described in SEQ ID NOs. 5 and 6. Amplification of a 1152 bp fragment indicated successful integration into the stt3 locus at the 5' end of the gene. Integration at the 3' end of stt3 was verified using the primers described in SEQ ID NOs. 7 and 8. Amplification of a 1752 bp fragment indicated successful integration into the stt3 locus at the 3' end of the gene. Quantitative PCR using the primers described in SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11 and 12, further analyzed transformants positive for integration into the stt3 locus, demonstrating complete deletion of the stt3 gene from them. Transformant strain C1, positive for construct integration into the stt3 locus and negative for the presence of the stt3 gene, was stored at -80°C and given strain number M3210.

[0148] Integration of the deletion construct into the cwh8 locus was demonstrated by two PCR reactions. Integration at the 5' end of the gene was verified using the primers described in SEQ ID NOs. 13 and 6. Amplification of a 1243 bp fragment indicated successful integration into the cwh8 locus at the 5' end of the gene. Integration at the 3' end of the gene was verified using the primers described in SEQ ID NOs. 14 and 8. Amplification of a 2009 bp fragment indicated successful integration into the cwh8 locus at the 3' end of the gene. Quantitative PCR using the primers described in SEQ ID NOs. 15 and 16, and SEQ ID NOs. 17 and 18, further analyzed transformants positive for integration into the cwh8 locus, demonstrating complete deletion of the cwh8 gene. Transformant strain C1, positive for construct integration into the cwh8 locus and negative for the presence of the cwh8 gene, was stored at -80°C and given strain number M3211.

[0149] [Table 3]

[0150] Strains M3210 and M3211 were cultured in a 1 L bioreactor for 7 days in a medium containing yeast extract and glucose as a carbon source, using a fed-batch process. The supernatant sample on day 7 was centrifuged three times at 13500 rpm for 15 minutes, and glycan analysis of total protein present in the supernatant was performed using the GlycoWorks™ RapiFluor-MS™ N-glycan Kit (Waters) according to the manufacturer's protocol. Strain M2864, which has nine protease deletions, was used as a control for N-glycan analysis. N-glycans could not be detected from the culture supernatant of the stt3 deletion strain, but the control strain showed a normal C1 glycan pattern (Figures 2A and 2B). The amount of N-glycans detected from the culture supernatant of the cwh8 deletion strain was approximately 10% of the amount of N-glycans in the control strain (Figures 3A and 3B).

[0151] Example 2 - Production of monoclonal antibodies in strains M3210 and M3211 To demonstrate the production of monoclonal antibodies without N-glycosylation and monoclonal antibodies with low N-glycosylation, the heavy and light chains of the therapeutic antibody nivolumab were expressed in M3210 and M3211 strains, respectively, which have stt3 or cwh8 deletions.

[0152] As described in Example 1, two pairs of expression cassettes were constructed on two separate plasmids. The 5' sequence of the construct contains a cbh1 5' flanking region fragment for integration, and the expression cassette in which the nivolumab light chain gene is fused to the C1 CBH1 signal sequence is between the bgl8 promoter and bgl8 terminator, and between the nia1 marker gene and the first half of the hygromycin marker gene. The 3' sequence of the construct contains the last half of the hygromycin marker, a direct repeat sequence from the bgl8 terminator, and the expression cassette in which the nivolumab heavy chain gene is fused to the CBH1 signal sequence from C1 is between the bgl8 promoter and chi1 terminator, as well as a cbh1 3' flanking region fragment for integration.

[0153] The 5' sequence of the nivolumab expression construct is described in SEQ ID NO: 19. The cbh1 5' adjacent sequence corresponds to positions 1-1,957 of SEQ ID NO: 19. The bgl8 promoter sequence corresponds to positions 1,966-3,357 of SEQ ID NO: 19. The sequence encoding the light chain fused to the C1 CBH1 signal sequence corresponds to positions 3,358-4,053 of SEQ ID NO: 19, with positions 3,358-3,408 encoding the CBH1 signal sequence and positions 3,409-4,053 encoding the light chain. This sequence was obtained by codon optimization of the human light chain gene for C1 and synthesis using a gene script. The synthesized sequence contained 40 bp flanks for the bgl8 promoter and terminator. The bgl8 terminator sequence corresponds to positions 4,054-4,520 of SEQ ID NO: 19. The nia1 marker gene corresponds to positions 4,537–8,651 of sequence number 19. The first half of the hygromycin marker gene corresponds to positions 8,660–10,360 of sequence number 19. The aforementioned fragments and the backbone vector pRS426 were assembled together using Gibson assembly to obtain the 5' sequence vector.

[0154] The 3' sequence of the nivolumab expression construct is described in SEQ ID NO: 20. Half of the hygromycin marker gene corresponds to positions 1-1,732 of SEQ ID NO: 20. The chi1 terminator sequence corresponds to positions 2,082-2,727 of SEQ ID NO: 20. The sequence encoding the heavy chain fused to the C1 CBH1 signal sequence corresponds to positions 2,736-4,109 of SEQ ID NO: 20, with positions 4,059-4,109 encoding the CBH1 signal sequence and positions 2,736-4,058 encoding the heavy chain. This sequence was obtained by codon optimization of the human heavy chain gene for C1 and synthesis using GenScript. It contains 40 bp flanks for the bgl8 promoter and chi1 terminator. The bgl8 promoter sequence corresponds to positions 4,110-5,501 of SEQ ID NO: 20. The 3' adjacent sequence corresponds to positions 5,510-6,266 of sequence number 20. The aforementioned fragment and backbone vector pRS426 were assembled together using Gibson assembly to obtain the 3' sequence vector.

[0155] Transformation of nivolumab expression plasmids into M3210 and M3211 strains, each carrying either the stt3 or cwh8 deletion, and selection of transformants were performed on selective medium plates containing 50 mg / l hygromycin. Transformants were screened by PCR to identify clones in which the cbh1 gene was replaced by the construct. The primers used for screening are shown in Table 2. Using the primers described in SEQ ID NOs. 21 and 22, accurate integration at the 5' end of the cbh1 gene was verified. Amplification of a 3592 bp fragment indicated successful integration into the cbh1 locus at the 5' end of the gene. Using the primers described in SEQ ID NOs. 23 and 24, accurate integration at the 3' end of the cbh1 gene was verified. Amplification of a 1477 bp fragment indicated successful integration into the cbh1 locus at the 3' end of the gene. We verified the complete deletion of the cbh1 gene by demonstrating the absence of amplification of a 500 bp fragment from the cbh1 open reading frame using primers from SEQ ID NOs. 25 and 26.

[0156] [Table 4]

[0157] As in Example 1, the constructed C1 strain was grown in liquid medium in a 24-well plate. Mycelium was removed by centrifugation at 3500 RPM for 10 minutes of 250 μl of the sample passed through a 0.65 μm MultiScreen filter plate (Merck Millipore). Nivolumab production by the transformants was confirmed by Western blotting. Transformants showing nivolumab production were purified by single colony plating on a selective medium plate as follows: Mycelium from the muscle was suspended in 800 μl of 0.9% NaCl-0.025% Tween 20 solution. Different dilutions of the suspension (10 -1 , 10 -2 and 10 -3A solution of 0.9% NaCl-0.025% Tween20 was prepared, and 100 μl of the dilution was plated onto a selective medium to obtain single colonies. Nivolumab production by the purified transformants was verified by 24-well plate culture and subsequent Western blot analysis, as in Example 1.

[0158] Purified transformants of the stt3 deletion strain M3210, which is positive for integration of the expression construct into the cbh1 locus and produces both the heavy and light chains of nivolumab, were stored at -80°C and given strain number M3480. Purified transformants of the cwh8 deletion strain M3211, which is positive for integration of the expression construct into the cbh1 locus and produces both the heavy and light chains of nivolumab, were stored at -80°C and given strain number M3481.

[0159] Strains M3480 and M3481 were cultured in a 1 L bioreactor for 7 days in a medium containing yeast extract and glucose as a carbon source, using a fed-batch process. Nivolumab produced by these strains was purified by passing it through a 1 ml MabSelect SuRe Protein A column (GE Healthcare) using an AKTA Start protein purification system (GE Healthcare) according to the manufacturer's protocol. Glycan analysis of the purified antibody was performed using the GlycoWorks® RapiFluor-MS® N-glycan kit (Waters) according to the manufacturer's protocol. Nivolumab purified from the fermentation supernatant of the non-glycolytic strain M3242 was used as a control in the glycan analysis. N-glycans could not be detected in nivolumab purified from the M3480 culture supernatant, but nivolumab purified from the control strain showed a normal C1 glycan pattern (Figures 4A and 4B). The amount of N-glycan detected in nivolumab purified from the culture supernatant of the cwh8 deletion strain M3481 was approximately 11% of the amount of N-glycan detected in nivolumab purified from the control strain (Figures 5A and 5B).

[0160] Peptide mapping of purified nivolumab was performed according to the following method: 100 μg of the sample underwent three first buffer exchanges with 50 mM ammonium bicarbonate using a Vivaspin 500 (10,000 MWCO, PES, Sartorius). Rapigest SF (Waters) was added to a final concentration of 0.1%, followed by dithiothreitol (DTT) to a final concentration of 5 mM. The sample was incubated at 60°C for 40 minutes. Next, iodoacetamide (IAM) was added to a final concentration of 15 mM, and the sample was incubated at room temperature in the dark for 40 minutes. Trypsin (Promega) solution was added to each sample until the final protease-to-protein ratio reached 1:50 (w / w), or 1 μl of trypsin (1 μg / ml) was added per 50 μg of protein. The sample was incubated overnight at 37°C. The reaction was stopped by adding 4 μl of 20% TFA, and the sample was incubated at 37°C for 30 minutes and centrifuged at 10000 rpm for 5 minutes. Finally, the sample was evaporated using SpeedVac and reconstituted with 50 μl of water / AcCN / TFA 20 / 80 / 0.1 (v / v / v). The following LC-MS conditions were used: Instruments: Acquity UHPLC system, Waters (Milford, MA, USA) and Waters Synapt G2-S MS system (Milford, MA, USA). Column: ACQUITY UPLC glycoprotein amide 300A, 1.7 μm, 2.1 × 150 mm (Waters) at 60°C. Solvent: A is 0.1% TFA in water, B is 0.1% TFA in acetonitrile. Gradient program: 0 min = 10% A, 70 min = 50% A, flow rate 0.2 ml / min. The mass spectrometry parameters used were positive polarity, with a cone voltage of 25V and capillary voltage of 3kV, a desolvation temperature of 350°C, and a source temperature of 120°C. MS was performed using an energy gradient of 25–40V. E Data was collected in the mode's m / z range of 50 to 2000. The data was processed using UNIFI software (Waters).

[0161] Nivolumab purified from the fermentation supernatant of the non-glycolytic strain M3242 was used as a control for peptide mapping analysis. According to the peptide mapping results, no N-glycans were bound to the heavy chain of nivolumab produced by the stt3 deletion strain M3480 (Table 3). A small amount of N-glycosylation of 26.6% was detected in the heavy chain of nivolumab produced by the cwh8 deletion strain M3481 (Table 4), while 95.8% N-glycosylation was detected in the heavy chain of nivolumab produced by the non-glycolytic C1 strain M3242 (Table 5).

[0162] [Table 5]

[0163] [Table 6]

[0164] [Table 7]

[0165] The foregoing description of specific embodiments has so sufficiently revealed the general nature of the invention that others may readily modify and / or adapt such specific embodiments for various uses without excessive experimentation and without departing from the general concept, by applying current knowledge. Therefore, such adaptations and modifications should and are intended to be understood within the scope of the meaning and equivalents of the disclosed embodiments. It should be understood that any expressions or technical terms used herein are for illustrative purposes only and not limiting. Means, materials, and steps for carrying out various disclosed chemical structures and functions can take on a wide variety of alternative forms without departing from the invention.

Claims

1. A genetically modified Thermothelomyces heterothallica fungus capable of producing heterologous proteins with low or no N-linked glycosylation, comprising at least one cell having reduced expression and / or activity of STT3 and / or CWH8, wherein the at least one cell comprises at least one exogenous polynucleotide encoding the heterologous protein.

2. The genetically modified fungus according to claim 1, wherein at least one of the cells has reduced expression and / or activity of STT3.

3. The genetically modified fungus according to claim 2, wherein the STT3 comprises the amino acid sequence of Thermoceromyces heterotalica STT3 described in Sequence ID No.

27.

4. The genetically modified fungus according to any one of claims 1 to 3, wherein at least one cell has reduced expression and / or activity of CWH8.

5. The genetically modified fungus according to claim 4, wherein the CWH8 comprises the amino acid sequence of Thermoceromyces heterotalica CWH8 described in Sequence ID No.

28.

6. A genetically modified fungus according to any one of claims 1 to 5, comprising at least one cell in which the expression and / or activity of STT3 and CWH8 is reduced.

7. The genetically modified fungus according to any one of claims 1 to 6, wherein the genetic modification includes deletion or disruption of the stt3 gene, causing failure to produce a catalytic subunit of the oligosaccharide transferase (OST) complex.

8. The genetically modified fungus according to any one of claims 1 to 7, wherein the genetic modification includes deletion or disruption of the cwh8 gene so as to fail to produce functional dolichol pyrophosphate phosphatase.

9. The genetically modified fungus according to any one of claims 1 to 8, wherein the fungus is Thermoselomyces heterotalica C1.

10. The genetically modified fungus according to any one of claims 1 to 9, wherein the heterologous protein is selected from the group consisting of antigens, therapeutic proteins, antibodies, enzymes, vaccines, and structural proteins.

11. The genetically modified fungus according to any one of claims 1 to 10, wherein the heterologous protein is a secreted protein.

12. The genetically modified fungus according to any one of claims 1 to 11, wherein the fungus is a strain further modified to delete one or more genes encoding an endogenous protease.

13. The genetically modified fungus according to any one of claims 1 to 12, wherein the fungus comprises at least one cell in which the expression and / or activity of at least five proteases is reduced.

14. A method for producing Thermoceromyces heterotalica fungi capable of producing proteins with little or no N-glycans, wherein: a) A step of reducing the expression and / or activity of the STT3 protein of the fungus; and / or b) A step of reducing the expression and / or activity of the CWH8 protein of the fungus. Methods that include...

15. a) deleting or disrupting the stt3 gene of the fungus so as to reduce the production of the functional catalytic subunit of the oligosaccharide transferase (OST) complex; and / or b) The step of deleting or disrupting the cwh8 gene of the fungus in such a way that it reduces the production of functional dolichol pyrophosphate phosphatase. The method according to claim 14, including the method described in claim 14.

16. The method according to claim 15, further comprising the step of introducing an exogenous polynucleotide encoding a heterologous protein into the fungus, thereby expressing the heterologous protein in which the fungus has little or no N-glycans.

17. A method for producing heterologous proteins that have little or no N-glycans: a) A genetically modified Thermoceromyces heterotalica fungus according to any one of claims 1 to 13, comprising the fungus containing an exogenous polynucleotide encoding a heterologous protein; b) the step of culturing the fungus under conditions suitable for expressing the heterologous protein; and c) Step of recovering the heterologous protein. Methods that include...

18. The method according to claim 17, wherein the heterologous protein is a heterologous mammalian protein recombinantly expressed in the fungus.

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