Recombinant fungal strains and methods for consistent protein production

Abstract

One or more particular embodiments of the present disclosure relate to, inter alia, recombinant (genetically modified) filamentous fungal cells (strains) that produce a protein of interest; methods and compositions for the design and construction of modified filamentous fungal cells that produce a protein of interest; methods and compositions for the expression / production / secretion / recovery, etc., of endogenous and / or heterologous proteins of interest (e.g., phytases, lipases, glucoamylases, phospholipases, esterases, cellulases, hemicellulases, xylanases, etc.) in modified filamentous fungal cells; methods and compositions for producing a protein of interest in a recombinant filamentous fungus, wherein the protein produced and secreted into the broth and / or recovered from the broth has a uniform and consistent N-linked glycosylation pattern and / or has reduced (undesirable) glycosylation of one or more proteins of interest;

Description

[Technical Field]

[0001] The present disclosure relates generally to the fields of biology, molecular biology, filamentous fungi, fermentation, genetics, glycoproteins, industrially relevant proteins, and protein production, etc. More particularly, the strains, compositions, and methods of the disclosure relate to genetic modifications in filamentous fungi that result in phenotypically altered recombinant (modified) strains, which are particularly suitable for growth in submerged culture (e.g., for large-scale production of proteins for industrial / commercial applications).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 476,079, filed December 19, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing Reference The contents of the electronic submission of the sequence listing text file entitled "NB42034-WO-PCT_SequenceListing.txt" was created on October 24, 2023, is 68KB in size, and is incorporated herein by reference in its entirety. [Background technology]

[0004] Filamentous fungi (e.g., Aspergillus spp., Penicillium spp., Talaromyces spp., Fusarium spp., Myceliophthora spp., Neurospora spp., and Trichoderma spp., etc.) are capable of expressing native and heterologous proteins at high levels and making them suitable for the mass production of proteins (e.g., enzymes, antibodies, receptors, peptides, etc.) and / or metabolites for industrial and commercial applications, such as pharmaceutical, animal health, food, beverage, and laundry and textile applications. Filamentous fungi are typically grown in submerged mycelial cultures in bioreactors (fermentors), which are adapted to introduce and distribute oxygen and nutrients into the culture medium (i.e., culture broth). For example, the filamentous fungus Trichoderma reesei (T. reesei; an anamorph of the fungus Hypocrea jecorina) is known to be an efficient producer of cellulase enzymes.

[0005] Thus, filamentous fungi have been exploited for their ability to produce proteins (e.g., enzymes) that are valuable in the production of everyday products such as cellulose-derived ethanol, textile processing, grain processing, detergents, fiber / pulp / paper, food additives, and feed additives. Similarly, filamentous fungi have been exploited for their ability to produce protein biopharmaceuticals (e.g., antibodies, antibody fragments, protein receptors, growth factors, etc.). For example, because recombinant gene expression in such fungal host strains is a common method for protein production, improving the protein productivity of fungal host strains is an important economic factor in protein production costs. In certain embodiments, proteins produced by filamentous fungi may contain post-translational modifications, including protein glycosylation at asparagine, threonine, and / or serine residues; proteins containing such glycosylation are referred to as glycoproteins. Notably, the glycosylation process consists of several biochemical modifications that correspond to the passage of the target glycoprotein along the secretory pathway. This process is well regulated, and the ultimate extent of protein glycosylation depends on many factors, including the structural features of the glycoprotein itself and the growth conditions.

[0006] As will be appreciated by those skilled in the art, recombinant expression of heterologous glycoproteins in filamentous fungal strains can be problematic and often results in glycoprotein products of less than satisfactory quality. For example, glycoproteins of less than satisfactory quality include glycoproteins with heterogeneous (inconsistent) glycosylation patterns, glycoproteins with reduced activity, and glycoproteins with reduced stability, which can add significant costs to downstream processing (e.g., glycoprotein product recovery and purification). In other embodiments, glycosylation (i.e., non-enzymatic glycosylation) of recombinant proteins produced in fungal strains can be problematic and often results in recombinant protein products of less than satisfactory quality. For example, glycosylation (non-enzymatic glycosylation) of proteins (e.g., enzymes) can be a substantial problem because it often reduces the activity of the protein (enzyme) product (Sutthirak et al., 2005). In addition, during many protein recovery processes, clarified (fermentation) broths must usually be diafiltered to remove any reactive free sugars to limit glycosylation. Subsequent steps in protein recovery (eg heat treatment to remove any (undesirable) background enzyme activity) may cause more free sugars to be released from the material.

[0007] Thus, as will be appreciated by those skilled in the art, novel compositions and methods for enhancing recombinant protein production in filamentous fungal strains are of significant commercial interest. In certain embodiments, there is an ongoing unmet need in the art for, among other things, the recombinant expression of heterologous proteins in fungal strains, and the recombinant expression of heterologous glycoproteins in fungal strains, where the recombinant proteins produced have uniform (consistent) glycosylation patterns, reduced glycosylation (i.e., non-enzymatic glycosylation), reduced downstream processing requirements, and the like. Summary of the Invention [Means for solving the problem]

[0008] As generally set forth and described below, certain embodiments of the present disclosure relate to, inter alia, recombinant filamentous fungal cells (strains) that produce a protein of interest, methods and compositions for designing and constructing recombinant (modified) fungal cells that produce a protein of interest, methods and compositions for expressing / producing an endogenous protein of interest (e.g., a lignocellulolytic enzyme, etc.) in recombinant filamentous fungal cells, methods and compositions for expressing / producing a heterologous protein of interest (e.g., a phytase, lipase, glucoamylase, phospholipase, esterase, cellulase, hemicellulase, xylanase, etc.) in recombinant filamentous fungal cells, and methods and compositions for producing a protein of interest in recombinant filamentous fungi, wherein the protein produced and / or recovered therefrom has a uniform and consistent N-linked glycosylation pattern and / or reduced (undesirable) glycosylation events of the protein of interest. Thus, in certain embodiments, the methods and compositions shown and described herein realize, among other things, significant cost savings associated with large-scale fermentation and downstream recovery of one or more proteins of interest produced in one or more recombinant filamentous fungal cells described herein.

[0009] Sequence Listing Free Text SEQ ID NO:1 is the nucleic acid (DNA) sequence of the wild-type Trichoderma mds1 gene encoding the native Mds1 protein of SEQ ID NO:2.

[0010] SEQ ID NO:2 is the amino acid sequence of the native Mds1 protein encoded by SEQ ID NO:1.

[0011] SEQ ID NO:3 is the DNA sequence of the wild-type Trichoderma mds2 gene encoding the native Mds2 protein of SEQ ID NO:4.

[0012] SEQ ID NO:4 is the amino acid sequence of the native Mds2 protein encoded by SEQ ID NO:3.

[0013] SEQ ID NO:5 is the DNA sequence of the wild-type Trichoderma gls2a gene encoding the native GIIα protein of SEQ ID NO:6.

[0014] SEQ ID NO:6 is the amino acid sequence of the native GIIα protein encoded by SEQ ID NO:5.

[0015] SEQ ID NO: 7 is a variant (truncated) GIIα Stop A variant Trichoderma DNA sequence (gls2a) encoding a protein Stop )

[0016] SEQ ID NO: 8 is a truncated GIIα encoded by SEQ ID NO: 7 Stop 1 is the amino acid sequence of the variant protein.

[0017] SEQ ID NO: 9 is a synthetic RNA sequence designated RGH2.

[0018] SEQ ID NO: 10 is the synthetic DNA gls2a recovery donor sequence.

[0019] SEQ ID NO: 11 is the synthetic RNA sequence designated LFP009.

[0020] SEQ ID NO: 12 is the synthetic RNA sequence designated LFP010.

[0021] SEQ ID NO: 13 is the synthetic DNA sequence designated LFP013.

[0022] SEQ ID NO: 14 is the synthetic DNA sequence designated LFP014.

[0023] SEQ ID NO: 15 is a synthetic RNA sequence designated TCg3.

[0024] SEQ ID NO: 16 is a synthetic RNA sequence designated TCg4.

[0025] SEQ ID NO: 17 is the synthetic DNA sequence designated TC128.

[0026] SEQ ID NO: 18 is the DNA sequence of the Aspergillus niger gls2a gene homolog.

[0027] SEQ ID NO:19 is the amino acid sequence of the A. niger GIIα protein encoded by SEQ ID NO:18.

[0028] SEQ ID NO: 20 is the DNA sequence of the A. niger mds1 gene homologue.

[0029] SEQ ID NO:21 is the amino acid sequence of the A. niger Mds1 protein encoded by SEQ ID NO:20.

[0030] SEQ ID NO: 22 is the DNA sequence of the A. niger mds2 gene homologue.

[0031] SEQ ID NO:23 is the amino acid sequence of the A. niger Mds2 protein encoded by SEQ ID NO:22.

[0032] SEQ ID NO:24 is the DNA sequence of the T. thermophilus gls2a gene homologue.

[0033] SEQ ID NO:25 is the amino acid sequence of the T. thermophilus GIIα protein encoded by SEQ ID NO:24.

[0034] SEQ ID NO: 26 is the DNA sequence of the T. thermophilus mds1 gene homologue.

[0035] SEQ ID NO:27 is the amino acid sequence of the T. thermophilus Mds1 protein encoded by SEQ ID NO:26.

[0036] SEQ ID NO:28 is the DNA sequence of the T. thermophilus mds2 gene homologue.

[0037]

[0038] SEQ ID NO:29 is the amino acid sequence of the T. thermophilus Mds2 protein encoded by SEQ ID NO:28.

[0039] SEQ ID NO: 30 is the sequence of an artificial RNA named LFP028.

[0040] SEQ ID NO: 31 is the sequence of an artificial RNA named LFP029.

[0041] SEQ ID NO: 32 is an artificial DNA sequence designated LFP030.

[0042] SEQ ID NO: 33 is the sequence of an artificial RNA named LFP031.

[0043] SEQ ID NO: 34 is the sequence of an artificial RNA named LFP032.

[0044] SEQ ID NO: 35 is the artificial DNA sequence designated LFP033.

[0045] SEQ ID NO: 36 is the DNA sequence of the wild-type T. reesei EndoT allele encoding the native endo-N-acetyl-β-D-glucosaminidase (EnGase) protein. [Brief explanation of the drawings]

[0046] [Figure 1] The amino acid sequences of the native Trichoderma Mds1 protein (FIG. 1A, SEQ ID NO: 2) and the native Trichoderma Mds2 protein (FIG. 1B, SEQ ID NO: 4) are shown.

[0047] [Figure 2]The amino acid sequences of the native GIIα protein (FIG. 2A, SEQ ID NO: 6) and the truncated GIIαStop variant protein (FIG. 2B, SEQ ID NO: 8) encoded by the mutant gls2aSTOP allele (SEQ ID NO: 7) are shown. As shown in FIG. 2A, the native GIIα protein (SEQ ID NO: 6) contains 964 amino acid residues, with the last 310 C-terminal residues of the native protein underlined. As shown in FIG. 2B, the variant (truncated) GIIαStop protein (SEQ ID NO: 8) contains 807 amino acid residues, with the truncated C-terminus containing 153 (frameshifted) amino acid residues underlined.

[0048] [Figure 3] Figure 3 shows SDS-PAGE analysis of diluted phytase produced in MTP cultures. The effect of restoring the gls2a (gls2aR) allele confirmed in three independent isolates (Phy-gls2aR) is shown relative to two technical replicates of the parental (Phy) strain. As shown in Figure 3, the left panel shows the phytase (Phy) parent strain and the modified gls2a-restored (gls2aR) strain without EndoH treatment, while the right panel shows the same phytase (Phy) parent strain and the modified gls2a-restored (gls2aR) strain with EndoH treatment.

[0049] [Figure 4] Electrospray ionization mass spectra (EIMS) of phytase produced from the Phy strain before (control) and after restoration of the truncated gls2aSTOP allele (gls2aR). Data shown are from supernatant samples obtained from the large-scale fermentor at 148 hours. Protein was detected in multiple charge states, as shown.

[0050] [Figure 5]1 shows the amino acid sequences of the T. reesei GIIα protein (SEQ ID NO: 6), the A. niger GIIα protein homolog (SEQ ID NO: 19), and the T. thermophilus GIIα protein homolog (SEQ ID NO: 25).

[0051] [Figure 6] 1 shows a CLUSTAL multiple sequence alignment of the T. reesei GIIα protein (SEQ ID NO: 6, designated "6"), the A. niger GIIα homolog (SEQ ID NO: 19, designated "19"), and the T. thermophilus GIIα homolog (SEQ ID NO: 25, designated "25").

[0052] [Figure 7] 1 shows the amino acid sequences of the T. reesei Mds1 protein (SEQ ID NO: 2), the A. niger Mds1 protein homolog (SEQ ID NO: 21), and the T. thermophilus Mds1 protein homolog (SEQ ID NO: 27).

[0053] [Figure 8] 1 shows a CLUSTAL multiple sequence alignment of the T. reesei Mds1 protein (SEQ ID NO: 2, designated "2"), the A. niger Mds1 homolog (SEQ ID NO: 21, designated "21"), and the T. thermophilus Mds1 homolog (SEQ ID NO: 27, designated "27").

[0054] [Figure 9] 1 shows the amino acid sequences of the T. reesei Mds2 protein (SEQ ID NO: 4), the A. niger Mds2 protein homolog (SEQ ID NO: 23), and the T. thermophilus Mds2 protein homolog (SEQ ID NO: 29).

[0055] [Figure 10] 1 shows a CLUSTAL multiple sequence alignment of the T. reesei Mds2 protein (SEQ ID NO: 4, designated "4"), the A. niger Mds2 homolog (SEQ ID NO: 23, designated "23"), and the T. thermophilus Mds2 homolog (SEQ ID NO: 29, designated "29").

[0056] [Figure 11] The annotated amino acid sequence positions of the native T. reesei Mds1 protein (SEQ ID NO: 2) and Mds2 protein (SEQ ID NO: 4) are shown. In particular, as shown in FIG. 11, the Mds1 protein contains 523 amino acid residues, with amino acid residues from about 43 to about 511 (SEQ ID NO: 2) shown as bolded residues. These amino acid positions (about 43 to about 511) comprise the glycosyl hydrolase family 47 (GH47) sequence domain. Similarly, as shown in FIG. 11, the Mds2 protein contains 794 amino acid residues, with amino acid residues from about 39 to about 286 (SEQ ID NO: 4) shown as underlined residues, and amino acid residue positions from about 292 to about 773 (SEQ ID NO: 4) shown as bolded residues. In particular, amino acid positions from about 39 to about 286 contain an N-terminal glycosyl hydrolase family 92 (GH92) sequence domain, and amino acid positions from about 292 to about 773 contain a glycosyl hydrolase family 92 (GH92; superfamily) sequence domain. DETAILED DESCRIPTION OF THE INVENTION

[0057] Certain embodiments described herein relate to recombinant (modified) filamentous fungal cells (strains) used for commercial-scale production of proteins (polypeptides) of interest. More particularly, the strains and methods of the disclosure relate to genetic modifications in filamentous fungi that result in recombinant (modified) strains with altered phenotypes, such variant strains being particularly suitable for growth in submerged culture (e.g., large-scale production of proteins for industrial / commercial applications). In certain embodiments, the disclosure provides, inter alia, recombinant filamentous fungal cells (strains) that produce proteins of interest, methods and compositions for expressing / producing endogenous and / or heterologous proteins of interest in recombinant filamentous fungal cells, methods and compositions for producing proteins of interest in recombinant filamentous fungi, where the proteins produced and recovered therefrom have uniform and consistent N-linked glycosylation patterns, and / or reduced (undesirable) protein glycosylation, and / or enhanced storage stability, and / or enhanced protein (enzyme) activity, etc.

[0058] I. Definition Before describing the present strains, compositions, and methods in further detail, the following terms and phrases are defined. Terms not defined are accorded their ordinary meanings as used in the art and known to those of ordinary skill in the art.

[0059] All publications and patents cited herein are hereby incorporated by reference.

[0060] Where a range of values ​​is presented, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within that range, is encompassed within the compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the compositions and methods, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the compositions and methods.

[0061] Certain ranges are indicated herein by numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number preceded by the term, as well as for numbers that are close to or approximately the number preceded by the term. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately may be a number that, in the context in which the number is presented, provides a substantially equivalent number to the specifically stated number. For example, the term "about" in reference to a numerical value refers to a range of -10% to +10% of the numerical value, unless the context clearly defines otherwise. In another example, the phrase "a pH value of about 6" refers to a pH value of 5.4 to 6.6, unless the pH value is clearly defined otherwise.

[0062] In accordance with the detailed description, the following abbreviations and definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, reference to "the dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0063] It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a prelude to the use of exclusive terminology, such as "solely," "only," "excluding," and "not including," or the use of "negative" limitations or qualifications thereof, in connection with the recitation of claim elements. For example, in certain embodiments, the qualification "the medium does not contain an inducing substrate" may be used to exclude inducing substrates such as cellulose, lactose, gentiobiose, and sophorose.

[0064] Furthermore, it should be noted that the term "comprising" as used herein means "including, but not limited to" the elements following the term "comprising." The elements following the term "comprising" are required or essential, but a composition including such elements may further include other non-essential or optional elements.

[0065] It should also be noted that, as used herein, the term "consisting of" means "including and limited to" the elements following the term "consisting of." Thus, the elements following the term "consisting of" are required or essential, and other elements are not present in the composition.

[0066] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the compositions and methods described herein. Any described method can be carried out in the order of events recited or in any other order which is logically possible.

[0067] As used herein, the term "ascomycota fungal cell" refers to any organism within the Ascomycota phylum in the kingdom Fungi. Examples of ascomycota fungal cells include, but are not limited to, filamentous fungi within the subdivision Pezizomcotina, such as Trichoderma species, Aspergillus species, Myceliophthora species, and Penicillium species.

[0068] As used herein, the term "filamentous fungi" refers to all filamentous fungal forms of the subdivision Eumycota and Oomycota, including, but not limited to, species of the genera Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma.

[0069] In certain embodiments, the filamentous fungus is a cell (strain) of a Trichoderma species, including, but not limited to, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride. As known to those skilled in the art, Trichoderma reesei was previously classified as "Hypocrea jecorina." Exemplary parent Trichoderma reesei strains include, but are not limited to, T. reesei strain QM6a (ATCC® 13631), T. reesei strain RL-P37 (NRRL accession number 15709), and T. reesei strain RUT-C30 (ATCC® 56765). For example, Trichoderma strains Rut-C30 and RL-P37 are mutagenized derivatives of T. reesei natural isolate QM6a (Le Crom et al., 2009; Sheir-Neiss and Montenecourt, 1984), with strain NG14 being their most recent common ancestor. Thus, in certain embodiments, an exemplary filamentous fungal strain may be derived from / obtainable from T. reesei strain RL-P37, which may contain a deletion (Δ) or loss-of-function variant of the T. reesei pyr2 gene (hereinafter abbreviated as "Δpyr2"), as generally described by Sheir-Neiss and Montenecourt (1984) and PCT Publication No. WO 2011 / 153449 (each of which is incorporated by reference herein in its entirety).

[0070] In certain embodiments, the filamentous fungus is selected from the group consisting of Aspergillus aculeatus, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, and Aspergillus terreus. The parent strain is a cell (strain) of Aspergillus species, such as A. terreus. Exemplary parent Aspergillus species strains include, but are not limited to, A. niger strain ATCC® 1015 and A. oryzae strain RIB40 (ATCC® 42149).

[0071] In other embodiments, the filamentous fungus is a cell (strain) of a Myceliophthora species, such as Myceliophthora thermophila (also known as Thermothelomyces thermophilus). Exemplary parent Myceliophthora species strains include, but are not limited to, M. thermophila strain ATCC® 42464.

[0072] In certain other embodiments, the fungal strains constructed herein utilize the Aspergillus nidulans amdS (acetamide) gene selection marker. More specifically, the amdS gene and its variants are generally known to those of skill in the art and are described, for example, in PCT Publication WO 2006 / 040358, which is incorporated herein by reference in its entirety. However, one of skill in the art can select any selection marker (e.g., auxotrophic marker, antibiotic resistance marker) that functions in the selected recombinant microbial cell, without limitation.

[0073] In certain embodiments, the recombinant fungal strains of the present disclosure are designed / constructed to express a heterologous reporter protein, including, but not limited to, a recombinant phytase (reporter) protein, a recombinant lipase (reporter) protein, and a recombinant glucoamylase (reporter) protein.

[0074] In certain embodiments, exemplary phytase (reporter) proteins include, but are not limited to, native or engineered (variant) phytases. For example, PCT Publication WO 2003 / 038111 generally describes purified enzymes with phytase activity from various filamentous fungal species (e.g., Penicillium spp., Fusarium spp., Humicola spp., Emericella spp.), as well as suitable methods for constructing and assaying recombinant fungal strains expressing / producing such phytase proteins. Similarly, PCT Publication Nos. WO 2008 / 097619, WO 2009 / 129489, and WO 2013 / 119470 (each of which is incorporated by reference in its entirety) describe engineered (variant) Buttiauxella sp. phytases, as well as related methods for constructing and assaying fungal strains that express / produce such mutant phytases.

[0075] In certain other embodiments, exemplary glucoamylase (reporter) proteins include, but are not limited to, native or engineered (variant) glucoamylases. For example, PCT Publication Nos. WO 2008 / 04589, WO 2009 / 067218, WO 2021 / 020852, and WO 2011 / 212095 generally describe methods for constructing and assaying fungal strains that express / produce glucoamylases.

[0076] In certain other embodiments, exemplary lipase (reporter) proteins include, but are not limited to, native or engineered (variant) lipases. For example, PCT Publication WO 2020 / 190782 generally describes methods for constructing and assaying fungal strains that express / produce lipases.

[0077] In certain other embodiments, the recombinant fungal strains of the present disclosure are designed, constructed, fermented, etc. for the expression and secretion of one or more heterologous proteins of interest as described in Section IV below.

[0078] In certain embodiments, a phytase (reporter) protein, a lipase (reporter) protein, and / or a glucoamylase (reporter) protein are referred to as exemplary "proteins of interest."

[0079] As used herein, the term "glycoprotein" can refer to a protein of interest that contains one or more oligosaccharide (carbohydrate / glycan) chains covalently attached via glycosidic bonds to one or more amino acid (residue) side chains (of the glycoprotein). In other embodiments, the term "glycoprotein" can refer to a protein that originally contained but subsequently lost the oligosaccharide side chains. For example, as will be understood by those skilled in the art, the covalently attached oligosaccharide (glycan) chains of a glycoprotein can be partially or completely cleaved by cellular enzymes and / or exogenously introduced enzymes.

[0080] As is generally known for filamentous fungal cells, N-linked glycans are initially derived from a tetrasaccharide composed of three monosaccharide building blocks, mannose (Man), glucose (Glc), and N-acetylglucosamine (GlcNAc), comprising the "Glc3Man9GlcNAc2" structure, with the terminal GlcNAc being transferred to an appropriately positioned asparagine residue on the nascent glycoprotein. Glycan processing during passage through the secretory pathway typically removes some of the sugar residues and, in some cases, adds additional sugar residues. For example, partially or completely processed glycans reported for the genus Trichoderma include GlcMan8GlcNAc2, GlcMan7GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2. In some cases, removal of nearly the entire glycan chain occurs after secretion, resulting in the production of proteins with only a single GlcNAc residue.

[0081] Similarly, O-linked glycans are attached to the hydroxyl groups of L-serine or L-threonine, which also occurs during the secretion process. The composition of O-linked glycans reported for fungi is highly variable, with numerous linear and branched configurations of oligosaccharides reported (Goto, 2007). O-linked oligosaccharides can contain Glc, Man, and two conformations of galactose (Gal) (designated Galp and Galf), and may additionally contain phosphate or sulfate linkages. For example, O-glycans reported for Trichoderma reesei include Man3, phosphoMan2, sulfateMan2, ManGlc, GlcGalp, and GlcManGalp.

[0082] As used herein, the phrase "high mannose (Man) structure" refers to an oligosaccharide having at least six mannose residues (Man), for example, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, GlcMan8GlcNAc2.

[0083] As used herein, the phrase "homogeneous N-linked glycan pattern" refers to a glycoprotein that comprises or consists of "Man5GlcNAc2" as the predominant N-linked glycan. In certain embodiments of the present disclosure, a glycoprotein comprising a homogeneous N-linked glycan pattern comprises at least about 75% Man5GlcNAc2 as the predominant N-linked glycan pattern. In other embodiments, a glycoprotein comprising a homogeneous N-linked glycan pattern comprises at least 70%-100% Man5GlcNAc2 as the predominant N-linked glycan pattern.

[0084] As used herein, the phrase "monoglucosylated structure" refers specifically to an N-linked glycan of a protein, where the monoglucosylated structure comprises or consists of a single glucose (Glc) residue, together with a variable number of mannose (Man), two GlcNAc, and optionally other residues (e.g., GlcMan9GlcNAc2).

[0085] The term "glycation" as used herein in phrases such as "protein glycosylation," "recombinant protein glycosylation," "protein glycosylation," and "glycation of glycoproteins" specifically refers to the "non-enzymatic glycosylation" of proteins (glycoproteins) as understood in the art. As described herein, glycosylation (non-enzymatic glycosylation) of many proteins of interest is a particularly undesirable (undesirable) outcome. For example, as generally described in Vetter and Indurthi (2011), even moderate protein glycosylation events can cause undesirable changes in protein structure.

[0086] As used herein, "reducing sugars" (e.g., glucose, mannose) present in fermentation broths, downstream protein recovery processes, protein preparations, etc. generally increase the level (amount) of protein glycation.

[0087] Thus, as used and described herein, "higher levels of reducing sugars" present in fermentation broths, downstream protein recovery processes, protein preparations, etc. generally correlate with higher levels of protein glycation, and such increased levels (amounts) of protein glycation are particularly undesirable, as exemplified by Vetter and Indurthi (2011).

[0088] As used herein, the terms "mannosidase protein" and "mannosidase" may be used interchangeably, and such mannosidases include "alpha-mannosidases" (e.g., alpha-mannosidase-1; Mds1, alpha-mannosidase 2; Mds2) and "glucosidases."

[0089] As used herein, phrases such as "mannosidase-encoding gene," "mannosidase-encoding gene," and "mannosidase gene" may be used interchangeably, and such genes encoding mannosidases include genes encoding "alpha-mannosidases" (e.g., Mds1, Mds2) and "glucosidases."

[0090] As used herein, the term "deficient" refers to a modified, mutated, or recombinant filamentous fungal cell / strain that does not produce detectable activity of one or more (several) mannosidase enzymes compared to a parent (control) filamentous fungal cell / strain when cultured under the same conditions, or alternatively, that produces preferably at least 25% less, more preferably at least 50% less, even more preferably at least 75% less, and most preferably at least 95% less of one or more mannosidase enzymes than the parental (control) filamentous fungal cell / strain when cultured under the same conditions. The level of one or more mannosidases produced by a filamentous fungal cell of the present disclosure can be determined using methods described herein or known in the art.

[0091] As described herein and further described in the Examples section below, a variety of methods and techniques are known and available for constructing one or more modified strains of the present disclosure.

[0092] As used herein, the wild-type Trichoderma reesei "alpha-mannosidase-1 gene" (hereinafter abbreviated as "mds1") comprises the nucleic acid (DNA) sequence set forth in SEQ ID NO: 1, and the wild-type mds1 gene encodes the native "alpha-mannosidase-1 protein" (hereinafter abbreviated as "Mds1") sequence set forth in SEQ ID NO: 2.

[0093] As used herein, the wild-type T. reesei "alpha-mannosidase 2 gene" (hereinafter abbreviated as "mds2") comprises the DNA sequence set forth in SEQ ID NO: 3, and the wild-type mds2 gene encodes the native "alpha-mannosidase 2 protein" (hereinafter abbreviated as "Mds2") sequence set forth in SEQ ID NO: 4.

[0094] As used herein, the wild-type T. reesei "glucosidase II alpha subunit" gene (hereinafter abbreviated as "gls2a" or "gls2α") comprises the DNA sequence set forth in SEQ ID NO:5, and the wild-type gls2a gene encodes the native "glucosidase II alpha subunit" protein (hereinafter abbreviated as "GIIα") sequence set forth in SEQ ID NO:6.

[0095] As used herein, a variant T. reesei "glucosidase II alpha subunit" (gls2a) gene containing a frameshift mutation at nucleotide position 1,965 (SEQ ID NO: 5) is referred to as "gls2a Stop" (hereinafter "gls2a Stop ") and is called gls2a Stop The DNA sequence of the allele is set forth in SEQ ID NO: 7 and encodes a truncated GIIα protein. For example, the mutant gls2a Stop The gene (nucleotide position 1,965 of SEQ ID NO:7) contains a frameshift mutation that results in a premature stop codon, resulting in the encoded variant GIIα protein (variant "GIIα Stop ") comprises the truncated amino acid sequence set forth in SEQ ID NO: 8. This frameshift mutation is present in T. reesei strain NG14, the most recent common ancestor of strains Rut-C30 and RL-P37.

[0096] As used herein, the Trichoderma strain designated "Phy" contains the introduced phytase expression cassette and gls2α. StopThe parental Trichoderma strain containing the alleles. The endogenous cellulase genes (i.e., cbh1, cbh2, egl1, egl2) were previously deleted from the genome.

[0097] As used herein, "Phy-gls2a R The modified Trichoderma strain, designated "Phy-gls2a," was derived from the Phy parent strain containing a phytase expression cassette. R The strain was recovered as glsa2 (gls2a R ) alleles.

[0098] As used herein, Trichoderma strains designated "GA" are those containing the introduced glucoamylase (GA) expression cassette and gls2α Stop The parental Trichoderma strain containing the alleles. The endogenous cellulase genes (i.e., cbh1, cbh2, egl1, egl2) were previously deleted from the genome.

[0099] As used herein, "GA-gls2a R The modified Trichoderma strain, designated "," is derived from the GA parent strain containing the GA expression cassette and is designated GA-gls2a. R The genus strains were recovered glsa2 (gls2a R ) alleles.

[0100] As used herein, the term "restored glsa2 allele" ("gls2a R ") is a single nucleotide base insertion that restores the wild-type reading frame, and gls2α Stop The restored gls2a allele (SEQ ID NO: 7) refers to the removal of the premature stop codon present in the allele. R The allele encodes the native GIIα protein of SEQ ID NO: 6. For example, in certain embodiments, the parent T. reesei cell / strain encodes a truncated GIIα StopThe variant gls2a allele encoding the protein (gls2a Stop The genetically modified T. reesei strain contains, may be derived from, or is obtained from a restored gls2a gene encoding the native GIIα protein. R Includes alleles.

[0101] As used herein, the modified Trichoderma strain designated "Phy-Δmds2" contains a phytase expression cassette and gls2α Stop Derived from the Phy parent strain containing the allele, the Phy-Δmds2 strain further contains a deletion (Δ) of the mds2 (Δmds2) gene.

[0102] As used herein, the Trichoderma strain designated "Lip" contains the introduced lipase expression cassette and gls2α. Stop The parental Trichoderma strain containing the alleles. The endogenous cellulase genes (i.e., cbh1, cbh2, egl1, egl2) were previously deleted from the genome.

[0103] As used herein, the modified Trichoderma strain designated "Lip-Δmds1" contains a lipase expression cassette and gls2α Stop Derived from the Lip parent strain containing the allele, the Lip-Δmds1 strain additionally contains a deletion (Δ) of the mds1 (Δmds1) gene.

[0104] As used herein, the phrase "Endo T gene product" refers to a secreted protein (i.e., endo-N-acetyl-β-D-glucosaminidase; abbreviated as "EnGase") that is a member of the glycoside hydrolase (GH) family of deglycosylating enzymes. For example, Stricker et al. (2012) (incorporated herein by reference in its entirety) described the expression, purification, and structural analysis of ENGase (Endo T) from the mesophilic fungus Hypocrea jecorina (anamorph Trichoderma reesei), and glycosylation analysis of cellulase secreted by an H. jecorina "Endo T knockout strain" demonstrated the in vivo function of ENGase. Glycan cleavage catalyzed by ENGase occurs between two base GlcNAc residues, leaving only a single GlcNAc residue on the protein.

[0105] As used herein, the phrase "Endo T deletion" allele (abbreviated "ETD" allele) specifically refers to a filamentous fungal strain having a genetic modification of the Endo T allele that renders the strain deficient in the production of native (functional) ENGase. More specifically, the Endo T allele of the recombinant (modified) T. reesei cells / strains described herein and in the Examples below has been genetically modified to render the strain completely (100%) deficient in the production of Endo T protein or an Endo T homolog. By reference to the wild-type T. reesei (H. jecorina) Endo T allele (SEQ ID NO: 36), in particular, one skilled in the art can identify related Endo T genes in other filamentous fungal strains of interest. For example, fungal strains containing the ETD allele retain larger N-linked glycan chains on secreted proteins due to an inability to cleave between two base GlcNAc residues (Stals et al., 2012).

[0106] As used herein, the Trichoderma strain designated "Cel" contains the introduced cellulase expression cassette and gls2α. Stop The parent Trichoderma strain containing the allele was constructed from a strain in which the endogenous cellulase genes (i.e., cbh1, cbh2, egl1, and egl2) had been previously deleted from the genome. The native cellulase-encoding genes (cbh1, cbh2, egl1, and egl2) were reintroduced as single integrated expression cassettes.

[0107] As used herein, "Cel-gls2a R The modified Trichoderma strain, designated "Cel-gls2a," was derived from the Cel parent strain, which contains a cellulase expression cassette. R The strain was recovered as glsa2 (gls2a R ) alleles.

[0108] As used herein, the term "Endo H" and related phrases such as "Endo H treatment" refer specifically to an enzymatic treatment that deglycosylates (removes) mannose (Man) from glycoproteins. In particular, enzymes with Endo H activity (i.e., endoglycosidase activity), such as "Endoglycosidase H" (Endo H, New England BioLabs), are recombinant glycosidases that cleave within the mannose-rich chitobiose core and within some hybrid oligosaccharides derived from N-linked glycoproteins.

[0109] As used herein, the terms "wild-type" and "native" are used interchangeably and refer to a gene, protein, fungal cell, or strain found in nature.

[0110] As used herein, the terms "recombinant" or "non-naturally occurring" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has at least one engineered genetic modification or that has been modified by the introduction of a heterologous nucleic acid molecule, or to a cell (e.g., a microbial cell) that has been modified so that expression of a heterologous or endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from or is the progeny of a non-naturally occurring cell that has one or more such modifications. Genetic modifications include, for example, modifications that introduce expressible nucleic acid molecules that encode proteins, or the addition, deletion, or substitution of other nucleic acid molecules, or other functional alterations of the genetic material of a cell. For example, recombinant cells can express genes or other nucleic acid molecules that are not found in the same or homologous form in native (wild-type) cells, or can achieve an altered expression pattern of an endogenous gene, e.g., that is overexpressed, underexpressed, minimally expressed, or not expressed at all.

[0111] As used herein, the term "gene" is synonymous with the term "allele" when referring to a nucleic acid that encodes and directs the expression of a protein or RNA. Because the vegetative forms of filamentous fungi are generally haploid, a single copy of a particular gene (i.e., a single allele) is sufficient to confer a particular phenotype.

[0112] As used herein, the term "gene" refers to a segment of DNA involved in producing a polypeptide (protein) chain, which may or may not include regions preceding and following the coding region (e.g., 5' untranslated (5' UTR) or "leader" sequence, 3' UTR or "trailer" sequence, promoter sequence, terminator sequence, etc.), and intervening sequences (introns) between individual coding segments (exons). For example, a gene (DNA) sequence of interest (GOI) can encode structural proteins and commercially important industrial proteins or peptides, such as enzymes (e.g., proteases, mannanases, xylanases, amylases, glucoamylases, cellulases, oxidases, phytases, lipases), etc. A gene of interest can be a naturally occurring gene, a mutated (modified) gene, or a synthetic gene.

[0113] As used herein, a "functional protein" is a protein that has an activity or function, such as enzymatic activity, binding function / activity (e.g., DNA binding), and surfactant properties, and that has not been mutated, truncated, or otherwise modified to eliminate or reduce the function or activity. Functional polypeptides can be thermally stable or unstable, as specified.

[0114] As used herein, a "functional gene" is a gene that can be used by cellular constituents to produce an active gene product, typically a protein. In contrast, a "non-functional gene" cannot be used by cellular constituents to produce an active gene product (i.e., a functional protein), or has a reduced ability to be used by cellular constituents to produce an active gene product (i.e., a functional protein).

[0115] As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene coding sequence (CDS; or open reading frame (ORF)). Promoters are generally appropriate for the host cell (e.g., fungal cell) in which the target gene is to be expressed. A promoter, along with other transcriptional and translational regulatory nucleic acid sequences (also referred to as "control sequences"), is essential for expressing a given gene. Generally, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences and terminator sequences, including core promoters and enhancer or activator or repressor sequences, transcriptional and translational start and stop sequences. In certain embodiments, the promoter is an inducible promoter, a constitutive promoter, a regulatable promoter, a synthetic promoter, a tandem promoter, and combinations thereof. In certain embodiments, the inducible promoter is an inducible cellulase gene promoter.

[0116] As used herein, the term "promoter activity" refers to the ability of a nucleic acid to direct transcription of a downstream (3') polynucleotide in a host cell. To test promoter activity, a (promoter) nucleic acid can be operably linked to a downstream polynucleotide to produce a recombinant nucleic acid. The recombinant nucleic acid can be introduced into a cell and transcription of the polynucleotide can be assessed. In certain cases, the polynucleotide can encode a protein, and transcription of the polynucleotide can be assessed by assessing the production of the protein in the cell.

[0117] As used herein, the term "operably linked" refers to a functional linkage between two or more nucleic acid sequences. Thus, a nucleic acid sequence is operably linked when it functionally relates to another nucleic acid sequence. For example, a promoter or terminator sequence is operably linked to a gene coding sequence (CDS) if it affects the transcription of the CDS. A ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. A nucleic acid sequence encoding a secretory leader (i.e., signal peptide) is operably linked to a nucleic acid sequence encoding a polypeptide (e.g., ORF) if it is expressed as a preprotein that participates in the secretion of the polypeptide. Generally, "operably linked" means that the DNA (nucleic acid) sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking (i.e., operably linking) two or more nucleic acid sequences can be accomplished using any method known to those of skill in the art.

[0118] As used herein, the phrases "lignocellulolytic enzymes," "cellulase enzymes," and "cellulases" are used interchangeably and include glycoside hydrolase (GH) enzymes, such as cellobiohydrolases, xylanases, endoglucanases, and β-glucosidases, which hydrolyze the glycosidic bonds of cellulose (hemicellulose) to produce sugars (e.g., glucose, xylose, arabinose, etc.).

[0119] As used herein, an "endoglucanase" protein may be abbreviated as "EG," a "cellobiohydrolase" protein may be abbreviated as "CBH," a "β-glucosidase" protein may be abbreviated as "BG," and a "xylanase" protein may be abbreviated as "XYL." Thus, as used herein, a gene (or ORF) encoding an EG protein may be abbreviated as "eg," a gene (or ORF) encoding a CBH protein may be abbreviated as "cbh," a gene (or ORF) encoding a BG protein may be abbreviated as "bg," and a gene (or ORF) encoding an XYL protein may be abbreviated as "xyl." In certain embodiments, cellobiohydrolases include enzymes classified under the Enzyme Commission number (EC 3.2.1.91), endoglucanases include enzymes classified under EC 3.2.1.4, endo-β-1,4-xylanases include enzymes classified under EC 3.2.1.8, β-xylosidases include enzymes classified under EC 3.2.1.37, and β-glucosidases include enzymes classified under EC 3.2.1.21.

[0120] As used herein, the term "cellulase gene promoter" includes, but is not limited to, cellobiohydrolase (cbh) gene promoter sequences, endoglucanase (eg) gene promoter sequences, β-glucosidase (bg) gene promoter sequences, and xylanase (xyl) gene promoter sequences.

[0121] As used herein, the terms "modification" and "genetic modification" are used interchangeably and include: (a) the introduction, substitution, or removal of one or more nucleotides in a gene or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of a gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation and / or upregulation, (f) directed mutagenesis, and / or (g) random mutagenesis of any one or more genes / DNA sequences disclosed herein.

[0122] As used herein, the phrases "modified filamentous fungal cell," "mutated filamentous fungal cell," "recombinant fungal cell," and "modified filamentous fungal strain," etc., may be used interchangeably and refer to a filamentous fungal cell derived from (i.e., obtained from) a parent or control filamentous fungal cell belonging to the subdivision Pezizomycotina. For example, a "modified" filamentous fungal cell may be derived from (obtained from) a parent or control filamentous fungal cell, and the modified cell contains at least one genetic modification not found in the parent or control cell.

[0123] As used herein, the terms "gene disruption," "gene disruption," "gene inactivation," and "gene inactivation" are used interchangeably and refer broadly to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., a functional protein). Exemplary methods of gene disruption include complete or partial deletion of any portion of a gene, including a polypeptide coding sequence, promoter, enhancer, or another regulatory element, or mutagenesis thereof (wherein mutagenesis encompasses substitutions, insertions, deletions, inversions, and any combinations and variations thereof that disrupt / inactivate the target gene and substantially reduce or prevent production of a functional gene product (i.e., a functional protein)).

[0124] As used herein, "gene deletion" refers to the removal of a gene from the genome of a host cell. When a gene contains regulatory elements (e.g., enhancer elements) that are not located immediately adjacent to the coding sequence of the gene, gene deletion refers to the deletion of some or all of the coding sequence and, optionally, adjacent enhancer elements, including, but not limited to, promoter and / or terminator sequences.

[0125] As used herein, "heterologous gene" refers to a polynucleotide (DNA) sequence in which at least a portion of the sequence is not native to, or does not exist in a natural form in, the cell into which it is introduced and / or expressed.

[0126] As used herein, a "heterologous nucleic acid construct" or "heterologous DNA sequence" has portions of the sequence that are not native to, or do not exist in a natural form in, the cell in which they are expressed.

[0127] As used herein, a "heterologous protein" is encoded by a heterologous gene, a heterologous nucleic acid (polynucleotide) sequence, a heterologous DNA sequence, and the like.

[0128] Thus, in certain embodiments, a heterologous gene, heterologous nucleic acid construct, heterologous DNA sequence, etc. encoding a protein of interest (POI) is introduced (e.g., transformed) into a filamentous fungal cell (strain). For example, a heterologous gene construct encoding a POI can be introduced into a filamentous fungal cell (strain) before, during, or after other genetic modifications described herein.

[0129] Heterologous, in reference to a regulatory sequence, refers to a regulatory sequence (e.g., a promoter, enhancer, terminator) that does not function in nature to regulate the same gene whose expression it currently regulates. Generally, a heterologous nucleic acid sequence is not endogenous to the cell or part of the genome in which it is present, but has been added to the cell by infection, transfection, transformation, microinjection, electroporation, or the like. A "heterologous" nucleic acid construct can contain a regulatory sequence / DNA coding sequence combination that is the same as or different from the regulatory sequence / DNA coding sequence combination found in the natural cell.

[0130] As used herein, the term "gene coding sequence" (abbreviated as "CDS") refers to a polynucleotide sequence that directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of a CDS are generally determined by an open reading frame (ORF), which usually begins with a start codon (ATG). Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences. For example, an ORF generally refers to a polynucleotide sequence (whether naturally occurring, non-naturally occurring, or synthetic) that includes a continuous reading frame consisting of (i) an initiation codon, (ii) a series of codons representing the amino acids of the encoded protein product, and (iii) a stop codon, and the ORF is read (or translated) in the 5' to 3' direction.

[0131] As used herein, the term "DNA construct" or "expression construct" refers to a nucleic acid sequence comprising at least two DNA polynucleotide fragments. DNA or expression constructs can be used to introduce nucleic acid sequences into fungal host cells. The DNA can be generated in vitro (e.g., by PCR) or by any other suitable technique. In some embodiments, the DNA construct comprises a sequence of interest (e.g., encoding a protein of interest). In certain embodiments, the polynucleotide sequence of interest is operably linked to a promoter and / or terminator. In some embodiments, the DNA construct further comprises at least one selectable marker. In further embodiments, the DNA construct comprises sequences homologous to a host cell chromosome. In other embodiments, the DNA construct comprises sequences heterologous to a host cell chromosome.

[0132] As used herein, "flanking sequence" refers to any sequence that is upstream or downstream of the sequence under consideration (e.g., for gene ABC, gene B is flanked by gene sequences A and C). In certain embodiments, the incoming sequence is flanked on both sides by homology boxes. In other embodiments, the incoming sequence and homology box comprise a unit that is flanked on both sides by stuffer sequences. In some embodiments, flanking sequences are present on only one side (3' or 5'), but in preferred embodiments, they are present on both sides of the sequence that they are flanking. The sequence of each homology box is homologous to a sequence in a filamentous fungal chromosome. These sequences direct where in the filamentous fungal chromosome the new construct will integrate and what portion of the chromosome, if any, will be replaced by the incoming sequence.

[0133] As used herein, the term "downregulation" of gene expression includes any method that results in lower (downregulated) expression of a functional gene product.

[0134] The term "vector" is defined herein as a polynucleotide designed to carry a nucleic acid sequence to be introduced into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage or viral particles, DNA constructs, and cassettes. Expression vectors can include regulatory sequences such as promoters, signal sequences, coding sequences, and transcription terminators.

[0135] As used herein, "expression vector" refers to a DNA construct containing a coding sequence operably linked to a suitable control sequence capable of expressing a protein in a suitable host. Such control sequences may include a promoter to initiate transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0136] As used herein, the term "secretory signal sequence" refers to a DNA sequence that encodes a polypeptide (i.e., a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through the secretory pathway of the cell in which it is synthesized. The larger polypeptide is typically cleaved to release the secretory peptide during transit through the secretory pathway.

[0137] As used herein, the terms "isolated" or "purified" refer to a filamentous fungal cell, nucleic acid, or polypeptide that has been removed from at least one component with which it is naturally associated.

[0138] As used herein, the term "protein of interest" (POI) refers to a polypeptide desired to be expressed in a filamentous fungal cell. Such proteins may be enzymes, substrate-binding proteins, surface-active proteins, structural proteins, and the like, and may be expressed at high levels and intended for commercialization. For example, as generally described below, POIs include phytases, glucoamylases, cellulases, hemicellulases, xylanases, peroxidases, proteases, lipases, phospholipases, esterases, cutinases, polyesterases, pectinases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, mannanases, α-glucanases, β-glucanases, hyaluronidases, chondroitinase, and the like. Examples of enzymes that can be used include, but are not limited to, enzymes such as thiaminase, laccase, amylase, glucoamylase, acetyl esterase, aminopeptidase, arabinase, arabinosidase, arabinofuranosidase, carboxypeptidase, catalase, nuclease, deoxyribonuclease, ribonuclease, epimerase, α-galactosidase, β-galactosidase, glucan lyase, endo-β-glucanase, glucose oxidase, glucuronidase, invertase, and isomerase.

[0139] The protein of interest (POI) can be encoded by an "endogenous" gene. For example, in certain embodiments, the POI is encoded by a gene endogenous to the filamentous fungal cell (strain), such as the wild-type genes encoding naturally occurring cellulases (e.g., cellobiohydrolases, xylanases, endoglucanases, and β-glucosidases) described above.

[0140] As used herein, the term "increased productivity" and variations thereof refers to an increase of at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% (e.g., greater than 20%) in production of a protein of interest by a modified (mutant) filamentous fungal cell relative to a parental (control) filamentous fungal cell when cultured under the same conditions (e.g., medium composition, temperature, pH, cell density, dissolved oxygen, time, etc.).

[0141] As used herein, the term "enhanced amount" and variations thereof, when used in phrases such as a recombinant cell "producing an 'enhanced amount' of a protein of interest," means an increase of at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% (e.g., more than 20%) of the amount of a protein of interest produced by a modified (mutant) filamentous fungal cell relative to a parental (control) filamentous fungal cell when cultured under the same conditions.

[0142] As used herein, the terms "polypeptide" and "protein" (and / or their respective plurals) are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein. Polymers can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The terms also encompass amino acid polymers that are modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component). Also included within this definition are polypeptides containing, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0143] As used herein, functionally and / or structurally similar proteins are considered to be "related proteins." Such proteins may be derived from organisms of different genera and / or species, or even different classifications (e.g., bacteria and fungi). Related proteins also encompass homologs as determined by primary sequence analysis, by secondary or tertiary structure analysis, or by immunological cross-reactivity.

[0144] As used herein, the phrase "substantially free of activity" or similar phrases means that the particular activity cannot be detected in the mixture or is present in an amount that does not interfere with the intended purpose of the mixture.

[0145] As used herein, the term "derived polypeptide" refers to a protein obtained or obtainable from a protein by the addition of one or more amino acids to either or both of the N-terminus and C-terminus, the substitution of one or more amino acids at one or many different sites in the amino acid sequence, the deletion of one or more amino acids at one or both termini of the protein or at one or more sites in the amino acid sequence, and / or the insertion of one or more amino acids at one or more sites in the amino acid sequence. Preparation of a protein derivative can be accomplished by modifying a DNA sequence encoding the native protein and transforming the DNA sequence into a suitable host and expressing the modified DNA sequence to form the derived protein.

[0146] Related (and derived) proteins include "variant proteins." Variant proteins differ from a reference / parent protein (e.g., a wild-type protein) by substitution, deletion, and / or insertion of a small number of amino acid residues. The number of different amino acid residues between a variant protein and a parent protein can be one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more amino acid residues. A variant protein can share at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more amino acid sequence identity with the reference protein. Variant proteins may also differ from the reference protein in selected motifs, domains, epitopes, conserved regions, and the like.

[0147] As used herein, the term "homologous" protein refers to a protein that has a similar activity, function, and / or structure as a reference protein. Homologues are not necessarily evolutionarily related. Thus, the term is intended to encompass identical, similar, or corresponding (i.e., with respect to structure and function) proteins obtained from different organisms. In some embodiments, it is desirable to identify homologues that have a similar quaternary structure, tertiary structure, and / or primary structure to the reference protein. For example, one or more gls2a, mds1, and / or mds2 genes encoding protein homologs comprising substantial amino acid sequence identity to the full-length T. reesei Gls2a, Mds1, and / or Mds2 proteins of the present disclosure from filamentous fungal strains such as A. niger, A. oryzae, and M. thermophila (T. thermophila) are readily identified in publicly available genome databases using methods further outlined in Example 4 below.

[0148] The degree of homology between sequences can be determined using any suitable method known in the art (see, for example, Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., 1984). For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) as implemented in the Needle program in the EMBOSS package (Rice et al., 2000), preferably version 3.0.0 or later. Optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the Needle label "longest identity" (obtained using the nobrief option) is used as the percent identity, calculated as follows: (Identical residues × 100) / (length of alignment − total number of gaps in alignment)

[0149] For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (Rice et al., 2000, supra), preferably version 3.0.0 or later. Optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version in NCBI NUC4.4) substitution matrix. The output of the Needle label "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment − total number of gaps in alignment)

[0150] As used herein, the phrases "substantially similar" and "substantially identical," in the context of at least two nucleic acids or polypeptides, typically mean that the polynucleotide or polypeptide comprises a sequence having at least about 40% identity, at least about 50% identity, at least about 60% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity or greater, relative to a reference (i.e., wild-type) sequence. Sequence identity can be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters.

[0151] As used herein, the terms "purified," "isolated," or "enriched" refer to a biomolecule (e.g., a polypeptide or polynucleotide) that has been altered from its native state by separation from some or all of the naturally occurring components with which it is naturally associated. Such isolation or purification can be achieved by art-recognized separation techniques, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation or other protein salting-out, centrifugation, size exclusion chromatography, filtration, microfiltration, ultrafiltration, gel electrophoresis, or gradient separation, to remove unwanted whole cells, cell debris, impurities, extraneous proteins, or enzymes in the final composition. Further components that impart additional benefits, such as activators, anti-inhibitors, desired ions, pH-adjusting compounds, or other enzymes or chemicals, can then be added to the purified or isolated biomolecule composition.

[0152] As used herein, a "protein preparation" is any material, typically a generally aqueous solution, that contains one or more proteins.

[0153] As used herein, the terms "recovered," "recovered," and "recovering" refer to the treatment or stabilization of a broth, or the at least partial separation of a protein from one or more soluble and / or insoluble components of a microbial broth, and / or from one or more solvents (e.g., water or ethanol) in the broth. Recovered proteins are often of higher purity than before the recovery process. However, in some embodiments, recovered proteins may be of the same or lower purity than before the recovery process.

[0154] As used herein, the terms "broth," "culture broth," "fermentation broth," and / or "whole fermentation broth" may be used interchangeably and refer to a preparation produced by cell fermentation that does not undergo processing steps after fermentation is complete. For example, whole fermentation broth is typically produced when filamentous fungal cells are grown to saturation and incubated under carbon-limited conditions that allow protein synthesis (e.g., protein expression and secretion into the cell culture medium). Typically, whole fermentation broth is unfractionated and includes spent cell culture medium, metabolic products, extracellular polypeptides, and microbial cells.

[0155] As used herein, the phrase "treated broth" refers to broth that has been conditioned by making changes to the chemical composition and / or physical properties of the broth. Broth "conditioning" can include one or more treatments or steps, such as filtration, diafiltration, cell lysis, pH modification, heating, cooling, addition of chemicals (e.g., calcium, salts, flocculants, reducing agents, enzyme activators, enzyme inhibitors, and / or surfactants), mixing, and / or holding the broth for a set period (e.g., 0.5 to 200 hours) without further processing.

[0156] As used herein, "aerobic fermentation" refers to growth in the presence of oxygen.

[0157] As used herein, the term "cell mass" refers to the cellular components (including intact and lysed cells) present in a liquid / submerged culture. Cell mass can be expressed as dry weight or wet weight.

[0158] As used herein, when comparing the expression / production of a protein of interest (POI) in an "unmodified" (parent or control) cell with the expression / production of the same POI in a "modified" (recombinant) cell, it will be understood that the "unmodified" (control) cell and the "modified" (recombinant) cell are grown / cultured / fermented under the same conditions (e.g., the same conditions of medium, temperature, pH, etc.). Thus, the POI of the present disclosure may be produced inside the host cell or may be secreted (or transported) into the culture medium.

[0159] It will be understood that the methods of the present disclosure are not limited to a particular order for obtaining modified (mutated) filamentous fungal cells (strains). Genetic modifications can be introduced into a parent strain at any step in the construction of the strain for the production of an endogenous protein of interest (POI) and / or a heterologous POI.

[0160] II. Glycoproteins As briefly described in the Background Art, filamentous fungi (e.g., Trichoderma species, Aspergillus species, and Myceliophthora species, etc.) are useful for producing proteins of interest and are therefore often utilized in the industrial production of recombinant proteins. In certain embodiments, recombinant (modified) fungal strains are particularly suitable for use in the production of glycoproteins. As generally understood by those skilled in the art, glycosylated proteins (glycoproteins) produced in filamentous fungal strains can be N-linked, e.g., glycans linked to the nitrogen (N) of the amide group of L-asparagine, and / or O-linked, e.g., glycans linked to the hydroxyl group (O) of L-serine or L-threonine. In most eukaryotes, N-linked glycans are initially synthesized within the lumen of the ER as lipid-linked tetrasaccharides (14 sugar units containing a "Glc3Man9GlcNAc2" precursor) that are transferred onto the appropriate L-asparagine (N-linked) residue of the nascent polypeptide chain (Kornfeld and Kornfeld, 1985). After transfer to the protein, the terminal α-1,2-linked glucose (Glc) is rapidly removed by α-glucosidase I (GI), while the two innermost α-1,3-linked glucose (Glc) residues are cleaved off by the action of α-glucosidase II (GII).

[0161] Alpha-glucosidase II proteins (abbreviated as "GII" proteins) are generally recognized as asymmetric non-globular heterodimers containing a catalytic alpha subunit (GIIα) and a beta subunit (GIIβ). Geysens et al. (2005) described the isolation and characterization of the gene encoding the alpha-glucosidase II alpha subunit (GIIα) from Trichoderma strain Rut-C30, where the gls2α gene encoding the GIIα-subunit in the hypercellulose-degrading strain Rut-C30 contained a frameshift mutation resulting in a truncated GIIα protein, and a unique monoglucosylated N-glycan pattern was observed on the protein produced by the strain. In particular, Geysens et al. (2005) concluded that the truncated GIIα protein was still able to hydrolyze the first α-1,3-linked glucose (Glc) residue, but not the innermost α-1,3-linked glucose (Glc2) residue from the Glc2Man9GlcNAc2 N-glycan ER structure. As described in Geysens et al. (2005), transformation of the Rut-C30 strain with a plasmid encoding the wild-type T. reesei GIIα-subunit significantly altered the glycosylation profile, reducing the amount of monoglucosylated structures and increasing the overall heterogeneity and amount of high-mannose N-glycans. Complete conversion to high-mannose carbohydrates (e.g., Man9GlcNAc2) was not obtained, which suggests that the endogenous mutant (truncated) GIIα Stop It was hypothesized that this was due to competition between the subunit and the introduced wild-type (native) GII α-subunit. A more recent crystallographic characterization of the fungal GII α-subunit was described by Satoh et al. (2016), which showed that the T. reesei gls2a frameshift mutation (gls2a STOP ) may have reduced contact with the GIIβ subunit.

[0162] As described herein and further described in the Examples section below, Applicants have demonstrated that the gls2a frameshift mutation (gls2a STOP ) were evaluated. More specifically, as described in the Examples, reporter strains derived from a common ancestor of the T. reesei (T. reesei) strain Rut-C30 were generated to express (native) cellulase or heterologous phytase reporter glycoproteins. For example, rather than providing an ectopic full-length copy of the gls2a allele as described by Geysens et al. (2005), Applicant has evaluated the specific role of the gls2a allele (i.e., allele gls2a) by repairing (i.e., restoring) the original (endogenous) frameshift mutation in each of the described reporter strains. R ) restored the correct reading frame.

[0163] Notably, as presented and described in Example 3, the N-glycan patterns of these reporter strains were remarkably uniform for both cellulase and phytase reporter strains, consisting primarily of Man5GlcNAc2, with no high-mannose structures detected. This result is in stark contrast to previous findings (Geysens et al. 2005), where gls2a STOP Instead of replacing with a revertant allele, gls2a STOP Strains containing both the .ALPHA. and wild-type versions were studied. When both alleles were present, the N-linked glycans contained significant amounts of monoglucosylated high-mannose chains.

[0164] As presented and described in Example 1, Applicants have demonstrated that gls2a STOP We found that replacing the β-glucan with a revertant allele resulted in a significant increase in glucoamylase production, suggesting that N-glycan processing in the secretory pathway may be rate-limiting for protein production in some cases.

[0165] Similarly, the post-production release of mannose (Man) from glycoproteins, oligosaccharides, and the like is a potential source of reducing sugars that can chemically react with surface lysine residues (a phenomenon known as glycation, protein glycation, and enzymatic glycation). For example, protein glycation adversely affects the storage stability of industrially produced enzymes, as shown in Sutthirak et al. (2005), and the enzymes described herein. However, the exact source and mechanism by which free mannose is produced are generally unknown. Therefore, characterization of the mechanism of mannose production will further facilitate the mitigation of mannose release, facilitate the mitigation of protein glycation events, mitigate loss of protein product activity, and enhance the storage stability of protein products.

[0166] Based on the above, understanding the sources of free mannose and the associated mechanisms of production of free mannose is highly desirable in the art. R The secretion of proteins with an overall reduced glycan content observed for the strains (Examples 1 and 3) was used to demonstrate the role of glycoproteins in the production of free sugars during post-fermentation recovery and processing (Example 2). Additionally, Applicant identified two 1,2-α-D-mannosidases (i.e., Mds1 and Mds2) in the medium during T. reesei fermentation. For example, alpha-mannosidase-1 (Mds1) was described by Maras et al. (2000). While this enzyme exhibited mannosidase activity when heterologously expressed in yeast (e.g., Pichia), no mannosidase activity was detected in the culture supernatant of the T. reesei host strain into which the gene was cloned.

[0167] Therefore, to determine the potential role of the two 1,2-α-D-mannosidases (Mds1 and / or Mds2) in the release of mannose in the soluble extract, the genes encoding these proteins were deleted, as described in the Examples below. Substrates for Mds1 and Mds2 may be secreted glycoproteins, cell wall oligosaccharides, and / or other substances. Notably, as described in the Examples, deletion of each mannosidase gene resulted in a reduction in free mannose levels, demonstrating their important role in mannose release. More specifically, deletion of the mannosidase genes resulted in a significant reduction in mannose release and a concomitant reduction in the amount of undesired protein glycation in the medium during fermentation.

[0168] Thus, as described herein, certain embodiments of the present disclosure relate, inter alia, to recombinant filamentous fungal cells (strains) that produce a protein of interest, methods and compositions for designing and constructing recombinant (modified) fungal cells that produce a protein of interest, methods and compositions for expressing / producing an endogenous protein of interest in recombinant filamentous fungal cells, methods and compositions for expressing / producing a heterologous protein of interest in recombinant filamentous fungal cells, methods and compositions for producing a protein of interest in recombinant filamentous fungi, whereby the protein produced and / or recovered therefrom has a uniform and consistent N-linked glycosylation pattern and / or a consistent monoglucosylation structure and / or reduced (undesirable) glycosylation events of the protein of interest, etc. Certain other embodiments therefore relate to proteins or glycoproteins produced (secreted) by the recombinant fungal strains of the present disclosure. In related embodiments, the strains, compositions, and methods of the disclosure provide proteins / glycoproteins with improved or enhanced whole broth processing properties, improved or enhanced downstream recovery and purification properties, improved or enhanced protein / glycoprotein product profiles such as stability, activity, shelf life, improved quality control (QC), reduced mannose (Man) release, etc.

[0169] III. Recombinant Nucleic Acids, Molecular Biology, and Methods for Constructing Modified Filamentous Fungal Strains As noted above, certain embodiments relate to recombinant (modified) fungal strains derived from parent strains containing native genes encoding functional Mds1 and / or Mds2 proteins and / or derived from parent strains containing mutated genes encoding truncated GIIα proteins. In certain embodiments, the modified strains comprise genetic modifications that render the modified strains deficient in the production of one or more native genes encoding functional Mds1 and / or Mds2 proteins. In other embodiments, the truncated GIIα strains of the present disclosure are Stop The variant gls2a gene encoding the protein (gls2a Stop ) is genetically modified with the gene to produce the variant gls2a Stop The wild-type gls2a gene (gls2a R ) with a restored copy. As described above, a variety of methods and techniques are available for constructing, screening, identifying, selecting, etc., one or more modified strains of the present disclosure.

[0170] Thus, in one or more embodiments of the present disclosure, the modified, mutant, or recombinant filamentous fungal cells / strains of the present disclosure are deficient in the production of one or more mannosidases described herein (i.e., compared to one or more control / parent cells). In certain embodiments, modified filamentous fungal cells deficient in the production of one or more mannosidases produce no detectable activity of one or more mannosidase enzymes compared to the parent (control) filamentous fungal cells / strain when cultured under the same conditions, or alternatively, produce preferably at least 25% less, more preferably at least 50% less, even more preferably at least 75% less, and most preferably at least 95% less of one or more mannosidase enzymes than the parent / control filamentous fungal cells when cultured under the same conditions. The level of one or more mannosidases produced by a filamentous fungal cell of the present disclosure can be determined using methods described herein and / or known in the art.

[0171] Thus, certain embodiments relate to modified fungal strains that include one or more genetic modifications and one or more introduced nucleic acids (e.g., expression cassettes, targeting vectors, etc.). In any of these embodiments, the modified strain, mutant strain, parent strain, and / or control strain may include additional genetic modifications described herein. Thus, certain embodiments relate to recombinant microbial strains, recombinant polynucleotides, plasmids, vectors, expression cassettes, etc. In certain embodiments, the modified (recombinant) filamentous fungal strains described herein express one or more proteins of interest (heterologous or endogenous).

[0172] Thus, in certain embodiments, one or more genetic elements, such as promoter sequences, gene coding sequences (CDS), 5'-UTR sequences, vectors, and polynucleotides, may be genetically modified, as generally understood by those of skill in the art. In certain embodiments, genetic modifications include, but are not limited to, (a) introduction, substitution, or removal of one or more nucleotides in a gene or introduction, substitution, or removal of one or more nucleotides in a regulatory element required for transcription or translation of a gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) gene overexpression (OE), (g) directed mutagenesis, and / or (h) random mutagenesis of any one or more genes disclosed herein.

[0173] As further described herein, one or more of the methods / techniques set forth above can be used to construct one or more genetically modified filamentous fungal strains of the present disclosure. Such methods are particularly suited to constructing modified filamentous fungal cells / strains that are defective in the production of one or more functional proteins. For example, in one or more specific embodiments of the present disclosure, the modified filamentous fungal cells are genetically modified to render the cells defective in the production of one or more mannosidases.

[0174] Thus, in certain embodiments, modified filamentous fungal cells of the present disclosure are constructed by reducing or eliminating expression of one or more of the genes set forth above using methods well known in the art, such as insertion, disruption, substitution, or deletion. The portion of the gene to be modified or inactivated can be, for example, a coding region or a regulatory element required for expression of the coding region. An example of such a regulatory or control sequence can be a promoter sequence or a functional portion thereof (i.e., a portion sufficient to affect expression of a nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, and the like.

[0175] In certain other embodiments, modified filamentous fungal cells can be constructed by gene deletion to eliminate or reduce expression of at least one of the above-described genes of this disclosure. Gene deletion techniques allow for the partial or complete removal of a gene, thereby eliminating expression or expressing a non-functional (or reduced activity) protein product. In such methods, gene deletion can be achieved by homologous recombination using a plasmid constructed to contain adjacent 5' and 3' regions flanking the gene. The flanking 5' and 3' regions can be introduced into cells, for example, on a temperature-sensitive plasmid in association with a second selectable marker, at a permissive temperature that allows the plasmid to establish itself in the cells. The cells are then shifted to a non-permissive temperature to select for cells with the plasmid integrated into the chromosome at one of the homologous flanking regions. Selection for integration of the plasmid is influenced by the selection of the second selectable marker. After integration, recombination events at the second homologous flanking region are stimulated by shifting the cells to a permissive temperature for several generations without selection. The cells are plated to obtain single colonies, which are then tested for the loss of both selectable markers. Thus, for example, by reference to one or more gene and / or protein sequences of the present disclosure, one of skill in the art can readily identify nucleotide regions within the coding sequence of a gene and / or the non-coding sequence of a gene that are suitable for complete or partial deletion.

[0176] In other embodiments, modified filamentous fungal cells of the present disclosure are constructed by introducing, substituting, or removing one or more nucleotides in a gene or a regulatory element required for its transcription or translation. For example, nucleotides can be inserted or removed to introduce a stop codon, remove a start codon, or cause a frameshift in the open reading frame. Such modifications can be achieved by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art. Thus, in certain embodiments, one or more genes of the present disclosure can be inactivated by complete or partial deletion.

[0177] In another embodiment, modified filamentous fungal cells can be constructed by the process of gene conversion, in which a nucleic acid sequence corresponding to a gene is mutated in vitro to generate a defective nucleic acid sequence, which is then transformed into a parent cell to generate the defective gene. The defective nucleic acid sequence replaces the endogenous gene through homologous recombination. It may be desirable for the defective gene or gene fragment to also encode a marker that can be used to select for transformants containing the defective gene. For example, the defective gene can be introduced on a non-replicating or temperature-sensitive plasmid in association with a selectable marker. Selection for integration of the plasmid is affected by selection for the marker under conditions that do not permit replication of the plasmid. Selection for a second recombination event resulting in gene replacement is affected by examining colonies for loss of the selectable marker and acquisition of the mutated gene. Alternatively, the defective nucleic acid sequence can contain an insertion, substitution, or deletion of one or more nucleotides of the gene, as described below.

[0178] In certain embodiments, modified filamentous fungal cells can be constructed via CRISPR-Cas9 editing. For example, a gene of interest can be modified, disrupted, deleted, or downregulated by a nucleic acid-guided endonuclease that finds the target DNA by binding to a guide RNA (e.g., Cas9 and Cpf1) or a guide DNA (e.g., NgAgo), which recruits the endonuclease to a target sequence on the DNA. The endonuclease can then generate a single- or double-strand break in the DNA. This target DNA break can then serve as a substrate for DNA repair and recombine with the prepared editing template to disrupt, delete, or modify the gene. For example, a gene encoding a nucleic acid-guided endonuclease (for this purpose, Cas9 from Streptococcus pyogenes) or a codon-optimized gene encoding a Cas9 nuclease can be operably linked to a promoter active in fungal cells and a terminator active in fungal cells to create a fungal Cas9 expression cassette. Similarly, one or more target sites unique to a gene of interest can be easily identified by those skilled in the art. For example, to construct a DNA construct encoding a gRNA directed to a target site within a gene of interest, a variable targeting domain (VT) would comprise the nucleotides of the target site 5' to the (PAM) protospacer adjacent motif (NGG) and fused to DNA encoding the Cas9 endonuclease recognition domain for Streptococcus pyogenes (S. pyogenes) Cas9 (CER). The combination of DNA encoding the VT domain and DNA encoding the CER domain thereby generates DNA encoding the gRNA. Thus, a fungal cell expression cassette for a gRNA is created by operably linking DNA encoding the gRNA to a promoter active in fungal cells and a terminator active in fungal cells. In other embodiments, purified Cas9 and gRNA can be obtained commercially, assembled in vitro, and introduced alone or together with an incoming DNA repair template.

[0179] In certain embodiments, the DNA break induced by the endonuclease is repaired / replaced by the incoming sequence. For example, a nucleotide editing template is provided so that the cell's DNA repair mechanism can use the editing template to accurately repair the DNA break generated by the above-mentioned Cas9 expression cassette and gRNA expression cassette. For example, about 500 bp of the target gene 5' can be fused to about 500 bp of the target gene 3' to generate an editing template, which is used by the fungal host's mechanism to repair the DNA break generated by the RNA-guided endonuclease (RGEN). A shorter stretch of nucleotides in the form of double-stranded DNA or single-stranded DNA can be used as an editing template.

[0180] The Cas9 expression cassette, gRNA expression cassette, or in vitro-formed Cas9-gRNA complex (RNP) and editing template can be co-delivered into filamentous fungal cells using a number of different methods (e.g., PEG-mediated protoplast transformation, protoplast fusion, electroporation, biolistics). Transformed cells are screened by PCR amplification of the target gene using forward and reverse primers. The primers can amplify the wild-type locus or the modified locus edited by RGEN.

[0181] For example, in certain embodiments, truncated GIIα Stop The variant gls2a allele encoding the protein (gls2a Stop A parent T. reesei strain containing the variant gls2a is genetically modified via such a CRISPR-Cas9 editing system. In particular, as described in the Examples, the variant gls2a Stop A parent T. reesei strain (RLP37) containing the allele was modified herein via CRISPR-Cas9 editing, wherein the resulting modified strain contained a restored gls2a gene encoding the native GIIα protein. RThose skilled in the art are well aware of suitable methods for introducing polynucleotides into filamentous fungal cells (e.g., Aspergillus species, Trichoderma species, etc.), where standard techniques for transforming filamentous fungi and culturing fungi (well-known to those skilled in the art) are used to transform the fungal host cells of the present disclosure. Thus, introduction of a DNA construct or vector into a fungal host cell includes techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection-mediated and DEAE-dextrin-mediated transfection), incubation with calcium phosphate DNA precipitates, high-velocity gun, gene gun or biolistic transformation with DNA-coated microparticles, and protoplast fusion. General transformation techniques are known in the art (see, e.g., Ausubel et al., 1987; Campbell et al., 2001). Expression of heterologous proteins in Trichoderma is described, for example, in U.S. Patent Nos. 6,022,725 and 6,268,328. For transformation of Aspergillus strains, see also Cao et al. (2000).

[0182] In other embodiments, modified filamentous fungal cells are constructed using established antisense technology, for example, using a nucleotide sequence complementary to a nucleic acid sequence of interest. For example, the expression of a functional gene by a filamentous fungal cell can be reduced (downregulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which can be transcribed in the cell and hybridize to the mRNA produced in the cell. Thus, under conditions in which the complementary antisense nucleotide sequence can hybridize to the mRNA, the amount of translated protein is reduced or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), and antisense oligonucleotides, all of which are well known to those skilled in the art.

[0183] In other embodiments, techniques can be used that essentially use classical mutagenesis followed by exhaustive screening to identify every specific point mutation in an organism. One such technique is called FIND-IT (Knudsen et al., 2022), in which genetically variable populations (derived from either natural variants or induced mutations) are pooled together and then systematically and repeatedly screened using a sensitive PCR approach to identify individuals containing the desired mutation.

[0184] In certain embodiments, gls2a does not insert the original deleted nucleotide. Stop Additional means of restoring the allele are likely to result in phenotypic restoration; for example, adding bases a few codons upstream or downstream results in a GIIα protein that retains wild-type function with only a few amino acid differences from the wild-type.

[0185] In other specific embodiments, the recombinant nucleic acid (or its polynucleotide expression cassette or its expression vector) further comprises one or more selectable markers. Selectable markers for use in filamentous fungi include, but are not limited to, alsl, amdS, hphB, pyr2, pyr4, pyrG, sucA, trpC, argB, bleomycin resistance markers, blasticidin resistance markers, pyrithiamine resistance markers, neomycin resistance markers, adenine pathway genes, and thymidine kinase markers. In a specific embodiment, the selectable marker is pyr2, the components and methods of use of which are generally described in PCT Publication WO 2011 / 153449.

[0186] Generally, transformation of Trichoderma species cells is typically performed within 10 5 ~10 7 / mL, especially 2 × 10 6 Permeabilized protoplasts or cells are used at a density of 100 μL / mL. The protoplasts or cells in a volume of 100 μL of an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCl2) are mixed with the desired DNA. Generally, a high concentration of polyethylene glycol (PEG) is added to the uptake solution. Additives such as dimethyl sulfoxide, heparin, spermidine, and potassium chloride may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Pat. Nos. 6,022,725 and 6,268,328 (both of which are incorporated by reference).

[0187] In certain embodiments, the mutant strain comprises a genetic modification that replaces or substitutes the native promoter sequence of an endogenous gene encoding a native protein of the present disclosure with a heterologous promoter sequence. For example, in certain embodiments, the mutant strain comprises a knock-in heterologous promoter sequence that drives expression of the endogenous gene encoding the native protein. In other embodiments, the mutant strain comprises a knock-out (or mutated) native promoter sequence of an endogenous gene encoding a functional protein, thereby rendering the mutant strain deficient in production of the native protein.

[0188] In other embodiments, the mutant (or modified) strain comprises one or more introduced nucleic acids that express or overexpress one or more proteins of interest. For example, in certain embodiments, the mutant strain comprises an introduced polynucleotide (expression cassette) comprising a heterologous promoter (pro) sequence operably linked upstream (5') to a downstream (3') nucleic acid encoding the protein of interest. Heterologous promoter (pro) sequences suitable for driving protein expression or overexpression include any promoter sequence known to those of skill in the art, with particularly preferred promoters including any promoter sequence capable of increasing protein expression in the desired fungal cell. In related embodiments, the cassette may further comprise a downstream (3') transcription terminator sequence operably linked to the gene CDS.

[0189] The promoter and / or terminator sequences used herein are not intended to be limiting, but rather are selected to be functional in the desired fungal cell / strain. For example, the promoter sequence can be any nucleotide sequence that exhibits transcriptional activity in filamentous fungal cells, including mutant / variant promoters, truncated promoters, tandem promoters, hybrid promoters, synthetic promoters, inducible promoters, regulatable promoters, conditional expression systems, and combinations thereof. In many cases, suitable promoters can be obtained from genes encoding extracellular or intracellular polypeptides that are native or heterologous (foreign) to the filamentous fungal cell. Examples of promoters suitable for driving expression of one or more regulatory genes of the present disclosure include, but are not limited to, the Trichoderma reesei cDNA1 promoter, eno1 promoter, pdc1 promoter, pki1 promoter, tef1 promoter, rp2 promoter, cbh1 promoter, cbh2 promoter, egl1 promoter, egl2 promoter, and other T. reesei promoters described in Fitz et al. 2018 (incorporated herein by reference in its entirety), the Aspergillus oryzae thiA promoter, amylase promoter, the A. nidulans gpdA promoter, and the A. niger glaA promoter.

[0190] In certain embodiments, the present disclosure relates to the expression / production of one or more proteins of interest that are endogenous to a filamentous fungal host cell, while in other embodiments, the present disclosure relates to the expression / production of one or more proteins of interest that are heterologous to a filamentous fungal host cell.

[0191] In certain embodiments, heterologous genes are cloned into intermediate vectors before being transformed into filamentous fungal (host) cells for expression. The intermediate vectors can be, for example, prokaryotic vectors such as plasmids or shuttle vectors. Expression vectors / constructs typically contain a transcription unit or expression cassette that contains all additional elements required for expression of the heterologous sequence. For example, a typical expression cassette contains a 5' promoter operably linked to the heterologous nucleic acid sequence encoding the POI and may further contain sequence signals required for efficient polyadenylation of the transcript, a ribosome binding site, and translation termination. Additional elements of the cassette may include an enhancer and, if genomic DNA is used as the structural gene, an intron with functional splice donor and acceptor sites.

[0192] In addition to a promoter sequence, the expression cassette may also contain a transcription termination region downstream of the structural gene to provide efficient termination. The termination region may be obtained from the same gene as the promoter sequence or from a different gene. While any fungal terminator is likely to be functional in the present invention, preferred terminators include those derived from the Trichoderma cbhI gene, the Aspergillus nidulans trpC gene, the Aspergillus awamori or Aspergillus niger glucoamylase gene, and / or the Mucor miehei carboxyl protease gene.

[0193] The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any conventional vector used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include bacteriophage λ and M13, as well as plasmids such as pBR322-based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ, as well as yeast 2μ plasmids and centromeric yeast plasmids. Epitope tags, such as c-myc, can also be added to recombinant proteins to provide convenient isolation methods.

[0194] Elements that can also be included in an expression vector include a replicon, a gene encoding antibiotic resistance to allow for selection of bacteria harboring the recombinant plasmid, or a unique restriction site in a non-essential region of the plasmid to allow for the insertion of heterologous sequences. Any of the many resistance genes known in the art may be suitable, so the particular antibiotic resistance gene selected is not dispositive. The prokaryotic sequence is preferably selected so as not to interfere with DNA replication or integration in the fungal host.

[0195] The transformation methods of the present disclosure can stably integrate all or part of the transformation vector into the genome of the filamentous fungus. However, transformation that results in the maintenance of a self-replicating extrachromosomal transformation vector is also contemplated. Any of the known procedures for introducing foreign (heterologous) nucleotide sequences into host cells can be used. These include calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, and any other known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells (see, e.g., Sambrook et al., supra). Agrobacterium-mediated transfection methods, such as those described in U.S. Pat. No. 6,255,115, are also useful.

[0196] After the expression vector is introduced into the cells, the transformed cells are cultured under conditions favoring expression of the gene under the control of the particular gene promoter used, the promoter being selected for optimal activity under selected appropriate fermentation conditions, etc. Large batches of transformed cells can be cultured as described herein. Finally, the product is recovered from the culture using standard techniques.

[0197] As further described and exemplified below (Example 4), applicants have screened appropriate gene / protein sequence databases to identify several filamentous genes encoding GIIα, Mds1, and Mds2 protein homologs, e.g., the T. reesei GIIα protein (SEQ ID NO: 6), the A. niger GIIα protein homolog (SEQ ID NO: 19; ATCC No. 1015 strain), and the T. thermophilus GIIα protein homolog (SEQ ID NO: 25; ATCC No. 42464 strain) are shown in Figure 5.

[0198] Similarly, the T. reesei Mds1 protein (SEQ ID NO: 2), the A. niger Mds1 protein homolog (SEQ ID NO: 21; ATCC strain No. 1015), and the T. thermophilus Mds1 protein homolog (SEQ ID NO: 27; ATCC strain No. 42464) are shown in Figure 7, and the T. reesei Mds2 protein (SEQ ID NO: 4), the A. niger Mds2 protein homolog (SEQ ID NO: 23; ATCC strain No. 1015), and the T. thermophilus Mds2 protein homolog (SEQ ID NO: 29; ATCC strain No. 42464) are shown in Figure 9.

[0199] More specifically, as described in Example 4 (see Figures 6, 8, and 10), the A. niger and T. thermophilus GIIα, Mds1, and Mds2 protein homologs share substantial sequence homology with the T. reesei GIIα, Mds1, and Mds2 proteins, respectively. For example, the A. niger genes encoding the Mds1 protein homolog (SEQ ID NO:21) and the Mds2 protein homolog (SEQ ID NO:23) contain approximately 51% and 60% amino acid sequence identity with the T. reesei Mds1 protein (SEQ ID NO:2) and Mds2 protein (SEQ ID NO:4), respectively.

[0200] Thus, in one or more embodiments of the present disclosure, by reference to the present specification, Examples 1-4, and Figures 5-11, one of skill in the art can readily construct modified filamentous fungal strains deficient in the production of one or more mannosidases shown and described herein. For example, as generally shown in Figure 11, the T. reesei Mdsl protein contains a total of 523 amino acid residues, with amino acid residues from about 43 to about 511 of SEQ ID NO:2 being shown as bolded residues. In particular, amino acid positions from about 43 to about 511 shown in SEQ ID NO:2 (Figure 11) contain a glycosyl hydrolase family 47 (GH47) sequence domain, members of which are alpha-mannosidases that catalyze the hydrolysis of terminal 1,2-linked alpha-D-mannose residues.

[0201] Similarly, as shown in Figure 11, the T. reesei Mds2 protein contains a total of 794 amino acid residues, with amino acid residues from about 39 to about 286 of SEQ ID NO: 4 shown as underlined residues and amino acid residue positions from about 292 to about 773 of SEQ ID NO: 4 shown as bolded residues. More specifically, as shown in Figure 11 (SEQ ID NO: 4), amino acid positions from about 39 to about 286 contain an N-terminal glycosyl hydrolase family 92 (GH92) sequence domain, and amino acid positions from about 292 to about 773 contain a glycosyl hydrolase family 92 sequence domain, members of this family being alpha-1,2-mannosidases, enzymes that remove alpha-1,2-linked mannose residues.

[0202] In certain embodiments, a gene or gene homologue encoding a mannosidase having an amino acid sequence homologous to the Mds1 protein of SEQ ID NO: 2 has been modified herein to render the strain deficient in the production of the Mds1 protein (or a homologue thereof), and / or a gene or gene homologue encoding a mannosidase having an amino acid sequence homologous to the Mds2 protein of SEQ ID NO: 4 has been modified herein to render the strain deficient in the production of the Mds2 protein (or a homologue thereof).

[0203] In certain embodiments, one or more genes (or genetic elements, e.g., promoters, gene coding sequences, 5'-UTRs, terminators, etc.) encoding one or more mannosidases have been genetically modified (e.g., via (a) introduction, substitution, or removal of one or more nucleotides in the gene or introduction, substitution, or removal of one or more nucleotides in a regulatory element required for transcription or translation of the gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) directed mutagenesis, and / or (h) random mutagenesis, etc., of any one or more of the genes disclosed herein). For example, one skilled in the art can reduce or completely eliminate production of functional Mdsl protein by mutagenizing, disrupting, deleting, or interfering (e.g., by RNAi) a portion of the gene encoding the GH47 (family) sequence domain of the Mdsl protein (Figure 11, bolded residues; or the entire GH47 domain). Similarly, one skilled in the art can reduce or completely eliminate production of functional Mds2 protein by mutagenizing, disrupting, deleting, or otherwise interfering with portions of the gene encoding the GH92 (family) N-terminal sequence domain (Figure 11, underlined residues; or the entire GH92 N-terminal sequence domain) and / or the GH92 (superfamily) sequence domain (Figure 11, bolded residues) of the Mds2 protein. In other embodiments, one skilled in the art can reduce or completely eliminate production of functional Mds1 and / or Mds2 protein by mutagenizing, disrupting, deleting, etc., portions of the gene encoding the (GH47; glycosyl hydrolase) active site of the Mds1 protein and / or the (GH92; glycosyl hydrolase) active site of the Mds2 protein.

[0204] IV. Protein of Interest As noted above, certain embodiments relate to genetically mutated and / or modified (recombinant) fungal cells comprising genetic modifications that express a gene encoding a protein of interest (POI). More particularly, certain embodiments relate to compositions and methods for the expression / production of such proteins of interest in the modified (mutant) fungal cells of the present disclosure. Thus, in certain embodiments, the recombinant fungal cells produce increased amounts of proteins of interest, including, but not limited to, enzymes, antibodies, receptor proteins, animal feed proteins, human food proteins, and biologics.

[0205] In certain embodiments, the protein of interest is encoded / expressed / produced by an endogenous filamentous fungal gene, such as an endogenous gene encoding a cellulase, endoglucanase, xylanase, etc. In certain other embodiments, the protein of interest is encoded / expressed / produced by a heterologous polynucleotide encoding the protein of interest. In certain embodiments, the protein of interest includes a glycosylated protein (glycoprotein).

[0206] In certain embodiments, the protein of interest is a cellulase, hemicellulase, xylanase, peroxidase, protease, lipase, phospholipase, esterase, cutinase, polyesterase, phytase, pectinase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, mannanase, α-glucanase, β-glucanase, hyaluronidase, chondroitinase, laminase, lactic acid bacteria ... The enzyme is selected from the group consisting of saccharide, amylase, glucoamylase, acetyl esterase, aminopeptidase, arabinase, arabinosidase, arabinofuranosidase, carboxypeptidase, catalase, nuclease, deoxyribonuclease, ribonuclease, epimerase, α-galactosidase, β-galactosidase, glucan lyase, endo-β-glucanase, glucose oxidase, glucuronidase, invertase, and isomerase.

[0207] In certain embodiments, the POI is selected from an Enzyme Commission (EC) number selected from the group consisting of EC1, EC2, EC3, EC4, EC5, and EC6.

[0208] Optimal conditions for protein production will vary with the choice of host cell and the choice of protein to be expressed, and such conditions can be readily ascertained by one skilled in the art by routine experimentation and / or optimization.

[0209] The protein of interest can be purified or isolated after expression. Depending on what other components are present in the sample, the protein of interest can be isolated or purified by various methods known to those skilled in the art. Standard purification methods include, but are not limited to, electrophoretic techniques, molecular techniques, immunological techniques, chromatographic techniques (including ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse-phase HPLC chromatography), and chromatofocusing. For example, the protein of interest can be purified using a standard anti-protein of interest antibody column. In relation to protein concentration, ultrafiltration and diafiltration techniques are also useful. The degree of purification required will vary depending on the intended use of the protein of interest. In some cases, protein purification is not necessary.

[0210] In certain other embodiments, various screening methods can be performed to confirm that the genetically modified fungal cells of the present disclosure produce high levels of the protein of interest. In certain embodiments, the protein of interest can be detected by its activity (e.g., enzymatic activity, binding activity, etc.) or chromatographic profile. In other embodiments, the expression vector can encode a polypeptide fusion to the target protein that functions as a detectable label, or the target protein itself can function as a selectable or screenable marker. The labeled protein can be detected via Western blotting, dot blotting (methods available on the Cold Spring Harbor Protocols website), ELISA, or whole-cell fluorescence and / or FACS if the label is GFP. For example, a hexa-histidine tag can be included as a fusion to the target protein, and the tag can be detected by Western blotting. If the target protein is expressed at sufficiently high levels, SDS-PAGE combined with Coomassie / silver staining can be performed to detect increased expression in the variant host cells over the parental (control) cells, in which case no label is required. Additionally, other methods can be used to confirm the enhanced levels of the protein of interest, such as detecting increased protein amounts per cell or per milliliter of fermentation medium using Coomassie Blue or BCA reagent-based protein separations or standard HPLC methods for total protein determination.

[0211] Determining the specific productivity is another method for assessing protein production. The specific productivity (Qp) can be calculated by the following formula: Qp=gP / gDCW·hr where "gP" is the grams of protein produced in the tank, "gDCW" is the grams of dry cell weight (DCW) in the tank, and "hr" is the fermentation time (hours) from the time of inoculation, including production time and growth time. Finally, if the protein of interest has enzymatic activity, its expression level can be calculated from the enzyme assay.

[0212] In certain other embodiments, the modified filamentous fungal cells exhibit increased total protein yield compared to the (unmodified) parent strain, where total protein yield is defined as the amount of protein (g) produced per gram of carbohydrate fed. Thus, total protein yield (g / g) as used herein can be calculated using the following formula: Yf=Tp / Tc where "Yf" is the total protein yield (g / g), "Tp" is the total protein produced during fermentation (g), and "Tc" is the total carbohydrate (g) fed during the fermentation (bioreactor) run. In certain embodiments, the increase in total protein yield of the modified strain (i.e., compared to the control strain) is at least about 0.1%, at least about 1%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% or more increase compared to the unmodified (parental) cell.

[0213] Total protein yield can also be described as carbon conversion efficiency / carbon yield, e.g., as the percentage (%) of supplied carbon that is incorporated into total protein. Thus, in certain embodiments, the modified filamentous fungal cell comprises an increased carbon conversion efficiency (e.g., an increased percentage (%) of supplied carbon that is incorporated into total protein) compared to the (unmodified) parent strain. In certain embodiments, the increased carbon conversion efficiency of the modified strain (i.e., compared to the control strain) is at least about 0.1%, at least about 1%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, or more, compared to the unmodified (parent) cell.

[0214] V. Fermentation Certain embodiments relate to compositions and methods for producing a protein of interest, including growing, culturing, or fermenting the modified (mutated) filamentous fungal cells of the present disclosure. Generally, fermentation methods known in the art are used to ferment the fungal cells. In some embodiments, the fungal cells are grown under batch, fed-batch, or continuous fermentation conditions. Classical batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and is not altered during the fermentation. At the start of the fermentation, the medium is inoculated with the desired organism. In this method, fermentation occurs without adding any components to the system. Typically, batch fermentation is considered "batch" with respect to the addition of nutrients, while factors such as pH and oxygen concentration are controlled. The broth and culture composition in a batch system are constantly changed until the point at which the fermentation is stopped. Within batch cultures, cells progress through a static lag phase to a high-growth logarithmic phase and eventually to a stationary phase where growth rate decreases or stops. If untreated, cells undergo apoptosis and ultimately die. Generally, in batch phase, the majority of product production occurs during the exponential phase.

[0215] A suitable variation on the standard batch system is the "fed-batch fermentation" system. In this variation of the typical batch system, substrate is added gradually as the fermentation progresses after the exponential phase has ended. Fed-batch systems are often used to avoid catabolic repression. The continuous supply of substrate allows the process to keep its concentration below a critical level that could result in inhibition of cellular metabolism and protein production. Batch and fed-batch fermentation are common and well known in the art.

[0216] Continuous fermentation is a system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant (high) density, with cells primarily in log-phase growth. In other systems, many factors affecting growth can be continuously altered while maintaining a constant cell concentration, as measured by medium turbidity. Continuous systems attempt to maintain steady-state growth conditions. Thus, cell loss due to medium removal should be balanced against the cell growth rate during fermentation. Methods for adjusting nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing product formation rates, are well known in the art of industrial microbiology.

[0217] Certain embodiments of the present disclosure relate to fermentation procedures for culturing fungi. Fermentation procedures for the production of cellulase enzymes are known in the art. For example, cellulase enzymes can be produced by solid-state or submerged culture, including batch, fed-batch, and continuous-flow processes. Cultivation is generally accomplished in a growth medium containing an aqueous inorganic salts medium, organic growth factors, carbon and energy source materials, molecular oxygen, and, of course, a starting inoculum of the filamentous fungal host to be used.

[0218] To ensure proper microbial growth, maximize the uptake of carbon and energy sources by the cells in the bioconversion process, and achieve maximum cell yield at maximum cell density in the fermentation medium, appropriate amounts of inorganic nutrients must be supplied in appropriate proportions in addition to the carbon and energy sources, oxygen, assimilable nitrogen, and microbial inoculum.

[0219] The composition of the aqueous mineral medium can vary widely, depending in part on the microorganism and substrate used, as is known in the art. In addition to nitrogen, the mineral medium should contain appropriate amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium in suitable soluble, absorbable, ionic, and complex forms, and certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, again in suitable soluble and absorbable forms, all of which are known in the art.

[0220] The fermentation process may be an aerobic process in which the necessary molecular oxygen is supplied by a molecular oxygen-containing gas, such as air, oxygen-enriched air, or substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel at a suitable oxygen partial pressure effective to support the vigorously growing microbial species.

[0221] Fermentation temperatures can vary somewhat, but for filamentous fungi such as Trichoderma reesei, temperatures are generally within the range of about 20°C to 40°C, and generally preferably within the range of about 25°C to 34°C.

[0222] Microorganisms also require an assimilable nitrogen source. The assimilable nitrogen source can be any nitrogen-containing compound or any compound capable of releasing nitrogen in a form suitable for metabolic utilization by microorganisms. While various organic nitrogen source compounds, such as protein hydrolysates, can be used, typically inexpensive nitrogen-containing compounds, such as ammonia, ammonium hydroxide, urea, and various ammonium salts, such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds, are utilized. Ammonia gas itself is convenient for large-scale operations and can be used by bubbling an appropriate amount through the aqueous fermentation product (fermentation medium). At the same time, such ammonia can also be used to assist in pH control.

[0223] The pH range in aqueous microbial fermentation should be within an exemplary range of about 2.0 to 10.0. For filamentous fungi, the pH is typically within the range of about 2.5 to 8.0, and for Trichoderma reesei, the pH is typically within the range of about 3.0 to 7.0. The preferred pH range for a microorganism depends to some extent on the medium used and on the specific microorganism, and can be adjusted somewhat, as can be easily determined by one skilled in the art.

[0224] Preferably, the fermentation is carried out in such a way that the carbon-containing substrate can be controlled as the limiting factor, thereby achieving good conversion of the carbon-containing substrate to products and avoiding contamination of the cells with significant amounts of unconverted substrate. The latter is not a problem with water-soluble substrates, as any traces remaining can be easily washed away. However, it can be a problem with water-insoluble substrates, necessitating additional product processing steps, such as appropriate washing steps.

[0225] As noted above, the time to reach this level is not critical and may vary depending on the particular microorganism and fermentation process being carried out, however, methods for determining the carbon source concentration in a fermentation medium and whether the desired carbon source level has been achieved are well known in the art.

[0226] Fermentation can be carried out as a batch or continuous operation, with fed-batch operation being highly preferred for ease of control, production of uniform amounts of product, and the most economical use of all equipment.

[0227] If desired, some or all of the carbon and energy source materials and / or some of the assimilable nitrogen source, such as ammonia, can be added to the aqueous mineral medium before it is fed to the fermentor.

[0228] Each stream introduced into the reactor is preferably controlled at a predetermined rate or according to need, which can be determined by monitoring the concentrations of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the fermentor exhaust gas, cell density as measured by dry cell weight, or light transmittance, etc. The feed rates of the various materials can be varied to obtain maximum production rate and / or maximum yield.

[0229] In a batch or, preferably, fed-batch operation, all equipment, reactors, or fermentation means, tanks or vessels, piping, and associated circulation or cooling devices are first sterilized, usually by using steam, for example, at about 121° C. for at least about 15 minutes. The sterilized reactor is then inoculated with a culture of the selected microorganism in the presence of all necessary nutrients, including oxygen, and a carbon-containing substrate. The type of fermentor used is not critical.

[0230] VI. Broth Conditioning and Protein Recovery Process As generally described above, certain embodiments of the present disclosure relate to the cultivation (fermentation) of filamentous fungal cells for the expression / production / secretion of a protein (or glycoprotein) of interest. Accordingly, certain embodiments relate to a fermentation broth obtained by fermenting filamentous fungal cells that express and secrete the protein into the broth. In certain other embodiments, the fermentation broth containing the glycoprotein of interest is subjected to one or more protein recovery processes. In certain embodiments, the present disclosure provides methods for recovering a protein from a fungal cell fermentation broth, e.g., obtaining and collecting a filamentous fungal cell fermentation broth containing the protein of interest, performing a filtration process to remove fungal cells, performing a diafiltration process to reduce the level of free sugars, heat-treating the broth at about 40°C for a sufficient time, and recovering the protein from the broth, wherein the recovered protein comprises a reduced level of glycosylation. In related embodiments, the protein expressed, secreted, and recovered from the broth retains a higher level of enzymatic activity and / or comprises enhanced thermostability during storage at about room temperature (e.g., about 20°C-22°C).

[0231] More specifically, the proteins described herein are expressed and secreted into a fermentation broth, and the broth at the end of fermentation (EOF) is subjected to one or more protein recovery processes (steps), such as a cell separation process, a protein concentration process, and a protein purification process. Protein recovery from the fermentation broth can be carried out by procedures known to those skilled in the art to obtain the desired protein preparation. For example, the broth will generally contain cellular debris, e.g., cells, various suspended solids, and other biomass contaminants, as well as the desired protein of interest. Those skilled in the art will recognize suitable protein recovery processes, including, but not limited to, conventional solid-liquid separation techniques (e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration), and other known processes for producing cell-free filtrate. The terms "cell separation" or "cell separation process" are not intended to be limiting and include any method of cell separation and / or broth clarification known to those skilled in the art.

[0232] Similarly, the terms "concentration" or "concentration process" are not meant to be limiting and include concentration methods known to those skilled in the art, such as ultrafiltration, evaporation, and centrifugation. Prior to crystallization, it may be preferable to further concentrate the fermentation broth or cell-free filtrate using techniques such as ultrafiltration, evaporation, or precipitation. Precipitation of the protein components of the supernatant or filtrate can be achieved with a salt, such as ammonium sulfate, followed by purification by various chromatographic procedures, such as ion exchange chromatography, affinity chromatography, or similar art-recognized methods.

[0233] Thus, as generally set forth above, protein preparations, glycoprotein preparations, etc. in accordance with the present disclosure may be recovered, purified, enriched, etc. using methods known to those of skill in the art (e.g., art-recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation (or other protein salt precipitation), centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or separation through a gradient to remove unwanted whole cells, cell debris, impurities, extraneous proteins, or enzymes in the final composition). Furthermore, components that impart additional benefits, such as activators, anti-inhibitors, desired ions, pH-adjusting compounds, or other enzymes or chemicals, can be added to the purified or isolated biomolecule composition thereafter.

[0234] Thus, in one or more preferred embodiments of the present disclosure, the fermentation broth containing one or more proteins of interest is collected. In related embodiments, the terminal fermentation broth is collected (harvested) and subjected to one or more recovery processes, including at least one heat treatment step. In certain embodiments, the heat treatment process is carried out for at least about 1 hour to about 5 hours. In related embodiments, the heat treatment process is carried out at a temperature of about 38.5°C to 41.5°C. In certain other embodiments, the heat treatment process includes heat treating the broth at 40°C for about 4 hours before collecting the broth. In certain embodiments, the broth may be cooled to about room temperature (20°C) or below.

[0235] VII. Exemplary Embodiments Non-limiting embodiments of the present disclosure include, but are not limited to:

[0236] 1. A modified filamentous fungal cell derived from a parent cell that contains an endogenous gene encoding a functional alpha-mannosidase protein, wherein the modified filamentous fungal cell contains a genetic modification that renders the cell deficient in the production of a functional alpha-mannosidase protein.

[0237] 2. A modified filamentous fungal cell derived from a parent cell that contains endogenous genes encoding at least two functional alpha-mannosidase proteins, the modified filamentous fungal cell comprising a genetic modification that renders the cell deficient in the production of at least two functional alpha-mannosidase proteins.

[0238] 3. The modified cell of embodiment 1 or embodiment 2, wherein the one or more endogenous genes encode a functional alpha-mannosidase protein comprising at least about 50% to 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:29.

[0239] 4. The modified cell according to any one of embodiments 1 to 3, comprising one or more endogenous genes encoding one or more proteins of interest, and / or comprising one or more heterologous genes encoding one or more heterologous proteins of interest.

[0240] 5. The modified cell according to embodiment 1 or embodiment 2, wherein the functional alpha-mannosidase protein selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 21, and SEQ ID NO: 27 comprises a glycosyl hydrolase family 47 (GH47) sequence domain.

[0241] 6. The modified cell according to embodiment 1 or embodiment 2, wherein the functional alpha-mannosidase protein selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 23, and SEQ ID NO: 29 comprises an N-terminal glycosyl hydrolase family 92 (GH92) sequence domain and a GH92 superfamily sequence domain.

[0242] 7. The modified cell of embodiment 1 or embodiment 2, wherein the genetic modification that renders the cell deficient in the production of a functional alpha-mannosidase protein is selected from the group consisting of: (a) introduction, substitution, or removal of one or more nucleotides in a gene encoding a functional alpha-mannosidase protein, and / or introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of a gene encoding a functional alpha-mannosidase protein; (b) alpha-mannosidase gene disruption; (c) alpha-mannosidase gene conversion; (d) alpha-mannosidase gene deletion; and (e) downregulation of the alpha-mannosidase gene.

[0243] 8. The modified cell of embodiment 7, wherein the genetic modification is selected from the group consisting of a non-functional GH47 protein family sequence domain, a non-functional GH47 protein family active site, a non-functional N-terminal GH92 protein family sequence domain, a non-functional GH92 superfamily sequence domain, and a non-functional GH92 protein family active site.

[0244] 9. The modified cell of embodiment 4, fermented under conditions suitable for the production and secretion of one or more proteins into the fermentation broth.

[0245] 10. The modified cells of embodiment 9, wherein the broth at the end of fermentation (EOF) is collected and stored at room temperature for at least 4 hours to about 5 days.

[0246] 11. The modified cell of embodiment 10, wherein the stored broth contains a reduced amount of mannose (Man) sugars compared to the amount of Man sugars present in the broth of the EOF of the parent cell when collected and stored under the same conditions.

[0247] 12. The modified cell of embodiment 9, wherein a heat-treatment step is performed on the end-of-fermentation (EOF) broth, and the heat-treated broth contains a reduced amount of mannose (Man) sugars compared to the amount of Man sugars present in the EOF broth of a parent cell that produces the same one or more proteins when fermented and heat-treated under the same conditions as the modified cell.

[0248] 13. The modified cell of any one of embodiments 9-12, wherein the one or more proteins are recovered from the broth.

[0249] 14. The modified cell of any one of embodiments 9 to 12, wherein the modified cell and the parent cell are fermented under the same conditions for at least about 96 hours to about 300 hours.

[0250] 15. The modified cell according to any one of embodiments 1-14, wherein the modified cell is selected from the group consisting of an Aspergillus spp. cell, an Emericella spp. cell, a Fusarium spp. cell, a Humicola spp. cell, a Mucor spp. cell, a Myceliophthora spp. cell, a Neurospora spp. cell, a Penicillium spp. cell, a Scytalidium spp. cell, a Thielavia spp. cell, a Tolypocladium spp. cell, and a Trichoderma spp. cell.

[0251] 16. The parent cells express truncated glucosidase II α-subunit (GIIα Stop ) protein encoding mutant glucosidase IIα (gls2a Stop The modified T. reesei cells were grown in Trichoderma reesei containing the α-glucosidase IIα (gls2a) allele, which encodes the native glucosidase II α-subunit (GIIα) protein. R 15. The modified cell of any one of embodiments 1-14, comprising a ) allele.

[0252] 17. The parent cell is a T. reesei strain selected from the group consisting of Rut-C30, RL-P37, and NG14 strains, or a mutant gls2a Stop 17. The modified cell of embodiment 16, which is an ancestral strain comprising the allele.

[0253] 18. The modified cell of embodiment 13, wherein the one or more proteins of interest produced comprise a uniform N-linked glycan pattern comprising greater than about 75% Man5GlcNAc2 compared to the N-linked glycan pattern of the same one or more proteins of interest produced by the parent cell, and the modified cell and parent cell are fermented under the same conditions, and the one or more proteins of interest are recovered under the same conditions.

[0254] 19. The modified cell of embodiment 4, wherein the one or more endogenous or heterologous genes encode proteins selected from the group consisting of lipase, glucoamylase, and phytase.

[0255] 20. The modified cell of embodiment 16, wherein the modified cell produces an increased amount of protein compared to the parent cell when fermented under the same conditions for the production of the protein.

[0256] 21. Truncated glucosidase II α-subunit (GIIα Stop ) protein encoding mutant glucosidase IIα (gls2a Stop Modified Trichoderma reesei cells derived from parental T. reesei cells containing the α-subunit (GIIα) allele, encoding the native glucosidase IIα (gls2a R ) modified cells containing the allele.

[0257] 22. The modified cell of embodiment 21, which produces one or more endogenous proteins of interest and / or produces one or more heterologous proteins of interest.

[0258] 23. The modified cell of embodiment 22, wherein the one or more proteins of interest are glycoproteins.

[0259] 24. The modified cell of embodiment 23, wherein the one or more proteins of interest comprise a uniform N-linked glycan pattern comprising greater than about 75% Man5GlcNAc2 compared to the N-linked glycan pattern of the same one or more proteins of interest produced by the parent cell, and the modified cell and the parent cell are fermented under the same conditions, and the one or more proteins of interest are recovered under the same conditions.

[0260] 25. The modified cell of embodiment 22, wherein the modified cell produces an increased amount of the protein of interest compared to the parent cell when fermented under the same conditions for production of the protein of interest.

[0261] 26. The parent cell is a T. reesei strain selected from the group consisting of Rut-C30, RL-P37, and NG14 strains, or a mutant gls2a Stop 22. The modified cell of embodiment 21, which is an ancestral strain comprising the allele.

[0262] 27. The modified cell of embodiment 21, further comprising a genetic modification that renders the cell deficient in the production of a functional alpha-mannosidase protein, or comprising a genetic modification that renders the cell deficient in the production of at least two functional alpha-mannosidase proteins.

[0263] 28. The modified cell of embodiment 27, fermented under conditions suitable for the production and secretion of one or more proteins into the fermentation broth.

[0264] 29. The modified cells of embodiment 28, wherein the broth at the end of fermentation (EOF) is collected and stored at room temperature for at least 4 hours to about 5 days.

[0265] 30. The modified cell of embodiment 29, wherein the stored broth contains a reduced amount of mannose (Man) sugars compared to the amount of Man sugars present in the broth of the EOF of the parent cell when collected and stored under the same conditions.

[0266] 31. The modified cell of embodiment 28, wherein a heat treatment step is performed on the broth at the end of fermentation (EOF), and the heat-treated broth contains a reduced amount of mannose (Man) sugars compared to the amount of Man sugars present in the broth at the EOF of a parent cell that produces the same one or more proteins when fermented and heat-treated under the same conditions as the modified cell.

[0267] 32. The modified cell of embodiment 31, wherein the one or more proteins are recovered from the treatment broth.

[0268] 33. The modified cell of embodiment 28, wherein the modified cell and the parent cell are fermented under the same conditions for at least about 96 hours to about 300 hours.

[0269] 34. The modified cell of embodiment 22, wherein the one or more proteins of interest are selected from the group consisting of lipase, glucoamylase, and phytase.

[0270] 35. A method for fermenting filamentous fungal cells for the production and recovery of a protein of interest (POI) in a fermentation broth containing reduced amounts of mannose (Man) sugars, comprising obtaining parent cells containing an endogenous gene encoding a functional alpha-mannosidase protein, genetically modifying the cells to be deficient in the production of the functional alpha-mannosidase protein, and fermenting the modified cells under conditions suitable for the production of the POI, wherein the end of fermentation (EOF) broth of the modified cells contains reduced amounts of Man sugars compared to the EOF broth of the parent cells fermented under the same conditions.

[0271] 36. A method for fermenting filamentous fungal cells for the production and recovery of a protein of interest (POI) in a fermentation broth containing reduced amounts of mannose (Man) sugars, comprising obtaining parent cells containing at least two endogenous genes encoding at least two functional alpha-mannosidase proteins, genetically modifying the cells to be deficient in the production of the at least two functional alpha-mannosidase proteins, and fermenting the modified cells under conditions suitable for the production of the POI, wherein the end of fermentation (EOF) broth of the modified cells contains reduced amounts of Man sugars compared to the EOF broth of the parent cells fermented under the same conditions.

[0272] 37. The method of embodiment 35, wherein the endogenous gene encodes a functional alpha-mannosidase protein comprising at least about 50% to 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:29.

[0273] 38. The method of embodiment 36, wherein the at least two endogenous genes encode functional alpha-mannosidase proteins comprising at least about 50% to 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:29.

[0274] 39. The method of embodiment 35 or embodiment 36, wherein the produced POI is secreted into the fermentation broth.

[0275] 40. The method of embodiment 35 or embodiment 36, wherein the filamentous fungal cell is selected from the group consisting of an Aspergillus spp. cell, an Emericella spp. cell, a Fusarium spp. cell, a Humicola spp. cell, a Mucor spp. cell, a Myceliophthora spp. cell, a Neurospora spp. cell, a Penicillium spp. cell, a Scytalidium spp. cell, a Thielavia spp. cell, a Tolypocladium spp. cell, and a Trichoderma spp. cell.

[0276] 41. The method of embodiment 35 or embodiment 36, wherein the modified cells and the parental cells are fermented under the same conditions for at least about 96 to about 300 hours.

[0277] 42. The method of embodiment 35 or embodiment 36, wherein the broth of the EOF is subjected to a heat treatment process.

[0278] 43. The method of embodiment 42, wherein the heat-treated EOF broth from the modified cells contains reduced amounts of Man sugars compared to the EOF broth of the parent cells subjected to the same heat treatment process.

[0279] 44. The parent cells express truncated glucosidase II α-subunit (GIIα Stop ) protein encoding mutant glucosidase IIα (gls2a Stop The modified T. reesei cells were grown in Trichoderma reesei containing the α-glucosidase IIα (gls2a) allele, which encodes the native glucosidase II α-subunit (GIIα) protein. R 37. The method of embodiment 35 or embodiment 36, comprising a ) allele.

[0280] 45. The method of embodiment 44, wherein the recovered POI comprises a uniform N-linked glycan pattern comprising greater than about 75% Man5GlcNAc2 compared to the N-linked glycan pattern of the same POI produced by the parental cells, and the modified cells and the parental cells are fermented under the same conditions.

[0281] 46. ​​The method of embodiment 44, wherein the modified cells produce an increased amount of POI compared to the parent cells when fermented under the same conditions for the production of POI.

[0282] 47. The parent cell is a T. reesei strain selected from the group consisting of Rut-C30, RL-P37, and NG14 strains, or a mutant gls2a Stop 45. The method of embodiment 44, wherein the ancestral strain comprises the allele.

[0283] 48. A method for producing a protein of interest (POI) containing a uniform N-linked glycan pattern, comprising: (a) preparing a truncated glucosidase II α-subunit (GIIα Stop ) protein encoding mutant glucosidase IIα (gls2a Stop The method involves obtaining Trichoderma reesei cells containing the α-subunit (GIIα) allele and converting the cells to a restored glucosidase IIα (gls2a) gene encoding the native glucosidase IIα-subunit (GIIα). R ) allele, fermenting the modified cells under conditions suitable for the production and secretion of a POI, and recovering the POI from the fermentation broth, wherein the recovered POI comprises a uniform N-linked glycan pattern comprising greater than 75% Man5GlcNAc2 compared to the N-linked glycan pattern of the same POI produced by a parental cell, wherein the modified and parental cells are fermented under the same conditions and the POI is recovered under the same conditions.

[0284] 49. The method of embodiment 48, wherein the modified cells produce increased amounts of the POI compared to the parent cells when fermented under the same conditions for the production of the POI.

[0285] 50. The parent cell is a T. reesei strain selected from the group consisting of Rut-C30, RL-P37, and NG14 strains, or a mutant gls2a Stop 49. The method of embodiment 48, wherein the ancestral strain comprises the allele.

[0286] 51. The method of embodiment 48, further comprising a genetic modification that renders the cell deficient in the production of a functional alpha-mannosidase protein, or comprising a genetic modification that renders the cell deficient in the production of at least two functional alpha-mannosidase proteins.

[0287] 52. The method of embodiment 48, wherein the modified cells secrete the POI into the fermentation broth.

[0288] 53. The method of embodiment 52, wherein the POI is recovered from the broth.

[0289] 54. The method of embodiment 48, wherein the modified cells and parental cells are fermented under the same conditions for at least about 96 to about 300 hours.

[0290] 55. The method of any one of embodiments 35, 36, or 48, wherein the cells comprise one or more introduced expression constructs encoding one or more heterologous proteins of interest.

[0291] 58. The method of embodiment 55, wherein the one or more expression constructs encode one or more proteins of interest selected from the group consisting of lipase, glucoamylase, and phytase. [Example]

[0292] Certain aspects of the present disclosure can be further understood in light of the following examples, which should not be construed as limiting. Modifications to materials and methods will be apparent to those skilled in the art. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).

[0293] Example 1 Genetic modification of recombinant fungal cells expressing heterologous glycoproteins As generally indicated above, the Trichoderma strains described in this example may be derived from publicly available strains (e.g., RutC-30, RLP37, etc.), which are well suited for the expression / production of endogenous cellulases and heterologous (recombinant) proteins of interest. More specifically, the particular Trichoderma reesei strain in this example is derived from T. reesei strain RLP37 (i.e., the mutant (frameshift) glsa2) following deletion of four native cellulase genes (cbh1, cbh2, egl1, egl2) as generally described in PCT Publication WO 2005 / 001036 (incorporated herein by reference). Stop The proteins were derived from various genomic DNA sequences (including alleles). Thus, in certain embodiments, Applicant has designed and constructed recombinant T. reesei cells / strains (e.g., strain RLP37) to express heterologous glycoproteins, including phytase (glycoprotein) reporter, glucoamylase (glycoprotein) reporter, and lipase (glycoprotein) reporter glycoproteins. More specifically, any suitable protein can be expressed / produced in one or more recombinant T. reesei cells of the present disclosure and further tested, screened, assayed, etc., as generally shown and described herein. Thus, in certain embodiments of the present disclosure, Applicant has screened three exemplary glycoproteins in parental T. reesei cells.

[0294] In certain embodiments, the applicant provides a mutant glsa2 Stop The heterologous phytase reporter protein was screened in the parent T. reesei strain (Phy) containing the allele and the homologous phytase reporter protein was produced and restored. R A modified T. reesei strain containing the allele (Phy-gls2a R In another embodiment, Applicants screened for heterologous phytase reporter proteins in a modified T. reesei strain (Phy-Δmds2) containing a deleted mds2 allele. In another embodiment, Applicants screened for homologous phytase reporter proteins in a modified T. reesei strain (Phy-Δmds2) containing a deleted mds2 allele. Stop Allele (GA-glsa2 Stop ) and produced the same heterologous glucoamylase reporter protein and recovered glsa2. R Allele (GA-gls2a R In yet another embodiment, Applicants screened heterologous lipase reporter proteins in a parent T. reesei strain (Lip) and a modified T. reesei strain containing a deleted mds1 allele (Lip-Δmds1).

[0295] More specifically, such heterologous phytase protein sequences and the genes encoding them are well known in the art and include, but are not limited to, the phytase and phytase variant sequences listed in PCT Publication Nos. WO 2008 / 097619, WO 2009 / 129489, and WO 2013 / 119470 (incorporated herein by reference in their entireties). Similarly, such heterologous glucoamylase protein sequences and the genes encoding them are well known in the art and include, but are not limited to, the glucoamylase and glucoamylase variant sequences listed in PCT Publication No. WO 2021 / 212095 (incorporated herein by reference in their entireties). Similarly, such heterologous lipase reporter protein sequences and the genes encoding them are well known in the art and include, but are not limited to, the glucoamylase and glucoamylase variant sequences listed in PCT Publication No. WO 2020 / 190782 (incorporated herein by reference in their entireties).

[0296] As briefly noted above, in certain other embodiments, to more directly compare data with T. reesei strains producing endogenous cellulases (cbh1, cbh2, egl1, egl2), a parent cellulase reporter strain designated "Cel" was constructed by reintroducing the cbh1, cbh2, egl1, and egl2 genes encoding the native cellulases as a single integrated expression cassette.

[0297] In the following examples, parent T. reesei strains were modified as described herein, and the resulting transformed (modified) strains were screened by microtiter plate (MTP) fermentation to confirm expression of cellulases or heterologous glycoproteins.

[0298] As demonstrated herein, all Trichoderma strains used in the following examples contain a deletion of the Endo T allele (SEQ ID NO: 36), which encodes a secreted endo-N-acetyl-β-D-glucosaminidase (a deglycosylating enzyme). For example, as generally described by Stals et al. (2012), mannosylglycoprotein endo-N-acetyl-β-D-glucosaminidase was shown to be responsible for the microheterogeneity observed for H. jecorina cellulases and hemicellulases. Here, deletion of the Endo T allele in H. jecorina eliminates this activity, thereby retaining larger N-linked glycan chains on secreted proteins.

[0299] A. Gls2a recovery As briefly described above, the glsa2 allele in certain Trichoderma strains has been found to have a frameshift mutation at nucleotide position 1,965, resulting in aberrant processing of N-linked glycans. Such a phenotype may affect the production of recombinant glycoproteins. Based on the foregoing, Applicant has identified a glsa2 allele (gls2a R ;SEQ ID NO: 5) restored the mutant (frameshift) glsa2 allele (glsa2 Stop ; SEQ ID NO: 7) is restored, and the Trichoderma strain contains an introduced phytase glycoprotein, an introduced glucoamylase glycoprotein, or an introduced cellulase reporter protein expression cassette (cbh1, cbh2, egl1, egl2) encoding the lignocellulolytic enzymes Cbh1, Cbh2, Egl1, and Egl2, respectively.

[0300] In particular, Cas9 nuclease and a custom synthetic guide RNA (designated "RHG2"; SEQ ID NO: 9) were obtained from Synthego (Menlo Park, California). The Cas9-sgRNA complex was assembled in vitro according to the manufacturer's protocol and used to transform a Trichoderma strain generally depicted in PCT Publication WO 2016 / 100568 (incorporated herein by reference in its entirety). Specific editing of the gls2a frameshift was achieved by including 200 pmoles of a 90-base-pair double-stranded donor DNA (SEQ ID NO: 10) assembled from two oligonucleotides. Transformant colonies were screened using PCR, and the products were Sanger sequenced (data not shown). While the donor fragment provided the missing base to repair the frameshift, it also contained a single-base change to further ensure that the donor fragment and resulting repair gene were no longer cleavable by Cas9. Base changes were made at the wobble positions to avoid altering the protein sequence, however in some cases this resulted in transformants containing only the frameshift repair.

[0301] B. Mds2 deletion As previously described, the mds2 gene was deleted to evaluate its role in the release of mannose from glycoproteins, cell wall oligosaccharides, and / or other substances during and after fermentation. In this example, Applicant deleted the T. reesei mds2 gene (SEQ ID NO: 3) in the previously described phytase reporter strain. More specifically, Cas9 nuclease and custom synthetic guide RNAs LFP009 (SEQ ID NO: 11) and LFP010 (SEQ ID NO: 12) were obtained from Synthego (Menlo Park, California). Cas9-sgRNA complexes were formed according to the manufacturer's protocol. Transformant colonies were screened using PCR, and the products were Sanger sequenced (data not shown). Deletion of mds2 due to non-homologous end joining (NHEJ) and oligo-mediated homologous recombination (HR oligos, LFP013 (SEQ ID NO: 13) and LFP014 (SEQ ID NO: 14), provided as a single-stranded mixture (100 pmoles each)) were both detected when cotransformed with a selectable marker targeting the other unjoined locus.

[0302] C. Mds1 deletion As previously described, the mds1 gene was deleted to evaluate its role in the release of mannose from glycoproteins, cell wall oligosaccharides, and / or other substances during and after fermentation. In this example, Applicant deleted the T. reesei mds1 gene (SEQ ID NO: 1) in the previously described lipase reporter strain. More specifically, Cas9 nuclease and custom guide RNAs TCg3 (SEQ ID NO: 15) and TCg4 (SEQ ID NO: 16), as well as tracrRNA, were obtained from Synthego (Menlo Park, California). The Cas9-sgRNA complex was formed according to the manufacturer's protocol. Both deletion of mds1 due to non-homologous end joining (NHEJ) and oligo-mediated homologous recombination (HR oligo TC128; SEQ ID NO: 17) (prepared as a single-stranded oligonucleotide mixture (100 pmoles)) were detected when cotransformed with a selectable marker targeting the other unlinked locus. Transformant colonies were screened using PCR and the products were Sanger sequenced (data not shown).

[0303] A summary of the strains constructed herein is shown in Table 1 below. [Table 1]

[0304] D. Protein Expression in Microtiter Plates (MTPs) Transformants and control strains were cultured generally as described in Example 3 of International Application PCT / US2022 / 075210 (incorporated herein by reference in its entirety), except that precultures were performed in 24-well plates (CytoOne, Catalog No. CC7672-754) for 24 hours, the production medium contained a 2.5% (w / v) glucose / sophorose mixture, and the lactose-slow-release microtiter plates were incubated at 27°C (5 days). Ten microliters of diluted filtrate was analyzed by SDS-PAGE (NuPAGE system, according to the manufacturer's protocol). As shown in Figure 3 (left panel; untreated), the modified Phy-gls2a strains were significantly more abundant than the parental Phy strains. R The phytase glycoprotein reporter produced by the strain showed a small downward shift in apparent molecular weight (MW). As shown in Figure 3 (right panel; with Endo H treatment), after treatment with endoglycosidase H (Endoh, New England BioLabs) to remove N-glycans, the protein band (MW) size was significantly different between the Phy parent strain and the modified Phy-gls2a strain. R (restored glsa2 allele) strains. R strain and Cel-gls2a R SDS-PAGE analysis of the strain also showed a small downward shift compared to the parental GA and cel strains, respectively (data not shown). The protein mobility shift indicated smaller glycan chains, which was subsequently confirmed in Example 3 below.

[0305] MTP samples were additionally assayed for enzyme activity and protein titer. The phytase reporter was assayed using p-nitrophenyl phosphate substrate (Thermo Scientific cat no. 34045, 10 mM in sodium acetate buffer, pH 5.5). Activity was measured spectrophotometrically (absorbance at 405 nm) after a pH shift with 2N NaOH. The glucoamylase (GA) reporter was assayed by detecting glucose released within 30 minutes from a maltodextrin solution (Sigma Aldrich cat no. 419672) in sodium acetate buffer (pH 4.3). After heat inactivation, glucose was measured spectrophotometrically using a D-glucose assay kit (K-GLUC, Megazyme, Bray, Ireland). The gls2a reporter was assayed using the following methods: R Modified glucoamylase reporter strain (GA-gls2a R ) compared to the parental strain (GA), higher activity titers were observed, but the phytase reporter was different between the parental (Phy) strain genotype and the modified (Phy-gls2a R These findings were confirmed in bioreactor experiments as described below. [Table 2]

[0306] E. Protein Expression in Large-Scale Fermenters The reporter proteins described herein were expressed in large-scale (approximately 14 L) bioreactors. These fermentations were performed as generally described in PCT Publication WO 2020 / 028126 (incorporated herein by reference in its entirety), with the glucose-sophorose feeding stage performed at 25°C. For the Phy, Lip, and Cel reporters, the modifications did not significantly alter the glycoprotein titer or yield. However, the GA reporter (GA-gls2a RRestoration of gls2a in the modified strain (GA-gls2a) resulted in a significant increase in most fermentation performance metrics, as shown in Table 3 below. In particular, protein yield, titer, and specific productivity (Qp) were determined after approximately 140 hours of fermentation, as shown in Table 3. Here, the modified strain (GA-gls2a R ) have increased protein yield (62%), increased titer (53%), and increased Qp, respectively, compared to the parent (GA) strain. [Table 3]

[0307] Example 2 Determination of mannose release during large-scale fermentation and glycoprotein production and recovery The reporter proteins described herein were expressed in a large-scale (approximately 14 L) bioreactor. After the fermentation period (approximately 200-300 hours), soluble proteins from the fermentation broth containing the phytase and lipase reporters were separated from the cells using standard protein recovery and processing methods known in the art. After cell separation, diafiltration was used to reduce the concentration of small molecules, including sugars, using standard approaches. Following this recovery process, the ultrafiltered sample was divided; one portion was heated at 40°C for 4 hours and then frozen at -20°C, while the other sample was immediately frozen at -20°C. Here, the amount of mannose in the sample before and after the final heat treatment step was measured using standard analytical procedures. In particular, proteins were removed from the sample by acetone precipitation. Sugars were then separated by HPLC using a Waters Xbridge column and quantified using a Thermo ISQ EM single quadrupole mass spectrometer. As genetic modifications were made in various parental strains (ie, Phy, Lip, and Cel parental strains), mannose release from each modified strain was compared to its appropriate parental (control) strain.

[0308] For example, the results of mannose release are shown in Table 4 below, which shows the results of the three screened genes (i.e., Δmds1, Δmds2, and gls2a R) are particularly suitable for reducing the amount of mannose released (e.g., by at least about 7% to 40%) during a recovery process that includes a heating step. Thus, as demonstrated herein, such a reduction in mannose release is particularly suitable for mitigating protein glycosylation events during downstream processing, recovery, and storage of such proteins produced in filamentous fungal strains. [Table 4]

[0309] Example 3 Determination of N-glycan composition by mass spectrometry In this example, proteins produced by the T. reesei phytase and cellulase reporter strains were characterized by protein and peptide mass spectrometry. Enzymatic digestion (Section A) allows characterization of N-glycans at specific positions in the polypeptide sequence, while intact mass analysis (Section B) allows quantitative assessment of the overall glycosylation state.

[0310] A. Detection of N-glycans on reporter protein peptides after enzymatic digestion Supernatant samples from fermenter or microtiter plate cultures, obtained by centrifugation or filtration, were prepared for mass spectrometry analysis using a filtration-assisted sample preparation method as follows: In a 1.7 ml Eppendorf tube, 200 μg of protein was combined with 5 μl of 0.2 M dithiothreitol and incubated for 30 min in an Eppendorf Thermo Mixer (50°C, 300 rpm). After the addition of 10 μl of 440 mM iodoacetamide, the tube was incubated for another 30 min at room temperature in the dark. Protein was then precipitated with 0.5 ml acetone. After centrifugation (14,000 rpm, 10 min), the supernatant was removed and the pellet was dried for 5 min in a chemical hood. The pellet was resuspended in 150 μl of 8 M urea (37°C, 5 min) and then filtered through a Millipore Microcon-30 filter (Ultracel YM-30 regenerated cellulose, 30,000 NMWL) at 14,000 rpm for 15 min. Ammonium bicarbonate (50 mM) was then applied three times (100 μl each) while centrifuging (14,000 rpm, 10 min). The collection vial was replaced, and ammonium bicarbonate (50 μM, 40 μl) and trypsin (0.1 mg / ml, 20 μl) were applied to the filter. This was then incubated at 37°C (300 rpm) for 12–18 h. The filter was washed two more times with 50 mM ammonium bicarbonate (14,000 rpm, 10 min each), and the protein was eluted with 10 μl of 0.1% formic acid (14,000 rpm, 10 min).

[0311] Protein digests were analyzed by an Ultimate 3000 Nano LC / Thermo Q-Exactive HF. The analytical column for the Nano LC was a Thermo PepMap RSLC C18 column with column dimensions of 75 μm × 50 cm, 2 μm particle size, and 100 Å pore size. Mobile phase A was 96% water, 4% acetonitrile, and 0.1% formic acid. Mobile phase B was 20% water, 80% acetonitrile, and 0.1% formic acid. The gradient profile was 4% B from 0 to 10 min, increasing to 45% B from 10 to 70 min; increasing to 99% B from 70.01 to 90 min, and holding at 99% B from 90 to 95 min. At 95.01 min, the gradient returned to 4% B. The total run time was 120 min at a flow rate of 0.3 μl / min. MS / MS was performed on a Thermo Q-Exactive HF. The full mass scan resolution was 60,000. The scan range was 380–2000 m / z. The AGC target was 1e6, with a maximum IT of 150 ms. The MS2 resolution was 15,000. The loop count was top 20, the NCE was 30, and the fixed initial mass was 100 m / z (the AGC target was 1e5, with a maximum IT of 100 ms). Glycosylation was analyzed using BioPharma Finder 3.5 software (ThermoFisher Scientific).

[0312] The data, expressed as percent relative abundance (%) for phytase-derived peptides and cellulase-derived peptides, are shown below in Tables 5 and 6, respectively. For both Phy and Cel proteins, gls2a (gls2a R Restoration of mds2 resulted in nearly uniform Man5GlcNAc N-glycan content at each of the analyzed positions. For comparison, deletion of mds2, assessed with the Phy reporter protein, which was found to reduce mannose release during downstream processing (Example 2), resulted in relatively little change in glycan composition in the fermentor and MTP samples. [Table 5] [Table 6]

[0313] B. Detection of N-glycans on intact phytase polypeptides In this example, fermentation supernatant samples were diluted in 0.1% formic acid to achieve a concentration of less than 10 g / L. Samples were then analyzed using a Thermo Scientific™ Vanquish liquid chromatography system equipped with a Waters BEH size-exclusion column coupled to a Thermo Scientific™ Q-Exactive Orbitrap HF mass spectrometer equipped with a HESI ion source. Protein separation was based on size by SEC using an isocratic mobile phase with 0.1% formic acid (aqueous). Mass spectrometric detection was achieved in positive ion ionization mode. The mass-to-charge (m / z) scan range varied depending on the charge state distribution observed for the protein of interest (e.g., m / z 1800 to m / z 7000). For analysis of phytase samples, the mass spectral resolution was set to 30,000. Thermo Scientific™ BioPharma Finder was used for data analysis. As shown in Figure 4, the distribution of masses varying by mannose / hexose residues was significantly higher in gls2a compared to the control strain. R It was found to be reduced in the fermented samples.

[0314] Example 4 Deletion of the mds1 and mds2 genes in other filamentous fungal cells As generally described above, in one or more embodiments of the present disclosure, one or more mds1 and / or mds2 genes are genetically modified in one or more filamentous fungal cells / strains of the present disclosure. This example describes the genetic modification of mds1 and mds2 gene homologs in recombinant A. niger and T. thermophilus cells. For example, A. niger genes encoding proteins containing approximately 51% and 60% amino acid sequence identity to the T. reesei Mds1 protein (Figures 7 and 8; SEQ ID NO: 2) and Mds2 protein (Figures 9 and 10; SEQ ID NO: 43), respectively, were identified in the genome sequence of the published reference strain A. niger ATCC® 1015.

[0315] In particular, these sequences can be used to design synthetic guide RNAs designated LFP028 (gRNA; SEQ ID NO:30) and LFP029 (gRNA; SEQ ID NO:31) that target the A. niger mds1 homolog (SEQ ID NO:20), and LFP031 (gRNA; SEQ ID NO:22) and LFP032 (gRNA; SEQ ID NO:34) that target the A. niger mds2 homolog (SEQ ID NO:22). For example, the above-mentioned Cas9 nuclease and custom synthetic guide RNAs can be obtained from suitable suppliers, such as Synthego (Menlo Park, California). In certain embodiments, synthetic donor DNA can be included to prepare homology-directed repair templates containing single- or double-stranded versions of the DNA sequences LFP030 (SEQ ID NO:32) and LFP033 (SEQ ID NO:35) for the mds1 and mds2 homologs, respectively. Transformants are obtained using unlinked selection markers (eg, hygromycin resistance, amdS, and pyrG, etc.).

[0316] In certain other embodiments, T. thermophilus genes encoding proteins with sequence homology to the T. reesei Mds1 protein (FIGS. 7 and 8; SEQ ID NO: 2) and Mds2 protein (FIGS. 9 and 10; SEQ ID NO: 4) were identified in the genome sequence of the published reference strain T. thermophilus ATCC® 42464. As described above for the A. niger gene homologs, one of skill in the art can readily construct one or more gRNAs targeting the T. thermophilus mds1 and / or mds2 genes in a similar manner.

[0317] In other embodiments, one of skill in the art can readily construct modified filamentous fungal strains deficient in the production of one or more of the mannosidases described herein. As shown in Figure 11, the T. reesei Mdsl protein contains 523 amino acid residues, with amino acid residues from about 43 to about 511 of SEQ ID NO:2 (bolded residues) comprising a glycosyl hydrolase family 47 (GH47) sequence domain, in which members of the family are alpha-mannosidases that catalyze the hydrolysis of terminal 1,2-linked alpha-D-mannose residues. Furthermore, as shown in FIG. 11, the T. reesei Mds2 protein contains 794 amino acid residues, with amino acid residues from about position 39 to about position 286 of SEQ ID NO: 4 (underlined residues) comprising an N-terminal glycosyl hydrolase family 92 (GH92) sequence domain, and amino acid residues from about position 292 to about position 773 of SEQ ID NO: 4 (bold residues) comprising a glycosyl hydrolase family 92 sequence domain, members of which are alpha-1,2-mannosidases, enzymes that remove alpha-1,2-linked mannose residues.

[0318] Based on the above, one skilled in the art can readily design and construct one or more modified filamentous fungal strains deficient in the production of Mdsl protein (or a homolog thereof) and / or deficient in the production of Mds2 protein (or a homolog thereof). In particular, as described above, one or more genes (or genetic elements, e.g., promoters, gene coding sequences, 5'-UTRs, terminators, etc.) encoding one or more mannosidases are genetically modified herein (including, but not limited to, mutagenesis, disruption, deletion, substitution, and interference with portions of the gene encoding the GH47 (family) sequence domain of the Mdsl protein) to reduce or completely eliminate production of functional Mdsl protein. In other embodiments, one or more genes (or genetic elements) encoding one or more mannosidases are genetically modified herein (including, but not limited to, by mutagenesis, disruption, deletion, substitution, interference, etc., of portions of the genes encoding the GH92 (family) N-terminal sequence domain and / or the GH92 superfamily sequence domain of the Mds2 protein) to reduce or completely eliminate production of functional Mds2 protein. In other embodiments, one or more active site (amino acid) residues of the Mds1 and / or Mds2 proteins are mutagenized, disrupted, deleted, etc. to reduce or completely eliminate production of functional Mds1 and / or Mds2 proteins.

[0319] Example 5 Assessment of reporter protein glycosylation and activity This example describes methods for evaluating one or more proteins produced by filamentous fungal cells of the present disclosure. More specifically, as generally indicated above, certain embodiments include a genetic modification that renders the cell deficient in the production of a functional alpha-mannosidase protein, and / or a restored glucosidase II alpha-subunit (gls2a) encoding the native glucosidase II alpha-subunit (GIIa). R) allele, e.g., as described above in Sections II and VI, modified filamentous fungal cells containing the gls2a allele, which is deficient in and / or restores the production of functional alpha-mannosidase. R The modified filamentous fungal cells carrying the allele can be cultured / fermented under conditions suitable for the production of one or more proteins, and the proteins can be recovered from the end-of-fermentation (EOF) broth. More specifically, after fermenting the modified fungal strain under conditions suitable for protein production and secretion, the EOF broth can be processed as described in Example 2. For example, after the EOF period (approximately 200-300 hours), the secreted proteins can be separated from the cells using standard protein recovery methods known to those skilled in the art. Similarly, such a cell separation process can be followed by a standard diafiltration process to reduce the concentration of small molecules (e.g., sugars), and the diafiltered protein sample can then be heat-treated for a sufficient period of time before being frozen at -20°C.

[0320] In one or more specific embodiments of the present disclosure, the modified filamentous fungal cells are fermented for at least about 180 hours to about 320 hours. In certain embodiments, the modified filamentous fungal cells are fermented for at least about 200 hours to about 300 hours. In certain other embodiments, the broth heat treatment process is carried out at a temperature of about 39.5°C to about 40.5°C. In certain embodiments, the broth heat treatment process is carried out at a temperature of at least 40°C. In certain other embodiments, the heat treatment process is for about 30 minutes to about 4.5 hours. In a related embodiment, the heat treatment process is at a temperature of about 40°C for about 4 hours.

[0321] Thus, following the preceding recovery and heat treatment processes, samples are formulated using compositions appropriate for the particular protein and stored at 25°C for six months, whereupon an aliquot of each sample is removed from storage monthly to measure the amount of free mannose (Man), and the reporter protein is analyzed for levels of glycosylation and / or protein (enzyme) activity and / or thermal stability. For example, Man levels can be measured as generally described above in Example 2. Similarly, as described herein, glycosylation of target proteins can be measured by one of several techniques well known in the art, including, but not limited to, changes in protein size as measured by mass spectrometry (Schmitt et al., 2005(a)), ultraviolet (UV)-excited blue fluorescence emission from glycated proteins (Schmitt et al., 2005(b)), and characterization of lysine and arginine side chain modifications (Schmitt et al., 2005(b)).

[0322] In related embodiments, the activity of one or more proteins (enzymes) can be assayed using methods known to those of skill in the art. For example, phytase activity can be measured using p-nitrophenyl phosphate as a substrate (see Example 1, Section D), and glucoamylase activity can be measured by the release of glucose from a maltodextrin solution (see Example 1, Section D).

[0323] Lipase activity can be measured using L-alpha-phosphatidylcholine (Avanti 441601G, Avanti Polar Lipids, USA) as a substrate dissolved in 50 mM HEPES buffer with 5 mM CaCl2 using Triton-X 100 as an emulsifier, as described in PCT Publication WO 2020 / 190782. For example, the amount of free fatty acids liberated during the enzymatic reaction can be measured using the NEFA kit (Wako Chemicals GmbH, Germany). Cellulase activity can be measured by measuring the reducing sugars released from pretreated corn stover or phosphoric acid-swollen cellulase, as described in AU2016 / 200955. In other embodiments, the thermal stability of the target protein can be measured by differential scanning calorimetry (DSC; Vetter and Indurthi, 2011). Thermal stability can also be evaluated by the degree of enzyme activity (e.g., phytase, glucoamylase, lipase, cellulase, etc.) remaining after heat-treating a sample for 10 minutes at temperatures of 40°C, 50°C, 60°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, and 98°C.

[0324] For example, one skilled in the art can perform one or more (several) of the above assays to identify cells containing modifications that render them deficient in the production of functional alpha-mannosidase protein and / or gls2a RThe level of free Man sugars can be assessed in heat-treated fermentation broth samples obtained from modified (recombinant) filamentous fungal cells containing the allele. Thus, any protein of interest can be expressed in one or more modified filamentous fungal cells of the present disclosure and assessed as described herein. More specifically, by reference to one or more modified and parent strains exemplified in Table 7 below, one skilled in the art can readily construct one or more modified filamentous fungal cells / strains for the production of other proteins of interest. For example, as contemplated and described herein, proteins of interest (e.g., phytase, glucoamylase, lipase, cellulase, etc.) produced by one or more modified cells of the present disclosure may exhibit reduced saccharification levels (i.e., compared to the corresponding parent), retain higher protein (enzyme) activity levels during extended storage at room temperature, and exhibit increased thermostability. [Table 7]

[0325] References AU2016 / 200955 specification PCT International Publication No. 2003 / 038111 Pamphlet PCT International Publication No. 2006 / 040358 Pamphlet PCT International Publication No. 2008 / 097619 Pamphlet PCT International Publication No. 2009 / 129489 Pamphlet PCT International Publication No. 2011 / 153449 Pamphlet PCT International Publication No. 2013 / 119470 Pamphlet PCT International Publication No. 2020 / 028126 Pamphlet PCT International Publication No. 2020 / 190782 Pamphlet PCT International Publication No. 2021 / 212095 Brochure Ausubel et al.,“Current Protocols in Molecular Biology”,Green Publishing Associates / Wiley Interscience,New York,Vol.1 and Vol.2,(1987;1988,1989). Geysens et al.,“Cloning and Characterization of the Glucosidase II Alpha Subunit Gene of Trichoderma reesei:a Frameshift Mutation Results in the Aberrant Glycosylation Profile of the Hypercellulolytic Strain Rut-C30”,Applied and Environmental Microbiology,Vol.71,No.6,pages 2910-2924,2005. Goto,“Protein O-Glycosylation in Fungi:Diverse Structures and Multiple Functions”,Biosci.Biotechnol.Biochem.,71,pages 1415-1427,2007. Knudsen et al.,“FIND-IT:Accelerated trait development for a green evolution”,Sci.Adv.8,eabq2266,2022. Kornfeld and Kornfeld,“Assembly of Asparagine-linked Oligosaccharides”,Ann.Rev.Biochem.,54,pages 631-664,1985. Le Crom et al.“Tracking the roots of cellulase hyperproduction by the fungus Trichderma reesei using massively parallel DNA sequencing”,Proc.Natl.Acad.Sci.U.S.A.,106,pages 16151-16156,2009 Sambrook et al.,Molecular Cloning,A Laboratory Manual,2 nd Edition,Cold Spring Harbor Laboratory Press,Cold Spring,New York,1989. Sambrook et al.,Molecular Cloning,A Laboratory Manual,4 th Edition,Cold Spring Harbor Laboratory Press,Cold Spring,New York,2012. Satoh et al.,“Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control”,Protein Science,Vol.25,pages 2095-2101,2016. Schmitt et al.,Analytical Biochemistry Volume 338,Issue 2,pages 201-215,2005(b). Schmitt et al.,Analytical Biochemistry,Volume 346,Issue 1,pages 101-106,2005(a) Sheir-Neiss and Montenecourt,“Characterization of the secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations”,Applied Microbiology and Biotechnology,20(1):46-53,1984. Stals et al.,“High Resolution Crystal Structure of the Endo-N-Acetyl-β-D-Glucosaminidase Responsible for the Deglycosylation of Hypocrea jecorina Cellulases”,PLoS ONE,Volume 7,Issue 7,2012. Sutthirak et al.,“Effect of glycation on stability and kinetic parameters of thermostable glucoamylase from Aspergillus niger”,Process Biochemistry,Vol.40,Issue 8,pages 2821-2926,2005. Vetter and Indurthi,“Moderate glycation of serum albumin affects folding,stability,and ligand binding”,Clinica Chimica Acta 412,pages 2105-2116,2011.

Claims

1. A modified filamentous fungal cell derived from a parent cell that contains an endogenous gene encoding a functional alpha-mannosidase protein and into which an expression cassette encoding a protein of interest (POI) has been introduced, the modified filamentous fungal cell comprising a genetic modification that renders the cell deficient in the production of the functional alpha-mannosidase protein.

2. 2. The modified cell of claim 1, wherein the endogenous gene encodes a functional alpha-mannosidase protein comprising at least about 50% to 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:

29.

3. The modified cell of claim 1 , fermented under conditions suitable for the production and secretion of said POI into a fermentation broth.

4. 2. The modified cell of claim 1, wherein an end of fermentation (EOF) broth is harvested and stored at room temperature for at least 4 hours to about 5 days, and the harvested and stored broth contains reduced amounts of mannose (Man) sugars compared to the amount of Man sugars present in the EOF broth of the parent cell when harvested and stored under the same conditions.

5. 2. The modified cell of claim 1, wherein the cell is selected from the group consisting of an Aspergillus spp. cell, an Emericella spp. cell, a Fusarium spp. cell, a Humicola spp. cell, a Mucor spp. cell, a Myceliophthora spp. cell, a Neurospora spp. cell, a Penicillium spp. cell, a Scytalidium spp. cell, a Thielavia spp. cell, a Tolypocladium spp. cell, and a Trichoderma spp. cell.

6. The parent cell expresses truncated glucosidase II α-subunit (GIIα Ｓｔｏｐ ) protein encoding a mutant glucosidase IIα (gls2a Ｓｔｏｐ a Trichoderma reesei cell containing a restored glucosidase IIα (gls2a) allele encoding a native glucosidase II α-subunit (GIIα) protein; Ｒ 6. The modified cell of claim 5, comprising a .

7. The modified cell of claim 6, wherein the modified cell produces an increased amount of the POI compared to the parent cell when fermented under the same conditions for the production of the POI.

8. Truncated glucosidase II α-subunit (GIIα Ｓｔｏｐ ) protein encoding a mutant glucosidase IIα (gls2a Ｓｔｏｐ Modified Trichoderma reesei cells derived from parental T. reesei cells containing the restored glucosidase IIα (gls2a) allele, encoding the native glucosidase II α-subunit (GIIα). Ｒ ) modified cells containing the allele.

9. 9. The modified cell of claim 8, which produces one or more endogenous proteins of interest and / or produces one or more heterologous proteins of interest.

10. 10. The modified cell of claim 9, wherein the modified cell produces increased amounts of the one or more proteins of interest compared to the parent cell when fermented under the same conditions as for the production of the one or more proteins of interest.

11. 10. The modified cell of claim 9, wherein the one or more proteins of interest are selected from the group consisting of lipase, glucoamylase, and phytase.

12. The modified cell of claim 8, comprising a genetic modification that renders the cell deficient in the production of a functional alpha-mannosidase protein, or comprising a genetic modification that renders the cell deficient in the production of at least two functional alpha-mannosidase proteins.

13. 1. A method for fermenting filamentous fungal cells for the production and recovery of a protein of interest (POI) in a fermentation broth containing reduced amounts of mannose (Man) sugars, comprising: (a) obtaining a parent filamentous fungal cell containing an endogenous gene encoding a functional alpha-mannosidase protein and introducing into said cell an expression cassette encoding a protein of interest (POI); (b) genetically modifying the cell so that it is deficient in the production of the functional alpha-mannosidase protein; (b) fermenting the modified cells under conditions suitable for the production of the POI; Including, The method wherein the end of fermentation (EOF) broth of the modified cells contains reduced amounts of Man sugars compared to the EOF broth obtained from the parent cells fermented under the same conditions.

14. 14. The method of claim 13, wherein the endogenous gene encodes a functional alpha-mannosidase protein comprising at least about 50% to 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27, and SEQ ID NO:

29.

15. 14. The method of claim 13, wherein the filamentous fungal cell is selected from the group consisting of an Aspergillus spp. cell, an Emericella spp. cell, a Fusarium spp. cell, a Humicola spp. cell, a Mucor spp. cell, a Myceliophthora spp. cell, a Neurospora spp. cell, a Penicillium spp. cell, a Scytalidium spp. cell, a Thielavia spp. cell, a Tolypocladium spp. cell, and a Trichoderma spp. cell.

16. The parent cell expresses truncated glucosidase II α-subunit (GIIα Ｓｔｏｐ ) protein encoding a mutant glucosidase IIα (gls2a Ｓｔｏｐ a Trichoderma reesei cell containing a restored glucosidase IIα (gls2a) allele encoding a native glucosidase II α-subunit (GIIα) protein; Ｒ 16. The method of claim 15, comprising a .) allele.

17. 1. A method for producing a protein of interest (POI) containing a uniform N-linked glycan pattern, comprising: (a) Truncated glucosidase II α-subunit (GIIα Ｓｔｏｐ ) protein encoding a mutant glucosidase IIα (gls2a Ｓｔｏｐ and obtaining Trichoderma reesei cells containing the native glucosidase IIα-subunit (GIIα) allele and transforming the cells with a restored glucosidase IIα (gls2a) allele encoding the native glucosidase IIα-subunit (GIIα). Ｒ ) genetically modifying the cells to express and encode the allele and fermenting the modified cells under conditions suitable for the production and secretion of the POI; (b) recovering said POI from the fermentation broth; Including, The method, wherein the recovered POI comprises a uniform N-linked glycan pattern comprising more than 75% Man5GlcNAc2 compared to the N-linked glycan pattern of the same POI produced by a parent cell, wherein the modified cells and the parent cell are fermented under the same conditions, and the POI is recovered under the same conditions.

18. 18. The method of claim 17, wherein the modified cells produce increased amounts of the POI compared to the parent cells when fermented under the same conditions for the production of the POI.

19. The method of claim 17, comprising a genetic modification that renders the cell deficient in the production of a functional alpha-mannosidase protein, or comprising a genetic modification that renders the cell deficient in the production of at least two functional alpha-mannosidase proteins.

20. 18. The method of claim 17, wherein the POI is a glycoprotein.

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