Glycoengineering of THERMOTHEMYCES HETEROTHALLICA

Abstract

In particular, provided are Thermothelomyces thermophila (Myceliomyces thermophila) genetically modified to produce glycoproteins having N-glycans of mammalian proteins, in particular human, companion animal and other animal proteins, which comprise a deletion or disruption of the alg3 gene, the expression of ER-targeted mannosidase 1 (alpha-1, 2-mannosidase), and the expression of the ER-targeted glucosidase 2 alpha subunit. The Th.hetero-thalica can further comprise a heterologous GlcNAc transferase 1 (GNT1), a GlcNAc transferase 2 (GNT2), an STT3 subunit of a heterologous oligoglycosyl transferase, and a galactosyl transferase, and can be used for preparing a transferase product.

Description

Technical Field

[0001] The present invention relates to genetically modified Thermothelomyces heterothallica (formerly known as Myceliophthora thermophila) in which the protein glycosylation pathway is engineered with minimal disruption to endogenous genes to produce proteins with N-glycans similar to mammalian proteins, particularly human proteins. Background Art

[0002] Most therapeutic proteins require glycosylation to ensure correct folding, function and activity. Glycosylation of therapeutic proteins is also particularly important for their immunogenicity. Therefore, such proteins cannot be produced in standard prokaryotic expression systems that lack necessary glycosylation machinery. Since glycosylation and other post-translational modifications are essential for therapeutic glycoproteins, most of them are currently produced in mammalian cells. However, the fermentation process based on mammalian cell culture (such as CHO, rodent or human cells) is usually very slow, requires expensive nutrients and cofactors (such as fetal bovine serum or specific growth factors), usually obtains low product titers, and is also susceptible to infection by infectious sources that may contaminate the resulting protein product. Therefore, more and more are turning to serum-free expression systems. Specifically, yeast and fungi are being developed as alternative protein expression systems.

[0003] As eukaryotic organisms, yeast and fungi are capable of post-translational modifications, including N- and O-glycosylation, but protein glycosylation in yeast and fungi differs greatly from that in mammalian cells. To overcome these problems, the possibility of reengineering the N-glycosylation pathway has been explored, particularly in species most commonly used for heterologous protein production (e.g., S. cerevisiae, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, and Aspergillus and Trichoderma species). However, protein yields still need to be improved, particularly in the need to improve the glycosylation step so that a high proportion of the produced proteins carry the desired glycoform, i.e., the glycoform of mammalian (particularly human and companion animal) proteins.

[0004] Parsaie Nasab et al., 2013, Appl Environ Microbiol., 79(3): 997-1007 describe a synthetic N-glycosylation pathway to produce recombinant proteins carrying human N-glycans in Saccharomyces cerevisiae. A Δalg3Δalg11 double mutant strain was used, which was further genetically modified to express artificial flippase, protozoan oligosaccharyltransferase, and human N-acetylglucosamine transferase I and II targeted to the Golgi apparatus. The results confirmed the presence of complex human N-glycan structures GlcNAc on secreted monoclonal antibodies recombinantly expressed in the mutant strain. 2 Man 3 GlcNAc 2 However, heterogeneity in N-linked glycans was observed due to interference by Golgi-localized mannosyltransferases.

[0005] The work of Parsaie Nasab et al. is also recorded in US 2011 / 0207214, which discloses cells modified to express lipid-linked oligosaccharide (LLO) flippase activity, which is capable of flipping LLO containing 1 mannose residue, 2 mannose residues and 3 mannose residues from the cytoplasmic side to the luminal side of intracellular organelles, and is further reviewed together with other related studies in De Wachter et al., 2018, "Engineering of Yeast Glycoprotein Expression" (Advances in Biochemical Engineering / Biotechnology, Springer, Berlin, Heidelberg).

[0006] US 7,029,872, US 7,326,681, US 7,629,163, US 7,981,660 disclose cell lines with genetically modified glycosylation pathways that allow them to perform a series of enzymatic reactions that mimic the processing of glycoproteins in humans. Eukaryotic organisms such as unicellular and multicellular fungi that normally produce high mannose N-glycans are modified to produce N-glycans such as Man 5 GlcNAc 2 or other structures along the human glycosylation pathway.

[0007] US 7,449,308 and US 7,935,513 disclose eukaryotic host cells with modified oligosaccharides, which can be further modified by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become a host strain for producing mammalian, such as human, therapeutic glycoproteins. The N-glycans produced in the engineered host cells have Man s GlcNAc 2 The core structure, which can then be further modified by heterologous expression of one or more enzymes such as glycosyltransferases, sugar transporters, and mannosidases, produces a human-like glycoprotein.

[0008] US 7,795,002 discloses eukaryotic host cells such as yeast and filamentous fungi that produce human-like glycoproteins characterized by having terminal β-galactose residues and substantially lacking fucose and sialic acid residues. Further disclosed is a method for catalyzing the transfer of galactose residues from UDP-galactose to an acceptor substrate in a recombinant eukaryotic host cell, which acceptor substrate can be used as a therapeutic glycoprotein.

[0009] US 8,986,949 discloses genetically engineered strains of non-mammalian eukaryotic organisms that express catalytically active endomannosidase genes to enhance the processing of N-linked glycan structures, with the overall goal of obtaining more human-like glycan distributions. In addition, the cloning and expression of novel human and mouse endomannosidases are disclosed.

[0010] US 9,359,628 discloses a genetically engineered strain of Pichia pastoris capable of producing proteins with smaller polysaccharides. Specifically, the genetically engineered strain is capable of expressing either or both of α-1,2-mannosidase and glucosidase II. The genetically engineered strain can be further modified so that the OCH1 gene is destroyed. A method for producing a glycoprotein with smaller polysaccharides using such genetically engineered strain of Pichia pastoris is also provided.

[0011] US 9,695,454 discloses a composition comprising a filamentous fungal cell (e.g., a Trichoderma fungal cell) having reduced protease activity and expressing a fucosylation pathway. Further described is a method for producing a glycoprotein having fucosylated N-glycans using a genetically modified filamentous fungal cell (e.g., a Trichoderma fungal cell) as an expression system.

[0012] Thermothelomyces heterothallica (Th. heterothallica) strain C1 (recently renamed from Myceliophthora thermophila, which in turn was renamed from Chrysosporium lucknowense) is a thermostable ascomycete filamentous fungus that produces high levels of cellulases, making it attractive for production of these and other enzymes on a commercial scale.

[0013] For example, U.S. Patent Nos. 8,268,585 and 8,871,493 disclose a transformation system for expressing and secreting heterologous proteins or polypeptides in the field of filamentous fungal hosts. A method for producing a large amount of polypeptides or proteins in an economical manner is also disclosed. The system includes a transformed or transfected fungal strain of Chrysosporium, more specifically Chrysosporium lucknowense, and a mutant or derivative thereof. A transformant containing a Chrysosporium coding sequence and an expression control sequence of a Chrysosporium gene is also disclosed.

[0014] Wild-type C1 was deposited under the Budapest Convention under the number VKMF-3500D on August 29, 1996. High cellulase (HC) and low cellulase (LC) strains have also been deposited, as described in U.S. Pat. No. 8,268,585. For example: strain UV13-6, deposited under the number VKM F-3632 D; strain NG7C-19, deposited under the number VKM F-3633 D; strain UV18-25, deposited under the number VKM F-3631 D.

[0015] Other improved C1 strains that have been deposited include: (i) HC strain UV18-100f (Δalp1Δpyr5), deposit number CBS141147; (ii) HC strain UV18-100f (Δalp1Δpep4Δalp2Δpyr5Δprt1), deposit number CBS141143; (iii) LC strain W1L#100I (Δchi1Δalp1Δalp2Δpyr5), deposit number CBS141153; and (iv) LC strain W1L#100I (Δchi1Δalp1Δpyr5), deposit number CBS141149.

[0016] EP 2505651 discloses an isolated fungus which has been mutated or selected to have low protease activity, wherein the fungus has less than 50% protease activity compared to the non-mutated fungus. The fungus belongs to the genus Chrysosporium, preferably it is a strain of Chrysosporium lucknowense.

[0017] WO 2021 / 094935 belonging to the applicant of the present invention discloses genetically modified Thermothelomycesheterothallica, in which the protein glycosylation pathway has been engineered to produce proteins with N-glycans similar to those in humans. WO 2021 / 094935 discloses the deletion or disruption of alg3 and alg11 genes, overexpression of flippase, and expression of heterologous GlcNAc transferase 1 (GNT1) and GlcNAc transferase 2 (GNT2).

[0018] There is a need for additional alternative expression systems for producing recombinant human, companion animal and other mammalian proteins that are capable of producing high yields of glycoproteins having N-glycans of mammalian proteins, particularly human and companion animal proteins, such that the proteins are suitable for therapeutic use in humans, companion animals and other mammals. Summary of the invention

[0019] The present invention provides Thermothelomyces heterothallica that is genetically modified to produce glycoproteins with N-glycans of mammalian proteins, particularly N-glycans of human proteins. The genetic modification of Th. heterothallica of the present invention includes the deletion or destruction of the alg3 gene, the heterologous expression or overexpression of the mannosidase 1 (α-1,2-mannosidase) targeting the ER and the glucosidase 2α subunit targeting the ER. The genetic modification of the Th. heterothallica may further include the expression of heterologous GlcNAc transferase 1 (GNT1) and GlcNAc transferase 2 (GNT2). In some embodiments, the genetic modification further includes the expression of the STT3 subunit of a heterologous oligosaccharyl transferase (OST). In other embodiments, the genetic modification further includes the expression of a heterologous galactosyl transferase. In some embodiments, the genetic modification further includes the overexpression of an endogenous flippase or the expression of a heterologous flippase.

[0020] The present invention is based, in part, on the discovery that genetically modified Th. heterothallica as disclosed herein produce glycoproteins in which desired mammalian / human N-glycans account for greater than 90% of the N-glycans present on the glycoprotein, and in some cases even greater than 95% or greater than 98% of the N-glycans. Moreover, when further modified to express heterologous mammalian glycoproteins (e.g., antibodies), genetically modified Th. heterothallica as disclosed herein produce high levels of heterologous glycoproteins in which desired mammalian / human N-glycans account for greater than 90% of their N-glycans. This is in contrast to expression systems described to date, which produce a wide range of variations in the N-glycans obtained. Remarkably, no significant negative impact on cell viability was observed for any of the modifications made.

[0021] It is further disclosed that the expression of ER-targeted mannosidase 1 (α-1,2-mannosidase) and ER-targeted glucosidase 2 α subunit does not reduce cell viability or the production level of heterologous glycoproteins. It is now disclosed that, unexpectedly, desired mammalian / human N-glycans are achieved in the produced glycoproteins without the need to disrupt the expression of endogenous α-1,2-mannosyltransferase (ALG11).

[0022] It is noteworthy that, unlike most fungi and yeasts, Th. heterothallica does not have highly mannosylated N-glycans that may contain up to 50 mannose residues, but rather has "oligomannose" N-glycans containing 3-9 mannose residues and hybrid N-glycans containing mannose and HexNAc residues whose structures have not yet been fully characterized. Since the structure and synthetic pathway of the hybrid N-glycans have not been fully characterized, it is unclear whether such glycans can be eliminated using the genetic modifications described herein. Surprisingly, the genetic modifications according to the present invention are sufficient to result in the substantial elimination of these structures, with more than 90% of the N-glycoforms being the desired mammalian / human N-glycans without the need to reduce the expression of alg11.

[0023] Advantageously, the Th. heterothallica cells of the invention produce high yields of protein. The protein levels obtained using the Th. heterothallica cells of the invention are much higher than those obtained using, for example, yeast.

[0024] Thus, the present invention provides a method for producing a glycoprotein having desired N-glycans suitable for therapeutic use in humans.

[0025] According to one aspect, the invention provides a Thermothelomyces heterothallica genetically modified to produce a glycoprotein having mammalian N-glycans, wherein the genetic modification comprises:

[0026] (i) the alg3 gene is deleted or destroyed, so that the Th. heterothallica cannot produce functional α-1,3-mannosyltransferase;

[0027] (ii) expression of ER-targeted mannosidase 1 (α-1,2-mannosidase); and

[0028] (iii) Expression of glucosidase 2α subunit targeted to the ER.

[0029] In some embodiments, the mannosidase 1 is Trichoderma reesei mannosidase 1. In other embodiments, the mannosidase 1 is Th. heterothallica mannosidase 1. In some embodiments, the glucosidase 2 alpha subunit is selected from Th. Heterothallica, T. reesei and Aspergillus niger glucosidase 2 alpha subunit. In other embodiments, the genetic modification further comprises the expression of a glucosidase beta subunit.

[0030] In some embodiments, the Trichoderma reesei mannosidase 1 targeted to ER comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the Trichoderma reesei mannosidase 1 targeted to ER is encoded by an exogenous polynucleotide introduced into the Th. heterothallica comprising the sequence set forth in SEQ ID NO: 1 or an analog or derivative thereof having at least 90% sequence identity.

[0031] In some embodiments, the Th. heterothallica glucosidase 2α subunit targeted to the ER comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the Th. heterothallica glucosidase 2α subunit targeted to the ER is encoded by an exogenous polynucleotide introduced into the Th. heterothallica comprising the sequence set forth in SEQ ID NO: 3 or an analog or derivative thereof having at least 90% sequence identity.

[0032] In some embodiments, the ER-targeted mannosidase 1 (α-1,2-mannosidase) and / or the ER-targeted glucosidase 2a subunit are integrated into the alp3 protease locus within the Th. Heterothallica genome. In certain embodiments, the ER-targeted mannosidase 1 (α-1,2-mannosidase) and the ER-targeted glucosidase 2α subunit are both integrated into the alp3 protease locus within the Th. Heterothallica genome.

[0033] In some embodiments, the genetic modification further comprises expression of heterologous GlcNAc transferase 1 (GNT1) and GlcNAc transferase 2 (GNT2).

[0034] In some embodiments, heterologous GNT1 and GNT2 according to the present invention are of animal origin.

[0035] In some embodiments, the animal-derived GNT1 according to the present invention comprises a heterologous Golgi localization signal.

[0036] In some embodiments, the animal-derived GNT1 according to the present invention is human GNT1. In some embodiments, the animal-derived GNT1 according to the present invention is human GNT1 comprising a heterologous Golgi localization signal.

[0037] In some embodiments, the animal-derived GNT1 according to the present invention is bovine GNT1. In some embodiments, the animal-derived GNT1 according to the present invention is bovine GNT1 comprising a heterologous Golgi localization signal.

[0038] In some embodiments, the heterologous Golgi localization signal according to the present invention is a Th. heterothallica Golgi localization signal. In some embodiments, the Th. heterothallica Golgi localization signal is from the Th. heterothallica protein KRE2.

[0039] In other embodiments, the heterologous Golgi localization signal according to the invention is a yeast Golgi localization signal. In some embodiments, the yeast Golgi localization signal is from the yeast protein KRE2.

[0040] In some embodiments, the animal-derived GNT2 according to the present invention is rat GNT2. In other embodiments, the animal-derived GNT2 according to the present invention is human GNT2.

[0041] In some embodiments, the animal-derived GNT1 is human GNT1, and the animal-derived GNT2 is rat GNT2. In some embodiments, the human GNT1 comprises a Th. heterothallica Golgi localization signal. In some embodiments, the Th. heterothallica Golgi localization signal is from C1 Th. heterothallica protein KRE2. In other embodiments, the human GNT1 comprises a yeast Golgi localization signal.

[0042] In some embodiments, the animal-derived GNT1 is human GNT1, and the animal-derived GNT2 is rat GNT2. In some embodiments, the human GNT1 comprises a Th. heterothallica Golgi localization signal. In some embodiments, the Th. heterothallica Golgi localization signal is from C1 protein KRE2. In other embodiments, the human GNT1 comprises a yeast Golgi localization signal.

[0043] In some embodiments, Th. heterothallica according to the present invention is genetically modified to overexpress an endogenous Th. heterothallica RFT1 flippase.

[0044] In other embodiments, Th. heterothallica according to the present invention is genetically modified to express a heterologous flippase, wherein the heterologous flippase is a yeast FLC2p flippase.

[0045] In some embodiments, the genetic modification according to the present invention further comprises the expression of a STT3 subunit of a heterologous oligosaccharyltransferase (heterologous STT3). In some embodiments, the heterologous STT3 according to the present invention is Leishmania major STT3.

[0046] In some embodiments, the genetic modification according to the present invention further comprises the expression of a heterologous galactosyltransferase. In some embodiments, the heterologous galactosyltransferase is a galactosyltransferase of animal origin.

[0047] In some embodiments, the animal-derived galactosyltransferase is a human galactosyltransferase. In some embodiments, the animal-derived galactosyltransferase according to the present invention is a human galactosyltransferase comprising a heterologous Golgi localization signal, such as a Th. heterothallica KRE2 Golgi localization signal.

[0048] In other embodiments, the animal-derived galactosyltransferase is a tropical clawed frog (Xenopus tropicalis) galactosyltransferase. In some embodiments, the animal-derived galactosyltransferase according to the present invention is a tropical clawed frog (Xenopus tropicalis) galactosyltransferase comprising a heterologous Golgi localization signal, such as a S. cerevisiae (S. cerevisiae) KRE2 Golgi localization signal.

[0049] In some embodiments, the Th. heterothallica is Th. heterothallica C1.

[0050] In some embodiments, the C1 is a strain modified to delete one or more genes encoding endogenous proteases.

[0051] In some embodiments, the C1 is a strain modified to delete a gene encoding an endogenous chitinase.

[0052] In some embodiments, the C1 is a strain selected from the following: wild-type C1 deposit number VKM F-3500 D, UV13-6 deposit number VKM F-3632 D, NG7C-19 deposit number VKM F-3633 D, UV18-25, deposit number VKM F-3631D, 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. Each possibility represents an independent embodiment of the present invention. According to certain embodiments, the C1 strain has reduced expression and / or activity of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 or more proteases. According to certain exemplary embodiments, the C1 strain has reduced expression or activity of ALP1, ALP2, PEP4, PRT1, SRP1, ALP3, PEP1, and MTP2 (ΔalpΔalp2Δpep4Δprt1Δsrp1Δalp3Δpep1Δmtp2).

[0053] According to some embodiments, the Th. heterothallica is capable of producing a heterologous mammalian glycoprotein having mammalian / human N-glycans that account for more than 90%, 95% or 98% of the N-glycans present on the glycoprotein.

[0054] In some embodiments, the Th. heterothallica is further genetically modified to express a heterologous mammalian glycoprotein. In some embodiments, the heterologous mammalian glycoprotein is an antibody or an antigen-binding fragment thereof.

[0055] According to some embodiments, the heterologous mammalian glycoprotein comprises more than 90% of the mammalian / human N-glycans of its total N-glycans. According to some embodiments, the heterologous mammalian glycoprotein comprises more than 92%, 94%, 96%, 98% or 99% of the mammalian / human N-glycans of its total N-glycans.

[0056] According to some embodiments, the heterologous mammalian glycoprotein comprises mammalian / human N-glycans in an amount that is at least 80%, 85%, 90%, 95% or more of the amount of N-glycans present in the same glycoprotein produced by mammalian / human cells.

[0057] According to another aspect, the present invention provides a method for producing Th. heterothallica producing a glycoprotein having mammalian N-glycans, the method comprising:

[0058] (a) deleting or disrupting the alg3 gene of the Th. heterothallica so that the Th. heterothallica cannot produce functional α-1,3-mannosyltransferase; and

[0059] (b) introducing an exogenous polynucleotide encoding ER-targeted mannosidase 1 (α-1,2-mannosidase) into the Th. heterothallica; and

[0060] (c) introducing into the Th. heterothallica an exogenous polynucleotide encoding the ER-targeted glucosidase 2α subunit.

[0061] According to some embodiments, the method further comprises the step of introducing into the Th. Heterothallica the following exogenous polynucleotides: an exogenous polynucleotide encoding an endogenous flippase to induce overexpression of the endogenous flippase in the Th. Heterothallica; or an exogenous polynucleotide encoding a heterologous flippase to induce expression of the heterologous flippase in the Th. Heterothallica.

[0062] According to some embodiments, the ER-targeted mannosidase 1 (α-1,2-mannosidase) and the ER-targeted glucosidase 2e subunit are introduced using a single exogenous polynucleotide encoding both enzymes.

[0063] In some embodiments, the mannosidase 1 is Trichoderma reesei mannosidase 1. In some embodiments, the glucosidase 2 alpha subunit is Th. heterothallica glucosidase 2 alpha subunit.

[0064] According to another aspect, the present invention provides a method for producing a glycoprotein having mammalian N-glycans, the method comprising:

[0065] (a) providing a genetically modified Th. heterothallica according to the present invention;

[0066] (b) culturing the Th. heterothallica under conditions suitable for expressing the glycoprotein; and

[0067] (c) recovering the glycoprotein.

[0068] In some embodiments, the glycoprotein is a heterologous mammalian glycoprotein recombinantly expressed in said (Th. heterothallica). In some specific embodiments, the glycoprotein is a human protein recombinantly expressed in said Th. heterothallica. In other embodiments, the glycoprotein is a companion animal protein recombinantly expressed in said Th. heterothallica. In some embodiments, the heterologous mammalian glycoprotein is an antibody or an antigen-binding fragment thereof.

[0069] According to some embodiments, the heterologous mammalian glycoprotein comprises 90%, 92%, 94%, 96%, 98% or 99% of the total N-glycans of the mammalian / human glycoprotein. According to other embodiments, the heterologous mammalian glycoprotein comprises at least 80%, 85%, 90%, 95% or more of the amount of N-glycans present in the same glycoprotein produced by a mammalian / human cell.

[0070] According to another aspect, the present invention provides a recombinant glycoprotein produced by the genetically modified Th. heterothallica according to the present invention, wherein the glycoprotein comprises GlcNAc 2 Man 3 GlcNAc 2 (G0) polysaccharide.

[0071] According to another aspect, the present invention provides a recombinant glycoprotein produced by the genetically modified Th. heterothallica according to the present invention, wherein the glycoprotein comprises Gal 1 GlcNAc 2 Man3 GlcNAc 2 (G1) Glycan, Gal 2 GlcNAc 2 Man 3 GlcNAc 2 (G2) glycans or combinations thereof.

[0072] In some embodiments, the recombinant glycoprotein produced by the genetically modified Th. heterothallica according to the present invention is a pharmaceutical grade glycoprotein.

[0073] According to some embodiments, the heterologous mammalian glycoprotein comprises 90%, 92%, 94%, 96%, 98% or 99% of the total N-glycans of the mammalian / human glycoprotein. According to other embodiments, the heterologous mammalian glycoprotein comprises at least 80%, 85%, 90%, 95% or more of the amount of N-glycans present in the same glycoprotein produced by a mammalian / human cell.

[0074] It should be understood that any combination of each of the aspects and embodiments disclosed herein is expressly included within the disclosed scope of the present invention.

[0075] These and other aspects and features of the present invention will become apparent from the following detailed description, examples, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1A -ID. Analysis of released N-glycans of protein A affinity purified Nivolumab from sugar modified strains. One chromatogram from each transformation is shown as an example. The results of the major N-glycans of all strains described in the text are shown in the corresponding tables below the chromatograms. Figure 1A - Strains M4855 and M4856 (M3291-based strains expressing T. reesei Mns1-HDEL and C1 gls2a-HDEL) grown in shake flasks. The chromatogram of M4855 and the table of the major released N-glycans for both M4855 and M4856 are shown. Figure 1B-1D - G1 / 2 strains M5129 to M5132 (M4855-based strains expressing the G1 / 2 machinery) grown in shake flasks. M5130 ( Figure 1B ) and M5132( Figure 1C ) and a table of the major N-glycans released from M5129 to M5132 ( Figure 1D ).

[0077] Figure 2Comparison of the amount of released N-glycans of nivolumab produced by strains M5129 to M5132 (M4855-based strains expressing the G1 / 2 mechanism) cultured in shake flasks. The amount of released N-glycans was normalized between samples using a fixed amount of internal standard added to each sample for analysis. The response % of the reference protein Opdivo was set to 100%.

[0078] Figures 3A-3D Analysis of released N-glycans of protein A affinity purified nivolumab from fermentation samples of three sugar-modified strains. The results of major N-glycans for all strains described in this article are shown in the corresponding tables below the chromatograms. Figure 3A - Starting strain M3291 with only alg3 deleted. Shown are the chromatograms of M3291 and the table showing the main N-glycans released under various fermentation conditions. Figure 3B-3D -G1 / 2 strains M5130 and M5132. M5130 ( Figure 3B ) and M5132( Figure 3C ) and a table showing the major N-glycans released under various fermentation conditions ( Figure 3D ).

[0079] Figure 4 Comparison of the amount of released N-glycans of nivolumab produced by strains M5130 and M5132 cultured under various fermentation conditions. The amount of released N-glycans was normalized between samples using a fixed amount of internal standard added to each sample for analysis. The response % of the reference protein Opdivo was set to 100%.

[0080] Figures 5A-5C Analysis of released N-glycans from total secreted proteins of sugar-modified strains. One chromatogram from each transformation is shown as an example. The results for the major N-glycans for all strains described in the text are shown in the table ( Figure 5C ) and chromatograms ( Figure 5A-5B M6589 and M6596 are strains with a G1 / 2 mechanism in which TrMns1-HDEL is under the ubiquitin-like protein promoter, while M6590 and M6597 are strains with a G1 / 2 mechanism in which TrMns1-HDEL is under the bg18 promoter. DETAILED DESCRIPTION

[0081] The present invention relates to the genetic modification of the fungus Thermothelomyces heterothallica, particularly strain C1, to produce glycoproteins having N-glycans of mammalian proteins, particularly N-glycans of human, companion animal and other mammalian proteins.

[0082] Glycoproteins produced by Th. heterothallica genetically modified as described herein are suitable for therapeutic use in humans, companion animals (eg, dogs, cats, and horses), and other mammals.

[0083] Protein glycosylation, the covalent attachment of oligosaccharides to the side chains of newly synthesized polypeptide chains in cells, is an ordered process in eukaryotic cells involving a series of enzymes that sequentially add and remove sugar moieties. N-glycosylation is a process that attaches oligosaccharides to the side chains of asparagine residues, specifically asparagine that occurs in the sequence Asn-Xaa-Ser / Thr, where Xaa represents any amino acid except Pro.

[0084] N-glycosylation is initiated in the endoplasmic reticulum (ER), where oligosaccharides Glc 3 Man 9 GlcNAc 2 Assembled on the lipid carrier pyrophosphate dolichol, and then transferred to the selected asparagine residue of the polypeptide that has entered the ER lumen. The biosynthesis of lipid-linked oligosaccharides requires the activity of several specific glycosyltransferases (such as ALG1, ALG2 and ALG3). It starts on the cytoplasmic side of the ER membrane and ends in the lumen, where oligosaccharyl transferase (OST) selects the NXS / T sequence of the nascent polypeptide and produces an N-glycosidic bond between the side chain amide of asparagine and the oligosaccharide. The flipping of lipid-linked oligosaccharides from the outside of the ER to the inside is carried out by the flip enzyme located on the ER membrane. After being transferred to the nascent polypeptide, the oligosaccharide is usually trimmed by glucosidase and mannosidase, and then the nascent glycoprotein is transferred to the Golgi apparatus for further processing.

[0085] The synthesis of pyrophosphate-bound oligosaccharides is essentially similar in all known eukaryotic organisms. However, the further processing of the oligosaccharides as the glycoprotein moves along the secretory pathway varies greatly between lower eukaryotic organisms such as fungi or yeast and higher eukaryotic organisms such as animals and plants. Therefore, the final composition of the sugar side chains is different between various organisms and depends on the host.

[0086] In microorganisms such as yeast, additional mannose and / or mannose phosphate sugars are often added, resulting in "high mannose" type N-glycans that may contain as many as 30-50 mannose residues.

[0087] In animal cells including humans, companion animals and other mammalian cells, nascent glycoproteins are transferred to the Golgi apparatus, where mannose residues are removed by Golgi-specific 1,2-mannosidases. As the protein passes through the Golgi apparatus, it is processed by a large number of modifying enzymes, including N-acetylglucosamine transferases (GnT I, GnTII, GnT III, GnT IV, GnT V, GnT VI), mannosidase II and fucosyltransferases, which add and remove specific sugar residues. Finally, the N-glycans are acted upon by galactosyltransferases (GalT) and sialyltransferases (ST), and the final glycoprotein is released from the Golgi apparatus. The N-glycans of animal glycoproteins have a double, three or four antennae structure, and may generally include galactose, fucose and N-acetylglucosamine. Typically, the terminal residues of the N-glycans are composed of sialic acid.

[0088] Unlike most fungi and yeasts, Th. heterothallica does not have highly mannosylated N-glycans, but rather has “oligomannose” glycans (Man 3 To Man 8-9 ) and hybrid glycans containing Man and HexNAc residues (Man 3 HexNac-Man 8 HexNAc). The exact structure of these hybrid glycans is not completely clear. The hybrid glycans have typical mannose residues, but also have unknown HexNAc attached through a bond that has not yet been characterized.

[0089] Since the structure and synthetic pathway of the hybrid glycans have not been fully characterized, it is not clear whether such glycans can be eliminated using the genetic modifications described herein. Surprisingly, the genetic modifications according to the present invention result in substantial elimination of these structures, with more than 90%, typically more than 98%, of the N-glycoforms being the desired mammalian / human glycans.

[0090] The present invention aims to genetically modify the N-glycosylation pathway in Th. heterothallica so that it produces a high percentage of mammalian N-glycans, particularly human N-glycans such as GlcNAc 2 Man 3 GlcNAc 2 ("G0"), GlcNAc 2 Man 3 GlcNAc 2 (Fuc)("FG0"), Gal 1-2 GlcNAc 2 Man 3 GlcNAc 2 ("G1" / "G2") and Gal1- 2 GlcNAc 2 Man 3 GlcNAc 2 (Fuc)("FG1" / "FG2") glycoprotein.

[0091] Specifically, in some embodiments, the genetic modification of the N-glycosylation pathway in Th. heterothallica comprises the following modifications:

[0092] 1. Deletion of the C1 alg3 gene (encoding α-1,3-mannosyltransferase);

[0093] 2. Expression of Trichoderma reesei mannosidase 1 (α-1,2-mannosidase) targeted to the ER;

[0094] 3. Expression of C1 glucosidase 2α subunit targeting ER;

[0095] 4. Expression of heterologous GlcNAc transferase 1 (GNT1);

[0096] 5. Expression of heterologous GlcNAc transferase 2 (GNT2); and

[0097] 6. Expression of heterologous galactosyltransferase 1 (GalT1).

[0098] Deletion of alg3 terminates the synthesis of N-glycan precursors at Man with 1 or 2 terminal glucose residues 5 GlcNAe 2 This glycan serves as a substrate for GNT1 and GNT2 introduced into Th. heterothallica. Additional genetic modifications may include the introduction of additional enzymes from the glycosylation pathway of humans, companion animals, and other mammals, such as galactosyltransferases and / or fucosyltransferases.

[0099] The above heterologous enzymes are expressed together with a targeting peptide so that the expressed enzymes are targeted to specific cellular compartments.

[0100] As used herein, when referring to an enzyme, it encompasses enzymatically active fragments thereof and enzymatically active variants thereof.

[0101] The present invention specifically relates to the engineering of the N-glycosylation pathway of Th. heterothallica. It is noteworthy that O-glycans may be present, or may be removed or altered by further genetic modification of the Th. heterothallica.

[0102] It will be appreciated that the genetic modification according to the present invention enables the genetically modified Th. heterothallica to grow at a sufficient rate for its intended use.

[0103] As used herein, "C1" or "Thermothelomyces heterothallica C1" or "Th. heterothallica C1" refers to Thermothelomyces heterothallica strain C1. Descriptions of the genus Thermothelomyces and its species can be found, for example, in Marin-Felix Y (2015. Mycologica 107(3): 619-632) and van den Brink J et al. (2012, Fungal Diversity 52(1): 197-207).

[0104] It is noteworthy that the aforementioned authors (Marin-Felix et al., 2015) proposed to split the genus Myceliophthora based on differences in optimal growth temperature, conidial morphology and details of the sexual reproduction cycle. According to the proposed criteria, C1 clearly belongs to the newly established genus Thermothelomyces (which contains the former thermotolerant Myceliophthora species) rather than the genus Myceliophthora (which still includes non-thermotolerant species). Since C1 can form ascospores with some other Thermothelomyces (formerly Myceliophthora) strains with opposite mating types, C1 is better classified as Th. heterothallica strain C1 rather than Th. thermophila C1.

[0105] It must also be recognized that fungal taxonomy has changed in the past, so the current names listed above may have previously had various old names except Myceliophthora thermophila (van Oorschot, 1977. Persoonia 9(3):403), which are now considered synonyms. For example, Thermothelomycesheterothallica (Marin-Felix et al., 2015. Mycologica, 3: 619-63) and Corynascusheterotchallica (von Arx et al., 1983), Thielavia heterothallica (von Klopotek, 1976. Archives of Microbiology 107(2), 223-224), Chrysosporium Lucknowense is synonymous with thermophile (von Klopotek, 1974. Archives of Microbiology 98(1), 365-369) and Sporotrichium thermophile (Alpinis 1963. Nova Hedwigia 5:74).

[0106] It should further be expressly understood that the present invention encompasses any strain containing a ribosomal DNA (rDNA) sequence showing 99% or greater homology to SEQ ID NO: 22, and all such strains are considered to be conspecifics with Thermothelomyces heterothallica.

[0107] SEQ ID NO: 22 is 99.98% identical to the rDNA sequence found on chromosome 7 of Th. heterothallica / thermophila (listed as Myceliophthora thermophila) ATCC 42464 rDNA sequence (ncbi.nlm.nih.gov / nucleotide / CP003008.1).

[0108] Th. heterothallica strain C1 (as Chrysosporium lucknowense strain C1) was deposited under the Budapest Convention under the number VKM F-3500 D on August 29, 1996.

[0109] The above terms also encompass genetically modified substrains derived from wild-type strains that have been mutated using random or site-directed methods, such as UV mutagenesis or by deleting one or more endogenous genes. For example, the C1 strain may refer to a wild-type strain modified to delete one or more endogenous genes encoding endogenous proteases and / or one or more genes encoding endogenous chitinases. For example, the C1 strains (substrains) encompassed by the present invention include: UV18-25, accession number VKM F-3631D; strain NG7C-19, accession number VKM F-3633 D; and strain UV13-6, accession number VKM F-3632 D. Other C1 strains that can be used according to 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.

[0110] When grown under suitable conditions, Th.heterothallica fungi (specifically strain C1) show higher biomass yields in general compared to yeast strains. Th.heterothallica fungi can grow in large volume three-dimensional (3D) liquid cultures as well as on solid media. Compared with conventional yeast and other fungi, several strains developed by the applicant of the present invention are less sensitive to feedback repression of glucose and other fermentable sugars present as carbon sources in fungal growth media, and can tolerate high feed rates of carbon sources, thereby obtaining high yields. In addition, some of these strains provide significantly reduced medium viscosity when grown in commercial fermenters, compared with the high viscosity obtained by wild-type Th.heterothallica fungi or other filamentous fungi known for protein production that are repressed with non-glucose. The low viscosity can be attributed to the morphological changes of the strains, from having long and highly interwoven hyphae in the parent strain to having short and less interwoven hyphae in the developed strains. Low medium viscosity is very advantageous in large-scale industrial production in fermenters. For example, Th. heterothallica C1 strain UV18-25 (Deposit No. VKM F-3631D), which shows reduced sensitivity to glucose repression, has been grown industrially to produce recombinant enzymes in volumes exceeding 100,000 liters.

[0111] In some embodiments, the C1 strain of the present invention is a strain modified to lack multiple (i.e., at least two) genes encoding endogenous proteases. In some embodiments, the C1 strain is a strain modified to lack at least four genes encoding endogenous proteases. In other embodiments, the C1 strain is a strain modified to lack at least five genes encoding endogenous proteases. In some specific embodiments, the C1 strain is a strain modified to lack at least six genes encoding endogenous proteases. In other specific embodiments, the C1 strain is a strain modified to lack at least eight genes encoding endogenous proteases. In other specific embodiments, the C1 strain is a strain modified to lack at least 8, 9, 10, 11, 12, 13, 14 or more genes encoding endogenous proteases. In certain exemplary embodiments, the C1 strain is a strain modified to lack at least 13 or 14 genes encoding endogenous proteases.

[0112] It should be expressly understood that the teachings of the present invention encompass mutants, derivative strains, progeny, clones and analogs of Th. heterothallica C1 strain, as long as such derivative strains, progeny, clones and analogs are capable of growing and producing proteins having N-glycans described herein when genetically modified according to the teachings of the present invention.

[0113] It should be clearly understood that the term "derivative strain" referring to a fungal strain encompasses any parent fungal strain with modifications that positively affect product yield, efficiency or efficacy, or any trait that improves the fungal derivative strain as a tool for producing heterologous proteins described herein with N-glycans of mammalian proteins, particularly human, companion animal and other mammalian proteins. The term "progeny" as used herein refers to the unmodified offspring from a parent fungal strain, such as a cell from a cell.

[0114] As used herein, "glycan" refers to an oligosaccharide chain that can be connected to a carrier such as an amino acid, a peptide, a polypeptide, a lipid or a reducing end conjugate. The present invention particularly relates to N-linked glycans ("N-glycans") that are coupled to polypeptide N-glycosylation sites such as -Asn-Xxx-Ser / Thr- through an N-linked bond to the side chain amide nitrogen of an asparagine residue (Asn), wherein Xxx is any amino acid residue except Pro. The present invention may also relate to glycans that are part of a precursor lipid structure of dolichol-phospho-oligosaccharide (Dol-PP-OS), 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 through its reducing end.

[0115] Monosaccharides commonly found in mammalian glycoproteins that constitute N-glycans include, but are not limited to, N-acetylglucosamine (abbreviated as "GlcNAc"), mannose (abbreviated as "Man"), glucose (abbreviated as "Glc"), galactose (abbreviated as "Gal"), sialic acid (abbreviated as "Neu5Ac"), and fucose (abbreviated as "Fuc").

[0116] N-glycans share a common pentasaccharide called the "core" structure. 3 GlcNAc 2 (Abbreviation: "Man 3 "). Important target glycan structures of the present invention include N-glycans having one GlcNAc residue on the terminal 1,3 mannose arm of the core structure and one GlcNAc residue on the terminal 1,6 mannose arm of the core structure. Such N-glycans include: GlcNAc 2 Man 3 GlcNAc 2 (called the "G0" glycoform), Gal 1-2 GlcNAc 2 Man 3 GlcNAc 2 (referred to as "G1" or "G2" glycoforms according to the number of galactose residues), and their core fucosylated glycoforms: GlcNAc 2 Man 3 GlcNAc 2 (Fuc)("G0F" or "FG0") and Gal 1-2 GlcNAc 2 Man 3 GlcNAc 2 (Fuc)("G1F" and "G2F", or "FG1" and "FG2").

[0117] The term "alg3 gene" refers to a gene encoding α-1,3-mannosyltransferase. The term "α-1,3-mannosyltransferase" refers to a gene encoding alpha-1,3-mannosyltransferase. 5 GlcNAc 2 -PP-polyphenol α-1,3-mannosyltransferase (EC 2.4.1.258), an ER-resident enzyme, catalyzes the following reaction:

[0118] Dolichol β-D-mannosyl phosphate + D-Man-ɑ-(1->2)-D-Man-α-(1->2)-D-Man-α-(1->3)-[D-Man-α-(1->6)]-D-Man-β-(1->4)-D-GlcNAc-β-(1->4)-D-GlcNAc-diphosphate dolichol

[0119] →

[0120] D-Man-a-(1->2)-D-Man-ɑ-(1->2)-D-Man-ɑ-(1->3)-[D-Man-α-(1->3)-D-Man-ɑ-(1->6)]-D-Man-β-(1->4)-D-GlcNAc-β-(1->4)-D-GlcNAc-diphosphodiol + diphosphodiol phosphate

[0121] In some specific embodiments, the "alg3 gene" is a gene encoding an alpha-1,3-mannosyltransferase of C1 (ortholog of the JGI M. thermophila genome (mycocosm. JGI.doe.gov), accession number 2310419).

[0122] The Th. heterothallica of the present invention is genetically modified by deletion or disruption of the alg3 gene, so that the Th. heterothallica cannot produce functional α-1,3-mannosyltransferase. The Th. heterothallica of the present invention does not exhibit detectable α-1,3-mannosyltransferase activity.

[0123] The term "mannosidase 1" (α-1,2-mannosidase) is abbreviated as "MDS1" or "MNS1", which catalyzes the following reaction:

[0124] 3xH 2 O+N4-(α-D-Man-(1→2)-α-D-Man-(1→2)-α-D-Man-(1→3)-[α-D-Man-(1→3)-[α-D-Man-(1→2)-α-D-Man-(1→6)]-α-D-Man-(1→6)]-β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-ɑ-D-GlcNAc)-L-asparagine-[protein](N-glucose Glycan mannose isomer 8A1,2, 3B1,3)→3β-D-mannose + N4-(α-D-Man-(1→3)-[α-D-Man-(1→3)-[α-D-Man-(1→6)]-ɑ-D-Man-(1→6)]-β-D-Man-(1→4)-β-D-GlcNAc-(1→4)-β-D-GlcNAc)-L-asparagine-[protein] (N-glucan mannose isomer 5A1,2).

[0125] An exemplary accession number for alpha-1,2-mannosidase is Uniprot Q9P8T8.

[0126] The term "glucosidase 2 alpha subunit" (GLS2-alpha or GLS2α) refers to an enzyme that sequentially cleaves the two innermost alpha-1,3-linked glucose residues from the Glc2Man9GlcNAc2 oligosaccharide precursor of an immature glycoprotein.

[0127] The term "flippase" (EC 7.6.2.1) refers to an enzyme that transfers lipid-linked glycan precursors from the cytosolic side to the luminal side of the ER during their synthesis in the ER.

[0128] The term "GlcNAc transferase 1", abbreviated as "GNT1" (also known as "GnTI"), refers to α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase (EC 2.4.1.101), a Golgi resident enzyme that transfers a GlcNAc residue from UDP-GlcNAc to the acceptor substrate Man 5 GlcNAc 2 , to produce GlcNAcMan 5 GlcNAc 2 In the present invention, in view of the deletion of alg3 and the expression of α-1,2-mannosidase and glucosidase 2α subunit, the synthesis of N-glycan precursors produces Man3GlcNAc2, and thus the glycan Man3GlcNAc2 serves as a substrate for GNT1, producing GlcNAcMan3GlcNAc2.

[0129] The term "GlcNAc transferase 2", abbreviated as "GNT2" (also known as "GnTII"), refers to α-1,6-mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase (EC 2.4.1.143), a Golgi resident enzyme that transfers GlcNAc residues from UDP-GlcNAc to GlcNAcMan 3 GlcNAc 2 to generate GlcNAc 2 Man 3 GlcNAc 2 .

[0130] The terms "STT3 subunit of oligosaccharyltransferase", "STT3 protein" or simply "STT3" are used interchangeably herein and refer to the dolichol-diphosphate oligosaccharide-protein glycosyltransferase subunit (EC: 2.4.99.18). It is the catalytic subunit of the oligosaccharyltransferase (OST) complex, catalyzing the catalytic conversion of defined glycans (Glc in eukaryotic organisms) to 3 Man 9 GlcNAc 2) The initial transfer of pyrophosphate from the lipid carrier polytrichol to the asparagine residue within the Asn-X-Ser / Thr consensus motif in the nascent polypeptide chain is the first step in protein N-glycosylation. The STT3 protein catalyzes the following reaction:

[0131] Dolichol diphosphate oligosaccharide + L-asparagine-[protein]

[0132] →

[0133] Polyterpenoid diphosphate + H + +N 4 -(Oligosaccharide-(1→4)-N-acetyl-pD-glucosamine-(1→4)-N-acetyl-β-D-glucosamine)-L-asparagine-[protein]

[0134] The term "galactosyltransferase" (EC 2.4.1.38) refers to a Golgi resident enzyme that transfers p-linked galactosyl residues to terminal N-acetylglucosamine.

[0135] The term "heterologous" when referring to a gene, enzyme, protein or peptide sequence such as a subcellular localization signal is used herein to describe a gene, enzyme, protein or peptide sequence that does not naturally occur or is expressed in C1. When referring to a subcellular localization signal, the term also describes a subcellular localization signal that is different from that naturally occurring in the corresponding protein.

[0136] The term "endogenous" when referring to a gene, enzyme, protein or peptide sequence, such as a subcellular localization signal, refers to a gene, enzyme, protein or peptide sequence that naturally occurs in C1.

[0137] When referring to polynucleotides, the term "exogenous" is used herein to describe synthetic polynucleotides that are exogenously introduced into C1 by transformation. The exogenous polynucleotides can be introduced into C1 in a stable or transient manner, thereby generating ribonucleic acid (RNA) molecules and subsequently polypeptide molecules.

[0138] Expression constructs and vectors

[0139] The terms "expression construct", "DNA construct" or "expression cassette" are used interchangeably herein and refer to an artificially assembled or isolated nucleic acid molecule that comprises a nucleic acid sequence encoding a target protein and is assembled so that the target protein is expressed in a target host cell. An expression construct typically comprises a suitable regulatory sequence operably linked to the nucleic acid sequence encoding the target protein. An expression construct may also comprise a nucleic acid sequence encoding a selection marker.

[0140] The terms "nucleic acid sequence", "nucleotide sequence" and "polynucleotide" are used herein to refer to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA) and modified forms thereof, either in the form of independent fragments or as components of larger constructs. A nucleic acid sequence may be a coding sequence, i.e., a sequence that encodes a final product, such as a protein, in a cell. A nucleic acid sequence may also be a regulatory sequence, such as a promoter.

[0141] The terms "peptide", "polypeptide" and "protein" are used herein to refer to polymers of amino acid residues. The term "peptide" generally 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.

[0142] Sequences (e.g., nucleic acid sequences and amino acid sequences) that are "homologous" to a reference sequence refer herein to the percent identity between the sequences, wherein the percent 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 an independent embodiment of the present invention. Homologs of the sequences described herein are encompassed within the present invention. Protein homologs are encompassed as long as they retain the activity of the original protein. Homologous nucleic acid sequences include changes associated with codon usage and the degeneracy of the genetic code. Sequence identity can be determined using nucleotide / amino acid sequence comparison algorithms known in the art.

[0143] The nucleic acid sequence encoding the polypeptide of the present invention can be optimized for expression. Examples of such sequence modifications include, but are not limited to, changing the G / C content to be closer to the content normally present in Th. heterothallica, and removing codons that are atypically present in this fungus, which is generally referred to as codon optimization.

[0144] The phrase "codon optimization" refers to the selection of suitable DNA nucleotides close to the codon usage in the target organism for use in a structural gene or fragment thereof, and / or refers to the process of modifying a nucleic acid sequence to enhance expression in the host cell by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the target host cell while maintaining the native amino acid sequence. Various species exhibit specific biases for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), which in turn is believed to depend on, in particular, the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of the selected tRNA in the cell generally reflects the most frequently used codons in protein synthesis. Therefore, genes can be customized on the basis of codon optimization to obtain optimal gene expression in a given organism. Thus, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified to utilize statistically preferred or statistically favored codons in the organism. The present invention specifically encompasses polynucleotides encoding the target enzymes disclosed herein that are codon-optimized for expression in Th. heterothallica.

[0145] The term "regulatory sequence" refers to DNA sequences that control the expression (transcription) of a coding sequence, such as promoters and terminators.

[0146] 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, the promoter is located in the 5' region of the transcribed sequence (i.e., before, upstream). The promoter can be derived from a natural source in its entirety, or can be composed of different elements derived from different promoters present in nature, or even comprise synthetic nucleotide segments. The promoter can be constitutive (i.e., promoter activation is not regulated by an inducing agent, so the transcription rate is constant) or inducible (i.e., promoter activation is regulated by an inducing agent). In most cases, the precise boundaries of the regulatory sequence have not yet been fully defined, and in some cases cannot be fully defined, so some variant DNA sequences may have the same promoter activity.

[0147] The term "terminator" refers to another regulatory DNA sequence that regulates the termination of transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence to be transcribed.

[0148] 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, the C1 promoter and C1 terminator are used, which refer to promoter and terminator sequences capable of directing gene expression in C1.

[0149] According to some embodiments, the Th. heterothallica promoter / terminator is derived from an endogenous gene of Th. heterothallica. According to other embodiments, the Th. heterothallica promoter / terminator is derived from an exogenous gene introduced into Th. heterothallica.

[0150] Suitable constitutive promoters and terminators include, for example, promoters and terminators of the following genes: C1 glycolytic genes, such as phosphoglycerate kinase gene (PGK) (Uniprot: G2QLD8, NCBI Reference Sequence: XM_003665967), glyceraldehyde 3-phosphate dehydrogenase (GPD) (Uniprot: G2QPQ8, NCBI Reference Sequence: XM_003666768), phosphofructokinase (PFK) (Uniprot: G2Q605, NCBI Reference Sequence: XM_003665969), CBI reference sequence: XM_003659879); or β-glucosidase 1 gene bgl1 (accession number: XM_003662656); or triosephosphate isomerase (TPI) (Uniprot: G2QBR0, NCBI reference sequence: XM_003663200); or actin (ACT) (Uniprot: G2Q7Q5, NCBI reference sequence: XM_003662111); or C1cbh1 promoter (GenBank AX284115) or C1 chi1 promoter (GenBank H1550986). Other promoters that can be used are the Aspergillus nidulans gpdA promoter, and the synthetic promoter described in Rantasalo et al. (2018 NAR 46 (18): e111). Synthetic promoters useful in the present invention are further described in WO 2017 / 144777. As exemplary terminators, the terminator of the C1 chitinase 1 gene chi1 (GenBank HI550986), the cellobiohydrolase 1 cbh1 (GenBank AX284115) or the yeast adh1 terminator can be used.

[0151] Exemplary promoter and terminator sequences and promoter / terminator pairs are provided in the Examples section below. In some embodiments, promoter sequences for the present invention are selected from promoter-8, bgl8 promoter, promoter-9, promoter-3, promoter-1 and TEF1A promoter as exemplified below. Each possibility represents an independent embodiment of the present invention.

[0152] The term "operably linked" means that a selected nucleic acid sequence is adjacent to a regulatory element (promoter or terminator) to allow the regulatory element to regulate the expression of the selected nucleic acid sequence.

[0153] The terms "localization signal", "localization sequence", "subcellular targeting peptide / signal / sequence", etc. are used interchangeably herein and refer to a short peptide sequence (usually 5-30 amino acids long) contained in a protein sequence (usually present at one end of the protein, such as the N-terminus), which directs the protein to a specific subcellular location within the cell. For example, a Golgi localization signal targets a protein to the Golgi apparatus. A "heterologous localization signal", such as a "heterologous Golgi localization signal", refers to a localization signal that is not naturally present in the protein. In some embodiments, "heterologous" refers to a localization signal from another organism.

[0154] In some embodiments, the localization signal of the protein expressed in Th. heterothallica according to the present invention is derived from an endogenous gene of Th. heterothallica. For example, in some embodiments, the Golgi localization signal from the C1 protein KRE2a (ortholog of the JGI M. thermophila genome (mycocosm.jgi.doe.gov), accession number 2300989) is used.

[0155] In other embodiments, the localization signal of the protein expressed in Th. heterothallica according to the present invention is derived from an exogenous gene (heterologous localization signal) of Th. heterothallica. For example, in some embodiments, the enzyme of animal origin expressed in Th. heterothallica according to the present invention is expressed using its own naturally occurring Golgi localization signal. As another example, in some embodiments, a Golgi localization signal from a yeast protein such as S. cerevisiae protein KRE2 (GenBank Accession No. CAA44516) is used.

[0156] According to some embodiments, the protein expressed in Th. heterothallica comprises an ER targeting sequence. In certain embodiments, the ER targeting sequence is the sequence HDEL.

[0157] As used herein, the term "in frame" when referring to one or more nucleic acid sequences means that the sequences are linked so that their correct reading frame is preserved.

[0158] The expression construct according to some embodiments of the present invention comprises a Th. heterothallica promoter sequence and a Th. heterothallica terminator sequence operably linked to a nucleic acid sequence encoding an enzyme (e.g., a flippase, GNT1, or GNT2). In some specific embodiments, the expression construct of the present invention comprises a C1 promoter sequence and a C1 terminator sequence operably linked to a nucleic acid sequence encoding an enzyme (e.g., a flippase, GNT1, or GNT2).

[0159] Specific expression constructs can be assembled by various methods, including conventional molecular biology methods, such as polymerase chain reaction (PCR), restriction endonuclease digestion, in vitro and in vivo assembly methods and gene synthesis methods or combinations thereof. Exemplary expression constructs and methods for their construction are provided in the Examples section below.

[0160] Deletion of alg3

[0161] Gene deletion techniques can partially or completely remove a gene, thereby eliminating its expression. In this method, gene deletion can be achieved by homologous recombination using a plasmid that has been constructed to continuously contain the 5' and 3' regions on both sides of the gene.

[0162] Gene deletion can also be performed by inserting a disruptive nucleic acid construct (also referred to herein as a deletion construct) into the gene. The disruptive construct may simply be a selection marker gene with 5' and 3' regions homologous to the gene. The selection marker can identify transformants containing the disrupted gene. Alternatively or in addition, the disruptive nucleic acid construct may contain one or more polynucleotides encoding heterologous proteins to be expressed in the host cell.

[0163] Exemplary deletion constructs for alg3 and procedures for performing the deletion are described, for example, in WO 2021 / 094935. The deletion can be confirmed using PCR using appropriate primers on both sides of the disruptive construct.

[0164] In some embodiments, Th. heterothallica of the present invention is genetically modified to express a heterologous flippase. In some specific embodiments, the heterologous flippase is a yeast flippase. In other specific embodiments, the yeast flippase is a Saccharomyces cerevisiae mutant flippase FLC2p, which is a C-terminal truncated version of the Saccharomyces cerevisiae ER localized flippase FLC2.

[0165] In other embodiments, the Th. heterothallica of the present invention is genetically modified to overexpress an endogenous Th. heterothallica RFT1 flippase. In some specific embodiments, the Th. heterothallica C1 is genetically modified to overexpress an endogenous C1 RFT1 flippase. Overexpression of RFT1 in the Th. heterothallica according to the present invention can be performed by introducing an exogenous polynucleotide encoding RFT1, wherein the exogenous polynucleotide comprises a nucleic acid sequence encoding RFT1 operably linked to a regulatory sequence operable in Th. heterothallica. An exemplary nucleotide sequence of rft1 is set forth in SEQ ID NO:7. An exemplary amino acid sequence of rft1 is set forth in SEQ ID NO:8.

[0166] In some exemplary embodiments, the Th. heterothallica is genetically modified to delete or disrupt alg3, express mannosidase 1 (α-1,2-mannosidase) and ER-targeted C1 glucosidase 2α subunit, and overexpress endogenous Th. heterothallica RFT1 flippase, and is further genetically modified to express animal-derived GNT1 and animal-derived GNT2 containing heterologous Golgi localization signals, such as human GNT1 and human GNT2 containing a Golgi localization signal from yeast protein KRE2.

[0167] In some exemplary embodiments, the Th. heterothallica is genetically modified to delete or disrupt alg3, express mannosidase 1 (α-1,2-mannosidase) and glucosidase 2 α subunit and express yeast FLC2p flippase, and is further genetically modified to express animal-derived GNT1 and animal-derived GNT2 containing a heterologous Golgi localization signal, for example, to express human GNT1 and rat GNT2 containing a Golgi localization signal from the Th. heterothallica protein KRE2.

[0168] In other exemplary embodiments, the Th. heterothallica is genetically modified by: deletion or disruption of alg3; expression of mannosidase 1 (α-1,2-mannosidase) and C1 glucosidase 2α subunit targeted to the ER; overexpression of endogenous Th. heterothallica RFT1 flippase; expression of human GNT1 and rat GNT2 containing the Th. heterothallica KRE2a Golgi localization signal; and expression of Leishmania major STT3. In some embodiments, the Th. heterothallica is further genetically modified by expression of human galactosyltransferase or Xenopus tropicalis galactosyltransferase.

[0169] In other exemplary embodiments, the Th. heterothallica is genetically modified by: deletion or disruption of alg3; expression of mannosidase 1 (α-1,2-mannosidase) and ER-targeted C1 glucosidase 2α subunit; overexpression of endogenous Th. heterothallica RFT1 flippase; expression of bovine GNT1 and rat GNT2 containing the Th. heterothallica KRE2 Golgi localization signal; and expression of Leishmania major STT3.

[0170] Mannosidase 1 (α-1,2-mannosidase) and glucosidase 2 α subunit

[0171] According to some embodiments, expression constructs for ER-targeted mannosidase 1 (α-1,2-mannosidase) and ER-targeted C1 glucosidase 2α subunit are integrated into the alp3 protease locus of Thermothelomyces heterothallica.

[0172] An exemplary nucleotide sequence of a Trichoderma reesei mannosidase 1 targeted to the ER is set forth in SEQ ID NO: 1. An exemplary amino acid sequence of a Trichoderma reesei mannosidase 1 targeted to the ER is set forth in SEQ ID NO:2.

[0173] An exemplary nucleotide sequence of the ER-targeted C1 glucosidase 2 alpha subunit (gls2a-HDEL) is set forth in SEQ ID NO: 3. An exemplary amino acid sequence of the ER-targeted C1 glucosidase 2 alpha subunit is set forth in SEQ ID NO:4.

[0174] GNT1 and GNT2

[0175] In some embodiments, the Th. heterothallica of the present invention is genetically modified to express heterologous GNT1 and GNT2. In some embodiments, the heterologous GNT1 and GNT2 are of animal origin. As used herein, "animal origin" encompasses mammalian origin, including, for example, companion animals (e.g., dogs and cats) and other mammals (e.g., horses). As exemplified below, animal origin includes, for example, rat origin. The term "animal origin" further encompasses human origin, as further exemplified below.

[0176] The heterologous GNT1 and GNT2 can be expressed in Th. heterothallica according to the present invention by introducing one or more exogenous polynucleotides encoding GNT1 and GNT2, the polynucleotide comprising a nucleic acid sequence encoding GNT1 and GNT2 operably linked to a regulatory sequence operable in Th. heterothallica. In some embodiments, the nucleic acid sequences encoding GNT1 and GNT2 are contained in a single polynucleotide introduced into the Th. heterothallica. In other embodiments, the nucleic acid sequences encoding GNT1 and GNT2 are contained in two different polynucleotides introduced into the Th. heterothallica.

[0177] In some embodiments, the GNT1 is expressed in the Th. heterothallica using its own naturally occurring Golgi localization signal. In other embodiments, the GNT1 is expressed in the Th. heterothallica using a heterologous Golgi localization signal.

[0178] In some embodiments, the heterologous Golgi localization signal is a yeast Golgi localization signal. In some specific embodiments, the heterologous Golgi localization signal is from the yeast protein KRE2α-1,2-mannosyltransferase. In some exemplary embodiments, the heterologous Golgi localization signal is from KRE2 of Saccharomyces cerevisiae.

[0179] In other embodiments, the heterologous Golgi localization signal is from a filamentous fungus. In some embodiments, the heterologous Golgi localization signal is from Th. heterothallica. In some specific embodiments, the heterologous Golgi localization signal is from the C1 homolog of the yeast protein KRE2.

[0180] In some embodiments, the GNT1 is human GNT1. In some embodiments, the human GNT1 introduced into Th. heterothallica comprises a heterologous Golgi localization signal. In some embodiments, the GNT1 is human GNT1 comprising a yeast Golgi localization signal. In some specific embodiments, the GNT1 is human GNT1 comprising a Golgi localization signal of the protein KRE2 from S. cerevisiae. An exemplary nucleotide signal of KRE2 signal-GNT1 is set forth in SEQ ID NO: 5. An exemplary amino acid sequence of KRE2 signal-GNT1 is set forth in SEQ ID NO: 6.

[0181] In some embodiments, the GNT2 is rat GNT2. GNT2 is typically expressed using its own naturally occurring Golgi localization signal. The amino acid sequence of rat GNT2 is set forth in SEQ ID NO: 16. An exemplary nucleic acid sequence of a polynucleotide encoding rat GNT2 for use in accordance with the present invention is set forth in SEQ ID NO: 15.

[0182] In other embodiments, the GNT2 is human GNT2. GNT2 is typically expressed using its own naturally occurring Golgi localization signal.

[0183] Exemplary but non-limiting combinations of GNT1 and GNT2 according to the present invention include:

[0184] - Human GNT1 and human GNT2 with yeast KRE2 Golgi localization signal:

[0185] - Human GNT1 and rat GNT2 with the yeast KRE2 Golgi localization signal;

[0186] - Human GNT1 and human GNT2 with the C1KRE2a Golgi localization signal;

[0187] - Human GNT1 and rat GNT2 with C1KRE2a Golgi localization signal;

[0188] -Bovine GNT1 and rat GNT2 with the C1 KRE2a Golgi localization signal.

[0189] Each combination represents a separate embodiment of the present invention.

[0190] Galactosyltransferase

[0191] In some embodiments, the Th. Heterothallica of the present invention is genetically modified to express a heterologous galactosyltransferase. In some embodiments, the heterologous galactosyltransferase is of animal origin.

[0192] The galactosyltransferase can be expressed in Th. heterothallica according to the present invention by introducing an exogenous polynucleotide encoding the galactosyltransferase, the polynucleotide comprising a nucleic acid sequence encoding the galactosyltransferase operably linked to a regulatory sequence operable in Th. heterothallica.

[0193] In some embodiments, the galactosyltransferase is expressed in the Th. heterothallica using its own naturally occurring Golgi localization signal. In other embodiments, the galactosyltransferase is expressed in the Th. heterothallica using a heterologous Golgi localization signal.

[0194] In some embodiments, the galactosyltransferase is a human galactosyltransferase (huGalT1). In some embodiments, the human galactosyltransferase introduced into Th. heterothallica comprises a heterologous Golgi localization signal. In some specific embodiments, the human galactosyltransferase comprises a S. cerevisiae KRE2 Golgi localization signal. The amino acid sequence of a human galactosyltransferase having a Golgi localization signal of protein KRE2 from S. cerevisiae is set forth in SEQ ID NO: 14. An exemplary nucleic acid sequence encoding a human galactosyltransferase having a Golgi localization signal of protein KRE2 from S. cerevisiae used in accordance with the present invention is set forth in SEQ ID NO: 13.

[0195] According to other embodiments, the galactosyltransferase is from Xenopus tropicalis (XtGalT1). In some embodiments, the galactosyltransferase from Xenopus tropicalis introduced into Th. heterothallica comprises a heterologous Golgi localization signal. In some specific embodiments, the galactosyltransferase from Xenopus tropicalis comprises a Golgi localization signal from the protein KRE2 of S. cerevisiae.

[0196] STT3 oligosaccharyltransferase

[0197] In some embodiments, the Th. heterothallica of the present invention is genetically modified to express the STT3 subunit of a heterologous oligosaccharyl transferase. In some specific embodiments, the heterologous STT3 is Leishmania major STT3. The amino acid sequence of Leishmania major STT3 is set forth in SEQ ID NO: 12. Leishmania major STT3 can be expressed in Th. heterothallica according to the present invention by introducing an exogenous polynucleotide encoding Leishmania major STT3, the polynucleotide comprising a nucleic acid sequence encoding Leishmania major STT3 operably linked to a regulatory sequence operable in Th. heterothallica. An exemplary nucleic acid sequence encoding Leishmania major STT3 used according to the present invention is set forth in SEQ ID NO: 11.

[0198] Genetically modified Th. heterothallica

[0199] According to the present invention, Th. heterothallica cells genetically modified to produce glycoproteins having N-glycans of mammalian proteins, particularly human and companion animal proteins, are produced by modifying, for example deleting, the endogenous gene of Th. heterothallica alg3 so that the gene cannot produce functional proteins, and expressing exogenous polynucleotides encoding various enzymes.

[0200] It should be understood that the genetic modification of Th. heterothallica disclosed herein does not necessarily require that every cell of the genetically modified Th. heterothallica be modified, as long as the desired result of producing glycoproteins having N-glycans of mammalian proteins, particularly human and companion animal proteins, disclosed herein is achieved.

[0201] In some embodiments, the Th. heterothallica is further genetically modified to express a heterologous mammalian glycoprotein. In some embodiments, the heterologous mammalian glycoprotein is an antibody or an antigen-binding fragment thereof.

[0202] In some exemplary embodiments, the Th. heterothallica is genetically modified to express nivolumab or an antigen-binding fragment thereof. In other exemplary embodiments, the Th. heterothallica is genetically modified to express a nivolumab light chain having a Th. heterothallica CBH1 signal sequence and a nivolumab heavy chain having a Th. heterothallica CBH1 signal sequence.

[0203] In some embodiments, the invention provides a genetically modified Th. heterothallica cell as disclosed herein.

[0204] The expression of the exogenous polynucleotide is carried out by introducing into the Th. heterothallica cell, in particular into the nucleus of the Th. heterothallica cell, an expression construct comprising a nucleic acid encoding the protein to be expressed in C1. In particular, the genetic modification according to the invention means introducing the expression construct into the host genome.

[0205] Introduction of the expression construct into Th. heterothallica cells, ie transformation of Th. heterothallica, can be performed by the method for transformation of filamentous fungi.

[0206] In order to facilitate the easy selection of transformed cells, selection markers can be transformed into the Th. heterothallica cells. "Selection marker" refers to a polynucleotide encoding a gene product that confers a phenotype of a particular type that does not exist in non-transformed cells, such as antibiotic resistance (resistance marker), the ability to utilize a certain resource (utilization / nutrition deficiency marker) or the expression of a reporter protein that can be detected, for example, by spectral measurement. Nutrient deficiency markers are usually preferred as a means of selection in the food or pharmaceutical industries. The selection marker can be on a separate polynucleotide co-transformed with the expression construct, or on the same polynucleotide of the expression construct. After transformation, according to the selected selection marker, positive transformants are selected by cultivating the C1 cells, for example, on a selective medium. In some cases, a split marker system is used, in which the selection marker is split in two plasmids, and only when the two plasmids are co-transformed and connected together by homologous recombination, a functional selection marker can be formed.

[0207] When a synthetic expression system is used, an expression cassette encoding an appropriate synthetic transcription factor (sTF) is introduced into the host cell.

[0208] The transformed DNA can be integrated into the Th. heterothallica chromosome by homologous recombination or non-homologous end joining. In order to facilitate targeted integration into a specific locus in the genome, the sequence corresponding to the target locus is incorporated into the same polynucleotide as the expression construct.

[0209] The selected clones are then grown and examined for production of proteins with the desired N-glycoforms. The genetically modified Th. heterothallica is cultured under suitable conditions. According to certain embodiments, the fungus is grown at a temperature of about 20°C to about 45°C and a medium pH of about 4.0 to about 8.0. A specific medium type may be selected based on regulatory requirements for the final product. The produced glycoproteins may be isolated and analyzed.

[0210] The expression of GNT1, GNT2 and optionally other enzymes such as galactosyltransferases in Th. heterothallica can be determined by structural analysis of the N-glycans produced by the C1.

[0211] The genetically modified Th. heterothallica according to the present invention produces G0 (Man 3 GlcNAc 2 ) as the final N-glycan structure or the intermediate N-glycan structure.

[0212] In some embodiments, Th. heterothallica genetically modified by deletion or disruption of alg3, expression of ER-targeted Trichoderma reesei mannosidase 1 (α-1,2-mannosidase) and ER-targeted C1 glucosidase 2α subunit, expression of optional heterologous flippase or overexpression of endogenous flippase, expression of heterologous GNT1 and GNT2, and expression of optional heterologous STT3 oligosaccharyltransferase produces secreted glycoproteins, wherein G0 accounts for at least 80% of the N-glycans on the secreted glycoprotein, preferably at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or even at least 95% of the N-glycans on the secreted glycoprotein. Each value represents an independent embodiment of the present invention.

[0213] In some embodiments, Th. heterothallica further genetically modified to express a heterologous galactosyltransferase produces a secreted glycoprotein wherein G1 and G2 (the sum of both G1 and G2) account for at least 75% of the N-glycans on the secreted glycoprotein, preferably at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or even at least 95% of the N-glycans on the secreted glycoprotein. Each value represents a separate embodiment of the present invention.

[0214] In some embodiments, the genetic modification of Th. heterothallica does not include expression of heterologous oligosaccharyltransferase (OST).

[0215] In order to more fully illustrate certain embodiments of the present invention, the following examples are provided. However, they should never be interpreted as limiting the broad scope of the present invention. Those skilled in the art can easily design many variations and modifications of the principles disclosed herein without departing from the scope of the present invention.

[0216] Example

[0217] Example 1 - Generation of a strain for producing nivolumab with humanized G1 / 2 glycans in three steps

[0218] The first step in generating a Thermothelomyces heterothallica C1 strain that produces human-type galactosylated glycans is to delete the alg3 gene from a strain carrying eight protease gene deletions and expressing nivolumab (described in WO2021 / 094935). After alg3 deletion, the next C1 glycoengineering step is to integrate ER-targeted Trichoderma reesei mannosidase 1 (α-1,2-mannosidase) and ER-targeted C1 glucosidase 2α subunit into the alp3 protease locus to trim the glycan precursors into DolP-GlcNAc suitable for synthesizing human-type glycans. 2 -Man 3 Then, expression cassettes containing human or bovine GNT1 (GlcNAc transferase I), rat GNT2 (GlcNAc transferase II), C1 flippase RFT1, oligosaccharyltransferase STT3 from Leishmania major, and human GalT1 (galactosyltransferase I) were integrated into the alp6 protease locus. Expression of these genes resulted in the expression of GlcNAc 2 Man 3GlcNAc residues are added to both branches of the glycan, resulting in G0 (GlcNAc2Man3GlcNAc2) glycans, followed by the addition of galactose residues to one or both branches of the G0 glycan, resulting in the production of G1 (G1cNAc2Man3GlcNAc2Gal) and G2 (GlcNAc2Man3MlcNAc2Gal2) glycans.

[0219] In the first transformation step, a DNA construct designed for integration into the alp3 locus (JGI M. thermophila genome database ID 2306020) and simultaneous expression of Trichoderma reesei (Tr)mns1-HDEL (JGI Trichoderma reesei genome database ID 45717) and C1gls2a-HDEL (JGI M. thermophila genome database ID 2125259) was constructed in two parts into two separate plasmids. HDEL is a four amino acid C-terminal ER localization signal. The first plasmid contained a fragment of the alp3 5′ flanking region for integration, an expression cassette for Tr-mns1-HDEL (wherein the gene is located between the C1 bgl8 promoter and terminator (JGI M. thermophila genome database ID 115968), a synthetic transcription factor (sTF, for the synthetic promoter in the 3′ plasmid), a direct repeat of the C1 cbh1 terminator (JGI M. thermophila genome database ID 109566), and the first 2 / 3 of the amdS marker gene (encoding acetamidase from Aspergillus nidulans). The second plasmid contained the last 2 / 3 of the amdS marker, a direct repeat fragment targeting the end of the sTF cassette (for removal of the amdS marker by recombination), a synthetic AnSES promoter (Rantasalo A et al., 2018, Universal gene expression system for fungi, Nucl. Acids Research 46(18):e111) and chi1 terminator (JGI M.thermophila genome database ID50608) for the expression cassette for C1 gls2a-HDEL, and the alp3 3' flanking region fragment for integration. The amdS marker fragments in the two plasmids overlap each other, and this region undergoes homologous recombination in C1 between the plasmids, while the 5' and 3' flanking region fragments recombine with the genomic DNA on both sides of the alp3 gene. Recombination between the selectable marker fragments functionalizes the marker gene and enables the transformants to grow under selection.

[0220] A construct containing a fragment of the alp3 5' flanking region, an expression cassette for Tr mns1-HDEL, sTF and the first 2 / 3 of the amdS marker is set forth in SEQ ID NO: 17 (pMYT1288). The 5' flanking sequence corresponds to positions 9-1196 of SEQ ID NO: 17. The bgl8 promoter sequence corresponds to positions 1202-2593 of SEQ ID NO: 17. The nucleic acid sequence encoding the genomic Trichoderma reesei mannosidase 1 with an artificial HDEL ER retention signal corresponds to positions 2594-4420 of SEQ ID NO: 17. The nucleic acid sequence encoding the T. reesei MNS1 with an HDEL signal is also set forth as SEQ ID NO: 1 (Tr MNS1-HDEL nt) and the amino acid sequence is SEQ ID NO: 2 (Tr MNS1-HDEL aa). The bgl8 terminator sequence corresponds to positions 4421-4887 of SEQ ID NO: 17. The nucleic acid sequence of the synthetic transcription factor cassette corresponds to positions 4901-6550 of SEQ ID NO: 17. The first 2 / 3 of the amdS marker gene corresponds to positions 7060-9126 of SEQ ID NO: 17. The fragments were generated by PCR, separated in an agarose gel, purified, and assembled by Gibson assembly ( HiFi DNA Assembly Cloning Kit, New England Biolabs) method was cloned into the backbone vector pRS426 to obtain the 5' arm expression vector pMYT1288, which was verified by sequencing.

[0221] A construct containing the last 2 / 3 of the amdS marker, an expression cassette for C1 gls2a-HDEL and a fragment of the alp3 3' flanking region for integration is set forth in SEQ ID NO: 18 (pMYT0721). The 2 / 3 of the amdS marker gene corresponds to positions 17-1738 of SEQ ID NO: 18. The synthetic AnSES promoter sequence corresponds to positions 2255-2747 of SEQ ID NO: 18. The sequence encoding C1 glucosidase 2α with an artificial HDEL ER retention signal corresponds to positions 2748-5760 of SEQ ID NO: 18. The nucleic acid sequence encoding C1 GLS2α with an HDEL signal is also set forth as SEQ ID NO: 3 (C1 gls2a-HDEL nt) and the amino acid sequence is SEQ ID NO: 4 (C1 gls2a-HDEL aa). The chi1 terminator sequence corresponds to positions 5761-6406 of SEQ ID NO: 18. The alp3 3' flanking sequence corresponds to positions 6415-7548 of SEQ ID NO: 18. The fragment was generated by PCR, isolated in an agarose gel, purified, and assembled by Gibson ( HiFi DNA Assembly Cloning Kit, New England Biolabs) method was cloned into the backbone vector pRS426 to obtain the 3' arm expression vector pMYT0721, which was verified by sequencing.

[0222] For the second transformation step, a DNA construct designed to integrate into the alp6 locus (JGI M. thermophila genome database ID 94536) and to simultaneously express GNT1 and GNT2 as well as RFT1, STT3 and GalT1 was constructed in two parts into two separate plasmids. The first plasmid contains the alp6 5′ flanking region fragment for integration, an expression cassette for GNT1 (where the human or bovine GNT1 gene fused to the C1 KRE2 Golgi localization signal (JGI M. thermophila genome database ID 2300989) is located between the C1 bgl8 promoter and bgl8 terminator), the C1 flippase RFT1 (JGIM. thermophila genome database ID 2307799) located between the promoter and terminator of the ubiquitin-like protein gene (JGIM. thermophila genome database ID 2315548), and the first 2 / 3 of the C1 pyr4 marker gene (JGIM. thermophila genome database ID 2311494). The second plasmid contains the last 2 / 3 of the C1 pyr4 marker gene, a direct repeat fragment targeting the beginning of the pyr4 cassette (for removal of the pyr4 tag by recombination), an inverted expression cassette for STT3 from Leishmania major between the promoter and terminator of the C1 hypothetical protein (JGI M. thermophila genome database ID 2315630), an expression cassette for human GalT1 fused to the Saccharomyces cerevisiae KRE2 Golgi localization signal between the promoter of the ubiquitin-like protein gene and the bgl8 terminator, the GNT2 gene from rat between the promoter of the translation elongation factor 1A (JGI M. thermophila genome database ID 2298136) and the terminator of the C1 hypothetical protein (JGI M. thermophila genome database ID 114107), and finally a fragment of the alp6 3' flanking region for integration. The C1 pyr4 marker fragments in the two plasmids overlap each other and this region undergoes homologous recombination in C1 between the plasmids, while the 5' and 3' flanking region fragments recombine with the genomic DNA on both sides of the alp6 gene. Recombination between the selectable marker fragments renders the marker gene functional and enables the transformants to grow under selection.

[0223] The first 5' construct containing the alp6 5' flanking region fragment, expression cassettes for human GNT1, C1 RFT1, and the first 2 / 3 of the C1 pyr4 marker gene is set forth as SEQ ID NO: 19 (pMYT1451). The 5' flanking sequence corresponds to positions 8-1156 of SEQ ID NO: 19. The bgl8 promoter sequence corresponds to positions 1165-2556 of SEQ ID NO: 19. The nucleic acid sequence encoding human GNT1 fused to the C1 KRE2 Golgi localization signal corresponds to positions 2557-4042 of SEQ ID NO: 19, wherein positions 2557-2821 encode the C1 KRE2 localization signal, and positions 2822-4042 encode human GNT1. The nucleic acid sequence encoding human GNT1 fused to the C1 KRE2 Golgi localization signal is also set forth as SEQ ID NO: 5 (C1 kre2-huGNT1nt). The nucleic acid sequence encoding human GNT1 was codon-optimized for M. thermophila C1 and synthesized by GenScript (USA). In the synthesized sequence, the region of amino acids 1-100 of human GNT1 was replaced by the C1 KRE2 Golgi localization signal (amino acids 1-70). The complete amino acid sequence of C1 KRE2-GNT1 is set forth as SEQ ID NO: 6 (C1 KRE2-huGNT1 aa). The bgl8 terminator sequence corresponds to positions 4046-4512 of SEQ ID NO: 19. The ubiquitin-like protein promoter sequence corresponds to positions 4513-5532 of SEQ ID NO: 19. The C1 flippase rft1 gene corresponds to positions 5533-7449 of SEQ ID NO: 19. The nucleic acid sequence encoding C1 RFT1 is also set forth as SEQ ID NO: 7 (C1 RFT1nt), and the amino acid sequence is SEQ ID NO: 8 (C1 RFT1 aa). The ubiquitin-like protein terminator sequence corresponds to positions 7458-7953 of SEQ ID NO: 19. The first 2 / 3 of the C1 pyr4 marker gene corresponds to positions 7969-9748 of SEQ ID NO:19.

[0224] A second 5' construct containing a fragment of the alp6 5' flanking region, expression cassettes for bovine GNT1, C1 RFT1, and the first 2 / 3 of the C1pyr4 marker gene is set forth as SEQ ID NO: 20 (pMYT1452). The 5' flanking sequence corresponds to positions 8-1156 of SEQ ID NO: 20. The bgl8 promoter sequence corresponds to positions 1165-2556 of SEQ ID NO: 20. The nucleic acid sequence encoding bovine GNT1 fused to the C1KRE2 Golgi localization signal corresponds to positions 2557-4051 of SEQ ID NO: 20, wherein positions 2557-2821 encode the C1 KRE2 localization signal and positions 2822-4051 encode bovine GNT1. The nucleic acid sequence encoding bovine GNT1 fused to the C1 KRE2 Golgi localization signal is also set forth as SEQ ID NO: 9 (C1 kre2-boGNT1 nt). The nucleic acid sequence encoding bovine GNT1 was codon optimized for M. thermophila C1 and synthesized by GenScript (USA). In the synthesized sequence, the region of amino acids 1-38 of bovine GNT1 was removed after cloning of the expression plasmid and replaced by the C1 KRE2 Golgi localization signal (amino acids 1-70). The complete amino acid sequence of C1 KRE2-GNT1 is set forth as SEQ ID NO: 10 (C1 KRE2-boGNT1 aa). The bgl8 terminator sequence corresponds to positions 4052-4518 of SEQ ID NO: 20. The ubiquitin-like protein promoter sequence corresponds to positions 4524-5543 of SEQ ID NO: 20. The C1 flippase RFT1 gene described for pMYT1451 corresponds to positions 5544-7460 of SEQ ID NO: 20. The ubiquitin-like protein terminator sequence corresponds to positions 7469-7964 of SEQ ID NO: 20. The first 2 / 3 of the C1 pyr4 marker gene corresponds to positions 7980-9759 of SEQ ID NO: 20. The fragments for the two 5' plasmids were generated by PCR, separated on an agarose gel, purified, and assembled by Gibson ( HiFi DNA Assembly Cloning Kit, New England Biolabs) was cloned into the backbone vector pRS426 to obtain the 5' arm expression vectors pMYT1451 and pMYT1452, which were verified by sequencing.

[0225] The construct containing the last 2 / 3 of the C1 pyr4 marker gene, an inverse expression cassette for STT3 from Leishmania major, an inverse expression cassette for human GalT1, an inverse expression cassette for the rat GNT2 gene, and an alp6 3' flanking region fragment for integration is set forth in SEQ ID NO: 21 (pMYT1453). The 2 / 3 of the C1 pyr4 marker gene corresponds to positions 17-1273 of SEQ ID NO: 21. The hypothetical protein (ID2315630) promoter sequence corresponds to positions 5965-4957 of SEQ ID NO: 21. The sequence encoding STT3 from Leishmania major was codon-optimized for M. thermophila C1 and synthesized by GenScript (USA), which corresponds to positions 4956-2383 of SEQ ID NO: 21. The nucleic acid sequence encoding the Leishmania major STT3 gene is also set forth as SEQ ID NO: 11 (LmSTT3nt). The complete amino acid sequence of Leishmania major STT3 is set forth as SEQ ID NO: 12 (LmSTT3 aa). The hypothetical protein (ID2315630) terminator sequence corresponds to positions 2374-1795 of SEQ ID NO: 21. The sequence encoding human GalT1 fused to the Sc KRE2 Golgi localization signal corresponds to positions 7704-6439 of SEQ ID NO: 21, wherein positions 7704-7405 encode the Sc KRE2 localization signal and positions 7404-6439 encode human GalT1. The ubiquitin-like protein promoter sequence corresponds to positions 8724-7705 of SEQ ID NO: 21. The nucleic acid sequence encoding human GalT1 fused to the ScKRE2 Golgi localization signal is also set forth as SEQ ID NO: 13 (Sckre2-huGalT1 nt). The nucleic acid sequence encoding human GalT1 was codon-optimized for M. thermophila C1 and synthesized by GenScript (USA). In the synthesized sequence, the region of amino acids 1-77 of human GalT1 was removed after expression plasmid cloning and replaced by the ScKRE2 Golgi localization signal (amino acids 1-100). The complete amino acid sequence of ScKRE2-GalT1 is set forth as SEQ ID NO: 14 (ScKRE2-huGalT1 aa). The bgl8 terminator sequence corresponds to positions 6438-5972 of SEQ ID NO: 21. The translation elongation factor 1A promoter sequence corresponds to positions 11618-10562 of SEQ ID NO: 21.The sequence encoding rat GNT2, codon optimized for M. thermophila C1 and synthesized by GenScript (USA), corresponds to positions 10561-9233 of SEQ ID NO: 21. The nucleic acid sequence encoding rat GNT2 is also set forth as SEQ ID NO: 15 (rat GNT2nt). The complete amino acid sequence of rat GNT2 is set forth as SEQ ID NO: 16 (rat GNT2aa). The hypothetical protein (ID114107) terminator sequence corresponds to positions 9232-8729 of SEQ ID NO: 21. The alp6 3' flanking sequence corresponds to positions 11626-12681 of SEQ ID NO: 21. The fragments were generated by PCR, separated in agarose gel, purified, and assembled by Gibson (. HiFi DNA Assembly Cloning Kit, New England Biolabs) method was cloned into the backbone vector pRS426 to obtain the 3' arm expression vector pMYT1453, which was verified by sequencing.

[0226] To obtain a strain for producing nivolumab with G1 / 2 N-glycans, the above expression plasmids were successively transformed into the pyr4-negative alg3 deletion strain M3291 for producing nivolumab (described in WO 2021 / 094935). In each transformation, a pair of one 5' arm expression vector and one 3' arm expression vector (cut from the expression plasmid backbone with MssI) were used. The pair was:

[0227] First round: SEQ ID NO: 17 (pMYT1288) (alp3 5' flanking region-Th Mns1-HDEL box-sTF box-2 / 3amdS) + SEQ ID NO: 18 (pMYT0721) (2 / 3amdS-C1 gls2a-HDEL box-alp3 3' flanking fragment)

[0228] Second round: SEQ ID NO: 19 (pMYT1451) or SEQ ID NO: 20 (pMYT1452) (alp6 5' flanking region - human or bovine GNT1 cassette - C1 RFT1 cassette - 2 / 3pyr4) + SEQ ID NO: 21 (pMYT1453) (2 / 3pyr4-LmSTT3 cassette - human GalT1 cassette - rat GNT2 cassette - alp6 3' flanking fragment)

[0229] C1 transformation and transformant selection were performed essentially as described in WO 2021 / 094935. For the first round of transformation, selection was based on a functional amdS gene and for the second round was based on a functional pyr4 gene. Transformants were screened by PCR to find clones in which the alp3 deletion site (first round) or the alp6 gene (second round) was replaced by the construct.

[0230] The correct integration of the plasmid / gene into the transformant genome was verified using specific primers. The first round transformants with the correct integration of the expression construct and further loss of the alp3 locus were stored as M4855 and M4856. Strain M4855 was used for the second round of transformation. The second round transformants with the correct integration of the construct and loss of the alp6 gene were stored as M5129 and M5130 (with human GNT1) and M5131 and M5132 (with cattle GNT1), respectively.

[0231] The constructed C1 strain from both transformations was grown in 250 ml shake flasks in 50 ml liquid medium as described for the shake flask culture of the parent strain M3291 in WO 2021 / 094935. The culture was carried out at 35°C and ~200 RPM for 4 days. The mycelium was removed by centrifugation and the culture was carried out using The culture supernatant was used for protein A affinity purification of nivolumab according to the manufacturer's instructions (Cytiva) using a Cytiva Start automated HPLC system and HiTrap MabSelectSure or HiTrap MabSelect PrismA pre-packed 1 ml columns. Peak fractions from all samples were analyzed for released N-glycans as described in Example 1 of WO 2021 / 094935. The results for all strains are summarized in Figure 1. Figure 1A It was shown that by expressing both TrMNS1-HDEL and C1 GLS2A-HDEL, the major N-glycan on nivolumab was Man3 (M3), with a content close to 90%, while there were only very few higher mannose structures (GlcNAc2Man4, GlcNAc2Man5) and almost no Hex6 (GlcNAc2Man5Glc), the latter of which is thought to block N-glycan modification to G0 and above. Figure 1B-1D It was shown that by using the alp6 targeted expression vector described in this example, very high levels of G1 (GlcNAc2Man3GlcNAc2Gal) and G2 (GlcNAc2Man3GlcNAc2Gal2) N-glycans were achieved on the target mAb. The conversion rate to human-type G1 / 2 N-glycans was 88-98%, i.e., almost complete conversion to human-type glycans using this method. In addition to very high levels of human-type N-glycans, Figure 2Also shown are the amounts of released N-glycans between samples normalized using a fixed amount of internal standard added to each sample for analysis. The percentages of released N-glycans from the different nivolumab samples were roughly at the same level as the reference Opdivo (commercial nivolumab produced in a mammalian system, Bristol-Myers Squibb), indicating that this approach to humanized glycosylation does not negatively affect the occupancy of N-glycosylation sites.

[0232] Next, C1 strains M3291, M5130, and M5132 were cultured in ambr250 or 1L bioreactors using a fed-batch method in a medium with yeast extract as an organic nitrogen source and glucose as a carbon source. The culture was carried out at 38°C for 7 days. After the end of the culture, the mycelium was removed by centrifugation at 4000g for 20 minutes, phenylmethylsulfonyl fluoride (PMSF) at a concentration of 1-2mM was added to inhibit the protease activity in the resulting culture supernatant, and the samples were stored at -80°C. Nivolumab was purified from the seventh-day fermentation sample using a protein A affinity purification method that was essentially the same as the above-mentioned shake flask sample purification. The peak fractions from all samples were analyzed for released N-glycans. Figure 3 summarizes the results for all three strains. Figure 3A It was shown that although the parent strain M3291 had more than 80% of the desired precursor Man3 (M3), it also had more than 3.5% Hex6 (M6), which is thought to prevent N-glycan humanization (conversion to G0 and further modification), and some higher mannose structures (Man4, Man5). Figure 3B-3D It was shown that the released N-glycans on the target mAb were nearly fully humanized, ie, 99% or more of the detected N-glycans belonged to the G0 to G2 species. Figure 4 The amount of released N-glycans between samples normalized using a fixed amount of internal standard (sialyl glycan peptide) added to each sample for analysis is shown. As in the shake flasks, the percentage of released N-glycans from both nivolumab strains with humanized N-glycans appeared to be at the same level as the reference Opdivo (commercial nivolumab produced in a mammalian system), further confirming that there was no negative impact on N-glycan site occupancy.

[0233] Example 2 - One-step generation of an empty strain with humanized G1 / 2 glycans

[0234] In this second step, all three sugar modification steps required for the generation of a Thermothelomyces heterothallica C1 strain producing human-type galactosylated glycans described in Example 1 were combined. Briefly, the deletion of the alg3 gene in a strain carrying a deletion of 14 protease genes and the kex2 protease gene under a weaker promoter was combined with the integration of ER-targeted Trichoderma reesei mannosidase 1 (ɑ-1,2-mannosidase), ER-targeted C1 glucosidase 2ɑ subunit, human GNT1 (GlcNAc transferase I), rat GNT2 (GlcNAc transferase II), oligosaccharyltransferase STT3 from Leishmania major, and human GalT1 (galactosyltransferase I) in the alg3 locus. The expression of these genes first results in the trimming of glycan precursors to DolP-GlcNAc2-Man suitable for the synthesis of human-type glycans. 3 , and then in the GlcNAc 2 Man 3 GlcNAc residues are added to the two branches of the glycan to produce G0(GlcNAc2Man 3 GlcNAc 2 ) glycans, and then galactose residues are added to one or both branches of the G0 glycan, resulting in the production of G1 (GlcNAc 2 Man 3 GlcNAc 2 Gal) and G2 (GlcNAc 2 Man 3 MlcNAc 2 Gal 2 ) polysaccharides.

[0235] DNA constructs designed for integration into the alg3 locus (JGI M. thermophila genome database ID 2310419) and simultaneous expression of Trichoderma reesei (Tr) mns1-HDEL (JGI T. reesei genome database ID 45717), C1gls2a-HDEL (JGI M. thermophila genome database ID 2125259), human GNT1, rat GNT2, and Leishmania major (Lm) STT3 and human GalT1 were constructed in two parts into two separate plasmids. HDEL represents a four amino acid C-terminal ER localization signal.

[0236] The first plasmid contained alg3 5′ flanking region fragment for integration, an expression cassette for GNT1 in which the human GNT1 gene was fused to the C1KRE2 Golgi localization signal (JGI M. thermophila genome database ID2300989) between the C1bgl8 promoter and the bgl8 terminator (JGI M. thermophila genome database ID115968), an expression cassette for human GalT1 fused to the Saccharomyces cerevisiae KRE2 Golgi localization signal located between the promoter of the ubiquitin-like protein gene (JGI M. thermophila genome database ID2315548) and the terminator of the C1 hypothetical protein (JGI M. thermophila genome database ID2302731), an expression cassette for human GalT1 located between the promoter of the ubiquitin-like protein gene (JGI M. thermophila genome database ID2315548) and the terminator of the C1 hypothetical protein (JGI M. thermophila genome database ID2302731), a synthetic AnSES promoter (Rantasalo A et al., 2018, A universal gene expression system for fungi, Nucl. Acids Research 46(18):e111) and chi1 terminator (JGIM.thermophila genome database ID50608), an expression cassette for C1 gls2a-HDEL, an expression cassette for a synthetic transcription factor (sTF, synthetic promoter for C1 gls2a-HDEL), and the first 2 / 3 of the pyr4 marker gene (JGIM.thermophila genome database ID2311494).

[0237] The second plasmid contains the last 2 / 3 of the C1 pyr4 marker gene, a direct repeat targeting the beginning of the pyr4 cassette (for removal of the pyr4 marker by recombination), an inverse expression cassette for STT3 from Leishmania major between the promoter and terminator of the C1 hypothetical protein (JGI M. thermophila genome database ID 2315630), an inverse expression cassette for Tr mns1-HDEL (wherein the gene is located between the C1 bgl8 promoter (JGI M. thermophila genome database ID 115968) or the promoter of the ubiquitin-like protein gene (JGI M. thermophila genome database ID 2315548) and the terminator of the ubiquitin-like protein gene (JGI M. thermophila genome database ID 2315548), a promoter for the translation elongation factor 1A (JGI M. thermophila genome database ID 2298136) and the terminator of the C1 hypothetical protein (JGI The plasmids were then cloned into a retro-expression cassette for the rat GNT2 gene between the plasmids (M. thermophila genome database ID 114107) and finally the alg3 3' flanking region fragment for integration. The C1pyr4 marker fragments in the two plasmids overlap each other, and this region undergoes homologous recombination in C1 between the plasmids, while the 5' and 3' flanking region fragments recombine with the genomic DNA on both sides of the alg3 gene. The recombination between the selectable marker fragments makes the marker gene functional and enables the transformant to grow under selection.

[0238] A construct containing alg3 5' flanking region fragment, expression cassettes for human GNT1, human GalT1, C1 gls2a-HDEL, sTF, and the first 2 / 3 of the pyr4 tag is set forth in SEQ ID NO: 23 (pMYT1974). The alg3 5' flanking sequence corresponds to positions 9-1010 of SEQ ID NO: 23. The bgl8 promoter sequence corresponds to positions 1018-2409 of SEQ ID NO: 23. The nucleic acid sequence encoding human GNT1 fused to the C1 KRE2 Golgi localization signal corresponds to positions 2410-3898 of SEQ ID NO: 23, wherein positions 2410-2674 encode the C1 KRE2 localization signal and positions 2675-3898 encode human GNT1. The nucleic acid sequence encoding human GNT1 fused to the C1 KRE2 Golgi localization signal is also set forth as SEQ ID NO: 5 (C1 kre2-huGNT1 nt). The nucleic acid sequence encoding human GNT1 was codon-optimized for M. thermophila C1 and synthesized by GenScript (USA). In the synthesized sequence, the region of amino acids 1-100 of human GNT1 was replaced by the C1 KRE2 Golgi localization signal (amino acids 1-70). The complete amino acid sequence of C1 KRE2-GNT1 is set forth as SEQ ID NO: 6 (C1 KRE2-huGNT1 aa). The bgl8 terminator sequence corresponds to positions 3899-4365 of SEQ ID NO: 23. The ubiquitin-like protein promoter sequence corresponds to positions 4374-5393 of SEQ ID NO: 23. The sequence encoding human GalT1 fused to the Sc-KRE2 Golgi localization signal corresponds to positions 5394-5693 of SEQ ID NO: 23, wherein positions 5394-5693 encode the Sc KRE2 localization signal and positions 5694-6659 encode human GalT1. The nucleic acid sequence encoding human GalT1 fused to the Sc KRE2 Golgi localization signal is also set forth as SEQ ID NO: 13 (Sckre2-huGalT1 nt). The nucleic acid sequence encoding human GalT1 was codon-optimized for M. thermophila C1 and synthesized by GenScript (USA). In the synthesized sequence, the region of amino acids 1-77 of human GalT1 was removed after cloning of the expression plasmid and replaced by the Sc KRE2 Golgi localization signal (amino acids 1-100). The complete amino acid sequence of ScKRE2-GalT1 is set forth as SEQ ID NO: 14 (ScKRE2-huGalT1 aa). The terminator sequence of the hypothetical protein (ID2302731) corresponds to positions 6660-7065 of SEQ ID NO:23.The synthetic AnSES promoter sequence corresponds to positions 7073-7565 of SEQ ID NO: 23. The sequence encoding C1 glucosidase 2α with an artificial HDEL ER retention signal corresponds to positions 7566-10578 of SEQ ID NO: 23. The nucleic acid sequence encoding C1GLS2α with an HDEL signal is also set forth as SEQ ID NO: 3 (C1 gls2a-HDEL nt) and the amino acid sequence is SEQ ID NO: 4 (C1 GLS2A-HDEL aa). The chi1 terminator sequence corresponds to positions 10579-11224 of SEQ ID NO: 23. The nucleic acid sequence of the synthetic transcription factor cassette corresponds to positions 11225-12874 of SEQ ID NO: 23. The first 2 / 3 of the C1pyr4 marker gene corresponds to positions 12888-14667 of SEQ ID NO: 23. The fragments were generated by PCR, separated in agarose gel, purified, and cloned stepwise into the backbone vector pRS426 using yeast recombination to obtain the final 5' arm expression vector pMYT1974, which was verified by sequencing.

[0239] Two parallel constructs containing the last 2 / 3 of the C1 pyr4 marker gene, a direct repeat sequence for marker removal, an inverted expression cassette for STT3 from Leishmania major, an inverted expression cassette for Tr Mns1-HDEL, an inverted expression cassette for the rat GNT2 gene, and an alg3 3' flanking region fragment for integration are set forth as SEQ ID NO: 24 (pMYT1963) and SEQ ID NO: 25 (pMYT1964), respectively. The last 2 / 3 of the C1 pyr4 marker gene corresponds to positions 17-1273 of SEQ ID NO: 24. The direct repeat fragment targeting the beginning of the pyr4 cassette corresponds to positions 1290-1786 of SEQ ID NO: 24. The hypothetical protein (ID2315630) promoter sequence corresponds to positions 5965-4957 of SEQ ID NO: 24. The sequence encoding the Leishmania major STT3 gene, codon-optimized for T. heterothallica C1 and synthesized by GenScript (USA), corresponds to positions 4956-2383 of SEQ ID NO: 24. The nucleic acid sequence encoding the Leishmania major STT3 gene is also set forth as SEQ ID NO: 11 (LmSTT3 nt). The complete amino acid sequence of L. major STT3 is set forth as SEQ ID NO: 12 (LmSTT3aa). The hypothetical protein (ID2315630) terminator sequence corresponds to positions 2374-1795 of SEQ ID NO: 24. The ubiquitin-like protein promoter sequence corresponds to positions 9316-8297 of SEQ ID NO: 24. The nucleic acid sequence encoding the genomic Trichoderma reesei mannosidase 1 with an artificial HDEL ER retention signal corresponds to positions 8296-6470 of SEQ ID NO: 24. The nucleic acid sequence encoding the Trichoderma reesei MNS1 with an HDEL signal is also set forth as SEQ ID NO: 1 (Tr mns1-HDEL nt) and the amino acid sequence is SEQ ID NO: 2 (Tr MNS1-HDEL aa). The ubiquitin-like protein terminator sequence corresponds to positions 6469-5974 of SEQ ID NO: 24. The translation elongation factor 1A promoter sequence corresponds to positions 12210-11154 of SEQ ID NO: 24. The sequence encoding the rat GNT2, codon-optimized for T. heterothallica C1 and synthesized by GenScript (USA), corresponds to positions 11153-9825 of SEQ ID NO: 24.The nucleic acid sequence encoding rat GNT2 is also set forth as SEQ ID NO: 15 (rat GNT2 nt). The complete amino acid sequence of rat GNT2 is set forth as SEQ ID NO: 16 (rat GNT2 aa). The hypothetical protein (ID114107) terminator sequence corresponds to positions 9824-9321 of SEQ ID NO: 24. The alg3 3' flanking sequence corresponds to positions 12218-13217 of SEQ ID NO: 24. The fragment was generated by PCR, isolated in an agarose gel, purified, and cloned stepwise into the backbone vector pRS426 using yeast recombination to obtain the final 3' arm expression vector pMYT1963, which was verified by sequencing. The fragment was generated by PCR, isolated in an agarose gel, purified, and cloned stepwise into the backbone vector pRS426 using yeast recombination to obtain the final 3' arm expression vector pMYT1963, which was verified by sequencing.

[0240] In SEQ ID NO:25 (pMYT1964), the last 2 / 3 of the C1 pyr4 marker gene corresponds to positions 17-1273. The direct repeat targeting the beginning of the pyr4 cassette corresponds to positions 1290-1786 of SEQ ID NO:25. The hypothetical protein (ID2315630) promoter sequence corresponds to positions 5965-4957 of SEQ ID NO:25. The sequence encoding STT3 from Leishmania major, codon-optimized for T. heterothallica C1 and synthesized by GenScript (USA), corresponds to positions 4956-2383 of SEQ ID NO:25. The nucleic acid sequence encoding the Leishmania major STT3 gene is also set forth as SEQ ID NO:11 (LmSTT3nt). The complete amino acid sequence of L. major STT3 is set forth as SEQ ID NO: 12 (LmSTT3 aa). The hypothetical protein (ID2315630) terminator sequence corresponds to positions 2374-1795 of SEQ ID NO: 25. The bgl8 promoter sequence corresponds to positions 9688-8297 of SEQ ID NO: 25. The nucleic acid sequence encoding the genomic Trichoderma reesei mannosidase 1 with an artificial HDEL ER retention signal corresponds to positions 8296-6470 of SEQ ID NO: 25. The nucleic acid sequence encoding the T. reesei MNS1 with an HDEL signal is also set forth as SEQ ID NO: 1 (Trmns1-HDEL nt) and the amino acid sequence is SEQ ID NO: 2 (Tr MNS1-HDEL aa). The ubiquitin-like protein terminator sequence corresponds to positions 6469-5974 of SEQ ID NO: 25. The translation elongation factor 1A promoter sequence corresponds to positions 12584-11528 of SEQ ID NO:25. The sequence encoding rat GNT2, codon-optimized for M. thermophila C1 and synthesized by GenScript (USA), corresponds to positions 11527-10199 of SEQ ID NO:25. The nucleic acid sequence encoding rat GNT2 is also set forth as SEQ ID NO:15 (rat GNT2 nt). The complete amino acid sequence of rat GNT2 is set forth as SEQ ID NO:16 (rat GNT2 aa). The hypothetical protein (ID114107) terminator sequence corresponds to positions 10198-9695 of SEQ ID NO:25. The alg3 3' flanking sequence corresponds to positions 12592-13591 of SEQ ID NO:25.The fragments were generated by PCR, separated in agarose gel, purified, and cloned stepwise into the backbone vector pRS426 using yeast recombination to obtain the final 3' arm expression vector pMYT1964, which was verified by sequencing.

[0241] To obtain two different empty strains (not expressing heterologous mammalian glycoproteins) with G1 / 2 N-glycans, the above expression plasmids were transformed in one step into pyr4 carrying a deletion of 14 protease genes and the kex2 protease gene under a weaker promoter. - strain. In each transformation, a pair of one 5' arm expression vector and one 3' arm expression vector (cut from the expression plasmid backbone with MssI) was used. The pair was: SEQ ID NO: 23 (pMYT1974; alg3 5' flanking fragment-human GNT1 box-human GalT1 box-C1 gls2a-HDEL box-sTF box-2 / 3 pyr4) + SEQ ID NO: 24 (pMYT1963) or SEQ ID NO: 25 (pMYT1964) (2 / 3pyr4-DR-LmSTT3 box-TrMns1-HDEL box-rat GNT2 box-alg3 3' flanking fragment). C1 transformation and transformant selection were performed essentially as described in WO 2021 / 094935. The selection of transformation was based on a functional pyr4 gene. Transformants were screened by PCR to find clones in which the alg3 gene was replaced by the construct. Correct integration of the plasmid / gene into the transformant genome was verified using specific primers. Transformants with correct integration of the construct and loss of the alg3 gene were stored as M6589 and M6596 (with the ubiquitin-like protein promoter for TrMns1-HDEL) and M6590 and M6597 (with the bgl8 promoter for TrMns1-HDEL), respectively.

[0242] The constructed C1 strains from both transformations were grown in 3.5 ml of liquid medium in a 24-well deep well plate as described for strain culture in WO 2021 / 094935. Cultivation was carried out for 4 days at 35°C, 800 RPM, and 80% humidity. The mycelium was removed by centrifugation, and the culture supernatant was analyzed for released N-glycans as described in Example 1 of WO 2021 / 094935. Figure 5 summarizes the results for all four strains. Figure 5 shows that by expressing all six genes that affect glycan structure from the alg3 locus, more than 80% of the major N-glycans on the total secreted proteins are human-type (N-glycans belonging to the G0 to G2 categories), and there is no detectable Hex6 (GlcNAc 2 Man 5Glc), which is thought to block N-glycan modification to G0 and above. It is also noteworthy that the amount of final G2 N-glycans is large. It has been found that the amount of human-type N-glycans (in %) on total secreted proteins is always lower than that observed on any purified target protein (e.g., nivolumab in Example 1).

[0243] sequence

[0244] SEQ ID NO: 1-Trichoderma reesei mns1-HDEL nt (coding sequence, i.e., introns removed, 1684 bp)

[0245] SEQ ID NO: 2-Trichoderma reesei MNS1-HDEL aa (527 aa)

[0246] SEQ ID NO: 3-C1 gls2a-HDEL nt (coding sequence, i.e., introns removed, 2961 bp)

[0247] SEQ ID NO: 4-C1 GLS2A-HDEL aa (986 aa)

[0248] SEQ ID NO: 5-C1 kre2-huGNT1 nt (coding sequence, i.e., introns removed, 1434 bp)

[0249] SEQ ID NO: 6-C1 KRE2-huGNT1 aa(477 aa)

[0250] SEQ ID NO: 7-C1rft1nt (coding sequence, i.e., introns removed, 1839 bp)

[0251] SEQ ID NO: 8-C1 RFT1 aa (612 aa)

[0252] SEQ ID NO: 9-C1 kre2-boGNT1 nt (coding sequence, i.e., introns removed, 1440 bp)

[0253] SEQ ID NO: 10-C1 KRE2-boGNT1 aa(479 aa)

[0254] SEQ ID NO: 11-LmSTT3 nt (2574bp)

[0255] SEQ ID NO: 12-LmSTT3 aa (857 aa)

[0256] SEQ ID NO: 13-Sckre2-huGalT1 nt(1266bp)

[0257] SEQ ID NO: 14-ScKRE2-huGalT1 aa(421 aa)

[0258] SEQ ID NO: 15—rat GNT2 nt (1329 bp)

[0259] SEQ ID NO: 16—rat GNT2 aa (442 aa)

[0260] SEQ ID NO: 17-pMYT1288(14648bp)

[0261] SEQ ID NO: 18-pMYT0721(13062bp)

[0262] SEQ ID NO: 19-pMYT1451(15270bp)

[0263] SEQ ID NO: 20-pMYT1452(15281bp)

[0264] SEQ ID NO: 21-pMYT1453(18195bp)

[0265] SEQ ID NO: 22-rDNA of Thermothelomyces heterothallica C1

[0266] SEQ ID NO: 23-pMYT1974(20189bp)

[0267] SEQ ID NO: 24-pMYT1963(18731bp)

[0268] SEQ ID NO: 25-pMYT1964(19105bp)

[0269] The above description of specific embodiments fully reveals the overall nature of the present invention, so that others can easily modify and / or adapt such specific embodiments to be suitable for various different applications by applying current knowledge without excessive experimentation and without departing from the general concept, and therefore, such adaptations and modifications should and are intended to be included in the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the phrases or terms used herein are for the purpose of description rather than limitation. The means, materials and steps for performing various disclosed chemical structures and functions can take various different alternative forms without departing from the present invention.

Claims

1. A Thermothelomyces heterothallica genetically modified to produce a glycoprotein having mammalian N-glycans, wherein the genetic modification comprises: (i) the alg3 gene is deleted or destroyed, so that the Th. heterothallica cannot produce functional α-1,3-mannosyltransferase; (ii) expression of ER-targeted mannosidase 1 (α-1,2-mannosidase); and (iii) Expression of glucosidase 2α subunit targeted to the ER.

2. Th. heterothallica according to claim 1, wherein the mannosidase 1 is a Trichoderma reesei mannosidase.

3. Th. heterothallica according to any one of claims 1 or 2, wherein the glucosidase 2ɑ subunit is the Th. heterothallica glucosidase 2α subunit.

4. Th. heterothallica according to any one of claims 1-3, wherein the genetic modification further comprises the expression of heterologous GlcNAc transferase 1 (GNT1) and GlcNAc transferase 2 (GNT2).

5. Th. heterothallica according to claim 4, wherein the heterologous GNT1 and GNT2 are of animal origin.

6. Th. heterothallica according to claim 5, wherein the GNT1 of animal origin is human GNT1. 7 . The Th. heterothallica according to claim 6 , wherein the GNT1 of animal origin is human GNT1 further comprising a Th. heterothallica Golgi localization signal.

8. Th. heterothallica according to claim 7, wherein the Th. heterothallica Golgi localization signal is derived from the C1 protein KRE2.

9. The Th. heterothallica according to claim 8, wherein the human GNT1 comprising the Golgi localization signal from the C1 protein KRE2 comprises the amino acid sequence set forth in SEQ ID NO:

6.

10. The Th. heterothallica according to claim 9, wherein the human GNT1 comprising the Golgi localization signal from the C1 protein KRE2 is encoded by an exogenous polynucleotide comprising the sequence set forth in SEQ ID NO: 5 introduced into the Th. heterothallica.

11. Th. heterothallica according to claim 5, wherein the GNT1 of animal origin is bovine GNT1.

12. The Th. heterothallica according to claim 11, wherein the GNT1 of animal origin is bovine GNT1 comprising a Th. heterothallica Golgi localization signal.

13. The Th. heterothallica of claim 12, wherein the bovine GNT1 comprising a Golgi localization signal from the Th. heterothallica protein KRE2 comprises the amino acid sequence set forth in SEQ ID NO:

10.

14. The Th. heterothallica according to claim 12, wherein the bovine GNT1 comprising the Golgi localization signal from the Th. heterothallica protein KRE2 is encoded by an exogenous polynucleotide comprising the sequence set forth in SEQ ID NO: 9 introduced into the Th. heterothallica.

15. Th. heterothallica according to claim 5, wherein the GNT2 of animal origin is rat GNT2.

16. The Th. heterothallica of claim 15, wherein the rat GNT2 comprises the amino acid sequence set forth in SEQ ID NO:

15.

17. The Th. heterothallica according to claim 15, wherein the rat GNT2 is encoded by an exogenous polynucleotide comprising the sequence set forth in SEQ ID NO: 16 introduced into the Th. heterothallica.

18. The Th. heterothallica according to claim 5, wherein the animal-derived GNT1 is human GNT1 comprising a Golgi localization signal from Th. heterothallica protein KRE2, and the animal-derived GNT2 is rat GNT2.

19. The Th. heterothallica according to claim 5, wherein the animal-derived GNT1 is bovine GNT1 comprising a Golgi localization signal from the Th. heterothallica protein KRE2, and the animal-derived GNT2 is rat GNT2.

20. Th. heterothallica according to any one of the preceding claims, wherein the genetic modification further comprises overexpression of an endogenous flippase or expression of a heterologous flippase.

21. The Th. heterothallica of claim 20, which is genetically modified to express an exogenous flippase, wherein the flippase is a Th. heterothallica RFT1 flippase.

22. The Th. heterothallica of claim 21, wherein the Th. heterothallica RFT1 flippase comprises the amino acid sequence set forth in SEQ ID NO:

8.

23. The Th. heterothallica according to claim 21, wherein the Th. heterothallica RFT1 flippase is encoded by an exogenous polynucleotide introduced into the Th. heterothallica comprising the sequence set forth in SEQ ID NO:

7.

24. The Th. heterothallica of claim 20, which is genetically modified to overexpress the endogenous Th. heterothallica RFT1 flippase.

25. Th. heterothallica according to any one of the preceding claims, wherein the genetic modification further comprises expression of the STT3 subunit of heterooligosaccharyltransferase.

26. Th. heterothallica according to claim 25, wherein the STT3 is Leishmania major STT3.

27. The Th. heterothallica of claim 26, wherein the Leishmania major STT3 comprises the amino acid sequence set forth in SEQ ID NO:

12.

28. The Th. heterothallica according to claim 26, wherein the Leishmania major STT3 is encoded by an exogenous polynucleotide comprising the sequence set forth in SEQ ID NO: 11 introduced into the Th. heterothallica.

29. Th. heterothallica according to any one of the preceding claims, wherein the genetic modification further comprises expression of a heterologous galactosyltransferase.

30. Th. heterothallica according to claim 29, wherein the heterologous galactosyltransferase is a galactosyltransferase of animal origin.

31. Th. heterothallica according to claim 30, wherein the galactosyltransferase of animal origin is human galactosyltransferase.

32. The Th. heterothallica of claim 31, wherein the human galactosyltransferase comprising the S. cerevisiae KRE2 Golgi localization signal comprises the amino acid sequence set forth in SEQ ID NO:

14.

33. The Th. heterothallica according to claim 31, wherein the human galactosyltransferase comprising the S. cerevisiae KRE2 Golgi localization signal is encoded by an exogenous polynucleotide comprising the sequence set forth in SEQ ID NO: 13 introduced into the Th. heterothallica.

34. The Th. heterothallica of any one of the preceding claims, wherein the Th. heterothallica is Th. heterothallica C1, wherein the C1 is a strain modified to delete one or more genes encoding endogenous proteases.

35. Th. heterothallica according to any one of the preceding claims, wherein the Th. heterothallica is Th. heterothallica C1, wherein the C1 is a strain modified to delete a gene encoding an endogenous chitinase.

36. Th. heterothallica according to any one of claims 1 to 35, wherein the Th. heterothallica is Th. heterothallica C1, wherein the C1 is a strain selected from the following: wild type C1 deposit number VKM F-3500D, UV13-6 deposit number VKM F-3632D, NG7C-19 deposit number VKM F-3633D, UV18-25 deposit number VKM F-3631D, 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.

37. The Th. heterothallica of any preceding claim, further genetically modified to express a heterologous mammalian glycoprotein.

38. Th. heterothallica according to claim 37, wherein the heterologous mammalian glycoprotein is an antibody or an antigen-binding fragment thereof.

39. A method for producing Th. heterothallica producing a glycoprotein having mammalian N-glycans, the method comprising: (a) deleting or disrupting the alg3 gene of the Th. heterothallica so that the Th. heterothallica cannot produce functional α-1,3-mannosyltransferase; and (b) introducing exogenous polynucleotides encoding ER-targeted mannosidase 1 (α-1,2-mannosidase) and ER-targeted glucosidase 2 α subunit into the Th. heterothallica.

40. The method according to claim 39, further comprising introducing into the Th. Heterothallica: an exogenous polynucleotide encoding an endogenous flippase to induce overexpression of the endogenous flippase in the Th. Heterothallica; or an exogenous polynucleotide encoding a heterologous flippase to induce expression of the heterologous flippase in the Th. Heterothallica.

41. A method for producing a glycoprotein having mammalian N-glycans, the method comprising: (a) providing a genetically modified Th. heterothallica according to any one of claims 1 to 40; (b) culturing the Th. heterothallica under conditions suitable for expressing the glycoprotein; and (c) recovering the glycoprotein.

42. The method of claim 41, wherein the glycoprotein is a heterologous mammalian glycoprotein recombinantly expressed in the Th. heterothallica.

43. The method of claim 41, wherein the heterologous mammalian glycoprotein is a human glycoprotein recombinantly expressed in said Th. heterothallica.

44. The method of claim 41, wherein the heterologous mammalian glycoprotein is an antibody or an antigen-binding fragment thereof.

45. A recombinant glycoprotein produced by the genetically modified Th. heterothallica according to any one of claims 1-39, wherein the glycoprotein comprises GlcNAc2Man3GlcNAc2(G0) glycans.

46. ​​A recombinant glycoprotein produced by the genetically modified Th. heterothallica according to any one of claims 1-39, wherein the glycoprotein comprises Gal1GlcNAc2Man3GlcNAc2(G1) glycans, Gal2GlcNAc2Man3GlcNAc2(G2) glycans, or a combination thereof.

47. The recombinant glycoprotein of claim 45 or claim 46, wherein the glycoprotein is of pharmaceutical grade.

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