Recombinant fungal strains and methods for their use in production of consistent proteins

Abstract

Certain one or more embodiments of the present disclosure are directed, inter alia, to recombinant (genetically modified) filamentous fungal cells (strains) that produce proteins of interest, methods and compositions for designing and constructing modified filamentous fungal cells that produce proteins of interest, for endogenous and / or heterologous proteins of interest (in modified filamentous fungal cells). The present invention relates to methods and compositions for expression / production / secretion / recovery and the like of recombinant filamentous fungi (e.g., phytase, lipase, glucoamylase, phospholipase, esterase, cellulase, hemicellulase, xylanase and the like), methods and compositions for the production of proteins of interest in recombinant filamentous fungi, and methods and compositions for the production of proteins of interest in recombinant filamentous fungi. Wherein the proteins produced and secreted into and / or recovered from the bouillon have a homogeneous and consistent N-linked glycosylation pattern, and / or a (unwanted) reduction in saccharification of one or more proteins of interest, and the like.

Description

Technical Field

[0001] The present disclosure generally relates to the fields of biology, molecular biology, filamentous fungi, fermentation, genetics, glycoproteins, industrial proteins, protein production, etc. More particularly, the strains, compositions, and methods of the present disclosure relate to genetic modifications in filamentous fungi that result in recombinant (modified) strains with altered phenotypes, where such recombinant strains 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

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 476,079, filed on December 19, 2022, which is hereby incorporated by reference in its entirety.

[0004] Reference to Sequence Listing

[0005] The content of the electronically submitted sequence listing of the text file named "NB42034-WO-PCT_SequenceListing.txt", created on October 24, 2023, and having a size of 68 KB, is hereby incorporated by reference in its entirety. Background Art

[0006] Filamentous fungi (e.g., Aspergillus species, Penicillium species, Talaromyces species, Fusarium species, Myceliophthora species, Neurospora species, Candida species, Trichoderma species, etc.) are capable of expressing high levels of native and heterologous proteins, making them very suitable for large-scale production of proteins (e.g., enzymes, antibodies, receptors, peptides, etc.) and / or metabolites for industrial and commercial applications (such as pharmaceutical applications, animal health applications, food applications, beverage applications, laundry and textile applications, etc.). Filamentous fungi typically grow in submerged culture of mycelia in bioreactors (fermenters) that are adjusted to introduce and distribute oxygen and nutrients into the culture medium (i.e., culture broth). For example, the filamentous fungus Trichoderma reesei (or T. reesei; anamorph of the fungus Hypocrea jecorina) is known to be a highly efficient producer of cellulases.

[0007] Accordingly, filamentous fungi are utilized due to their ability to produce proteins (e.g., enzymes) that are valuable in the production of commodities such as cellulosic (derived) ethanol, textile products, cereal products, detergents, fiber / pulp / paper, food additives, feed additives, etc. Similarly, filamentous fungi are utilized for their ability to produce protein biologics (e.g., antibodies, antibody fragments, protein receptors, growth factors, etc.). For example, recombinant gene expression in such fungal host strains is a common method for producing proteins, and thus, an increase in the protein productivity of the fungal host strain is an important economic factor in the cost of protein production. In certain embodiments, proteins produced by filamentous fungi may contain post-translational modifications, including protein glycosylation on asparagine, threonine, and / or serine residues, and proteins containing such glycosylation are referred to as glycoproteins. In particular, the glycosylation process consists of multiple biochemical modifications commensurate with the target glycoprotein along the secretory pathway. Although this process is well-regulated, the ultimate extent of protein glycosylation depends on many factors, including the structural characteristics of the glycoprotein itself, growth conditions, etc.

[0008] As will be appreciated by those skilled in the art, recombinant expression of heterologous glycoproteins in filamentous fungal strains can be problematic, often resulting in glycoprotein products of less than satisfactory quality. For example, glycoproteins of less than satisfactory quality include glycoproteins with non-uniform (inconsistent) glycosylation patterns (which can add significant costs to downstream processing, e.g., recovery and purification of glycoprotein products), glycoproteins with reduced activity, glycoproteins with reduced stability, etc. In other embodiments, glycation (i.e., non-enzymatic glycosylation) of recombinant proteins produced in fungal strains can be problematic, often resulting in recombinant protein products of less than satisfactory quality. For example, glycation (non-enzymatic glycosylation) of proteins (e.g., enzymes) can be a significant problem as it often reduces the activity of the protein (enzyme) product (Sutthirak et al., 2005). Additionally, during the recovery process of many proteins, it is often necessary to diafilter the clarified (fermentation) broth to remove any reactive free sugars, thereby limiting glycation. Subsequent steps in protein recovery (e.g., heat treatment to remove any (undesired) background enzyme activity) can result in the release of more free sugars from the material.

[0009] Accordingly, as will be appreciated by those skilled in the art, novel compositions and methods for enhancing the production of recombinant proteins in filamentous fungal strains have important commercial significance. In certain embodiments, there remains a continuing and unmet need in the art, particularly related to the recombinant expression of heterologous proteins in fungal strains, the recombinant expression of heterologous glycoproteins in fungal strains, etc., where the recombinant proteins produced have uniform (consistent) glycosylation patterns, reduced glycation (i.e., non-enzymatic glycosylation), reduced downstream processing requirements, etc. SUMMARY OF THE INVENTION

[0010] As outlined and described below, certain embodiments of the present disclosure particularly relate 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 (e.g., lignocellulose-degrading enzymes, etc.) in recombinant filamentous fungal cells, methods and compositions for expressing / producing a heterologous protein of interest (e.g., phytase, lipase, glucoamylase, phospholipase, esterase, cellulase, hemicellulase, xylanase, etc.) in recombinant filamentous fungal cells, 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 the (undesired) glycosylation events of the protein of interest are reduced, etc. Thus, in certain embodiments, the methods and compositions described and illustrated herein particularly provide significant cost savings, which are related to the large-scale fermentation and downstream recovery of one or more proteins of interest produced in one or more of the recombinant filamentous fungal cells described herein.

[0011] Biological Sequence Description

[0012] 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.

[0013] SEQ ID NO:2 is the amino acid sequence of the native Mds1 protein encoded by SEQ ID NO:1.

[0014] 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.

[0015] SEQ ID NO:4 is the amino acid sequence of the Mds2 protein encoded by SEQ ID NO:3.

[0016] 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.

[0017] SEQ ID NO:6 is the amino acid sequence of the native GIIα protein encoded by SEQ ID NO:5.

[0018] SEQ ID NO:7 is the mutant Trichoderma DNA sequence (gls2a Stop encoding a variant (truncated) GIIα Stop ) protein.

[0019] SEQ ID NO:8 is a truncated GIIα encoded by SEQ ID NO:7 Stop amino acid sequence of the variant protein.

[0020] SEQ ID NO:9 is a synthetic RNA sequence named RGH2.

[0021] SEQ ID NO:10 is a synthetic DNA gls2a restoration donor sequence.

[0022] SEQ ID NO:11 is a synthetic RNA sequence named LFP009.

[0023] SEQ ID NO:12 is a synthetic RNA sequence named LFP010.

[0024] SEQ ID NO:13 is a synthetic DNA sequence named LFP013.

[0025] SEQ ID NO:14 is a synthetic DNA sequence named LFP014.

[0026] SEQ ID NO:15 is a synthetic RNA sequence named TCg3.

[0027] SEQ ID NO:16 is a synthetic RNA sequence named TCg4.

[0028] SEQ ID NO:17 is a synthetic DNA sequence named TC128.

[0029] SEQ ID NO:18 is the DNA sequence of the Aspergillus niger gls2a gene homolog.

[0030] SEQ ID NO:19 is the amino acid sequence of the Aspergillus niger GIIα protein encoded by SEQ ID NO:18.

[0031] SEQ ID NO:20 is the DNA sequence of the Aspergillus niger mds1 gene homolog.

[0032] SEQ ID NO:21 is the amino acid sequence of the Aspergillus niger Mds1 protein encoded by SEQ ID NO:20.

[0033] SEQ ID NO:22 is the DNA sequence of the Aspergillus niger mds2 gene homolog.

[0034] SEQ ID NO:23 is the amino acid sequence of the Aspergillus niger Mds2 protein encoded by SEQ ID NO:22.

[0035] SEQ ID NO: 24 is the DNA sequence of a homolog of the T. thermophilus gls2a gene.

[0036] SEQ ID NO:25 is the amino acid sequence of the Thermus thermophilus GIIα protein encoded by SEQ ID NO:24.

[0037] SEQ ID NO: 26 is the DNA sequence of a homolog of the Thermus thermophilus mds1 gene.

[0038] SEQ ID NO:27 is the amino acid sequence of the Thermus thermophilus Mds1 protein encoded by SEQ ID NO:26.

[0039] SEQ ID NO: 28 is the DNA sequence of a Thermus thermophilus mds2 gene homolog.

[0040] SEQ ID NO:29 is the amino acid sequence of the Thermus thermophilus Mds2 protein encoded by SEQ ID NO:28.

[0041] SEQ ID NO: 30 is an artificial RNA sequence designated LFP028.

[0042] SEQ ID NO: 31 is an artificial RNA sequence designated LFP029.

[0043] SEQ ID NO: 32 is an artificial DNA sequence designated LFP030.

[0044] SEQ ID NO: 33 is an artificial RNA sequence designated LFP031.

[0045] SEQ ID NO: 34 is an artificial RNA sequence designated LFP032.

[0046] SEQ ID NO: 35 is an artificial DNA sequence designated LFP033.

[0047] SEQ ID NO: 36 is the DNA sequence of the wild-type Trichoderma reesei Endo T allele encoding the native endo-N-acetyl-β-D-glucosaminidase (ENGase) protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Presentation of the native Trichoderma Mds1 protein ( Figure 1 A, SEQ ID NO: 2) and native Trichoderma Mds2 protein ( Figure 1 B, amino acid sequence of SEQ ID NO: 4).

[0049] Figure 2 Present the amino acid sequences of the native GIIα protein ( Figure 2 A, SEQ ID NO:6) and the truncated GIIα STOP variant protein encoded by the mutant gls2a Stop allele (SEQ ID NO:7) ( Figure 2 B, SEQ ID NO:8). As Figure 2 shown in A, 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 Figure 2 shown in B, the variant (truncated) GIIα Stop protein (SEQ ID NO:8) contains 807 amino acid residues, with the truncated C-terminal containing 153 (frameshift) amino acid residues, which are underlined.

[0050] Figure 3 Show the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis of the diluted phytase produced in MTP cultures. Show the restoration effect of the gls2a (gls2a R ) allele confirmed in two (2) technical replicates of three (3) independent isolates (Phy-gls2a R ) and the (Phy) parental strain (Parent). As Figure 3 shown, the left panel shows the phytase (Phy) parental strain untreated with Endo H and the modified gls2a restored (gls2a R ) strain, while the right panel shows the same phytase (Phy) parental strain treated with Endo H and the modified gls2a restored (gls2a R ) strain.

[0051] Figure 4 Show the electrospray ionization mass spectrometry (EIMS) of the phytase produced by the Phy strain before (control) and after (gls2a STOP ) restoration of the truncated gls2a R allele. The presented data are from supernatant samples obtained from a large fermenter at the one hundred and forty-eight (148)-hour time point. As shown, the protein was detected in multiple charge states.

[0052] Figure 5 Show the amino acid sequences of the Trichoderma reesei GIIα protein (SEQ ID NO:6), the Aspergillus niger GIIα protein homolog (SEQ ID NO:19), and the Thermus thermophilus GIIα protein homolog (SEQ ID NO:25).

[0053] Figure 6 Shows the CLUSTAL multiple sequence alignment of Trichoderma reesei GIIα protein (SEQ ID NO: 6, labeled "6"), Aspergillus niger GIIα homolog (SEQ ID NO: 19, labeled "19"), and Thermus thermophilus GIIα homolog (SEQ ID NO: 25, labeled "25").

[0054] Figure 7 Shows the amino acid sequences of Trichoderma reesei Mds1 protein (SEQ ID NO: 2), Aspergillus niger Mds1 protein homolog (SEQ ID NO: 21), and Thermus thermophilus Mds1 protein homolog (SEQ ID NO: 27).

[0055] Figure 8 Shows the CLUSTAL multiple sequence alignment of Trichoderma reesei Mds1 protein (SEQ ID NO: 2, labeled "2"), Aspergillus niger Mds1 homolog (SEQ ID NO: 21, labeled "21"), and Thermus thermophilus Mds1 homolog (SEQ ID NO: 27, labeled "27").

[0056] Figure 9 Presents the amino acid sequences of Trichoderma reesei Mds2 protein (SEQ ID NO: 4), Aspergillus niger Mds2 protein homolog (SEQ ID NO: 23), and Thermus thermophilus Mds2 protein homolog (SEQ ID NO: 29).

[0057] Figure 10 Shows the CLUSTAL multiple sequence alignment of Trichoderma reesei Mds2 protein (SEQ ID NO: 4, labeled "4"), Aspergillus niger Mds2 homolog (SEQ ID NO: 23, labeled "23"), and Thermus thermophilus Mds2 homolog (SEQ ID NO: 29, labeled "29").

[0058] Figure 11 Presents the annotated amino acid sequence positions of native Trichoderma reesei Mds1 (SEQ ID NO: 2) and Mds2 (SEQ ID NO: 4) proteins. In particular, as Figure 11 shown, the Mds1 protein contains 523 amino acid residues, where the amino acid residues from approximately position 43 to approximately position 511 (SEQ ID NO: 2) are represented by bold residues, and these amino acid positions (approximately 43 to approximately 511) contain a glycoside hydrolase family 47 (GH47) sequence domain. Similarly, as Figure 11As shown, the Mds2 protein contains 794 amino acid residues, wherein the amino acid residues from about position 39 to about position 286 (SEQ ID NO:4) are represented by underlined residues, and the amino acid residue positions from about position 292 to about position 773 (SEQ ID NO:4) are represented by bold residues. In particular, the amino acid positions from about position 39 to 286 contain an N-terminal glycoside hydrolase family 92 (GH92) sequence domain, and the amino acid positions from about position 292 to 773 contain a glycoside hydrolase family 92 (GH92; superfamily) sequence domain. Detailed Description of the Invention

[0059] As described herein, certain embodiments relate to recombinant (modified) filamentous fungal cells (strains) for use in the production of a target protein (polypeptide) on a commercial scale. More particularly, the strains and methods of the present disclosure relate to genetic modifications in filamentous fungi that result in recombinant (modified) strains with altered phenotypes, wherein such variant strains are particularly suitable for growth in submerged culture (e.g., for the large-scale production of proteins for industrial / commercial applications). In certain embodiments, the present disclosure particularly provides recombinant filamentous fungal cells (strains) that produce a target protein, methods and compositions for expressing / producing endogenous and / or heterologous target proteins in recombinant filamentous fungal cells, methods and compositions for producing a target protein in recombinant filamentous fungi, wherein the proteins produced and recovered therefrom have a uniform and consistent N-linked glycosylation pattern, and / or reduced (undesired) protein glycosylation, and / or enhanced storage stability, and / or enhanced protein (enzyme) activity, etc.

[0060] I. Definitions

[0061] Before further describing the strains, compositions, and methods of the present invention in detail, the following terms and phrases are defined. Terms not defined shall conform to the conventional meanings known and used by those skilled in the art.

[0062] All publications and patents cited in this specification are incorporated herein by reference.

[0063] In cases where a range of values is provided, it is to be understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limits of that range and any other stated or intervening value in that stated range is encompassed within the compositions and methods of the present invention. 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 of the present invention, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either one or both of those included limits are also included in the compositions and methods of the present invention.

[0064] This document provides certain ranges, where the term "about" precedes a numerical value. The term "about" is used herein to provide textual support for the exact number that follows it and for numbers that are close to or approximate the number that follows it. When determining whether a number is close to or approximate to a particularly recited number, an unrecited number that is close to or approximate to the particularly recited number can be a number that is substantially equivalent to the particularly recited number in the context in which it is presented. For example, with respect to a numerical value, the term "about" means a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context. In another example, the phrase "a pH value of about 6" means a pH value ranging from 5.4 to 6.6, unless the pH value is otherwise specifically defined.

[0065] According to this specific embodiment, the following abbreviations and definitions apply. It should be noted that the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an enzyme" includes multiple such enzymes, and reference to "a dose" includes reference to one or more doses known to those skilled in the art and their equivalents, etc.

[0066] It should be further noted that claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as a premise for the use of exclusive terms related to the recitation of claim elements, such as "alone", "only", "exclude", "do not include", etc., or the use of "negative" limitations or "conditions". For example, in certain embodiments, the condition "wherein the culture medium does not contain an inducing substrate" can be used to exclude inducing substrates, such as cellulose, lactose, gentiobiose, sophorose, etc.

[0067] It should be further noted that, as used herein, the term "comprising" means "including but not limited to" one or more components that follow the term "comprising". The components that follow the term "comprising" are necessary or mandatory, but a composition comprising one or more components can also include other non-mandatory or optional one or more components.

[0068] It should also be noted that, as used herein, the term "consisting of" means including and limited to one or more components that follow the term "consisting of". Thus, the components that follow the term "consisting of" are necessary or mandatory, and no other components are present in the composition.

[0069] After reading this disclosure, it will be apparent to those skilled in the art that each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any one of several other embodiments without departing from the scope or spirit of the compositions and methods of the invention described herein. Any of the methods described may be carried out in the order of events recited or in any other order that is logically possible.

[0070] As used herein, the term "ascomycete fungal cell" refers to any organism in the phylum Ascomycota within the kingdom Fungi. Examples of ascomycete fungal cells include, but are not limited to, filamentous fungi of the subclass Pezizomycotina, such as species of Trichoderma, Aspergillus, Myceliophthora, Penicillium, and the like.

[0071] As used herein, the term "filamentous fungi" refers to all filamentous forms of the subphyla Fungi and Oomycota. For example, filamentous fungi include, but are not limited to, Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and species of Trichoderma.

[0072] In certain embodiments, the filamentous fungus is a Trichoderma species cell (strain), including but not limited to Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, and the like. As is known to those skilled in the art, Trichoderma reesei was previously classified as "Hypocrea jecorina". Exemplary parental Trichoderma reesei strains include, but are not limited to, Trichoderma reesei strain QM6a( 13631), Trichoderma reesei strain RL-P37 (NRRL deposit number 15709), and Trichoderma reesei strain RUT-C30( 56765). For example, Trichoderma strains Rut-C30 and RL-P37 are mutagenized derivatives of the Trichoderma reesei natural isolate QM6a (Le Crom et al., 2009; Sheir-Neiss and Montenecourt, 1984), with strain NG14 being the last common ancestor. Thus, in certain embodiments, exemplary filamentous fungal strains can be derived from / obtained from Trichoderma reesei strain RL-P37, which can contain a deletion (Δ) or loss-of-function variant of the Trichoderma reesei pyr2 gene (hereinafter abbreviated as "Δpyr2"), as generally described in Sheir-Neiss and Montenecourt (1984) and PCT Publication No. WO 2011 / 153449, each of which is incorporated herein by reference in its entirety.

[0073] In certain embodiments, the filamentous fungus is an Aspergillus species cell (strain), such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, etc. Exemplary parental Aspergillus species strains include but are not limited to Aspergillus niger strain 1015, Aspergillus oryzae strain RIB40( 42149), etc.

[0074] In other embodiments, the filamentous fungus is a Myceliophthora species cell (strain), such as Myceliophthora thermophila (also known as Thielavia terrestris), etc. Exemplary parental Myceliophthora strains include but are not limited to Myceliophthora thermophila strain 42464.

[0075] In certain other embodiments, the fungal strain thus constructed is selected using the Aspergillus nidulans amdS (acetamide) gene marker. More specifically, the amdS gene and its variants are generally known to those skilled in the art, as described, for example, in PCT Publication No. WO 2006 / 040358, which is incorporated herein by reference in its entirety. However, those skilled in the art are not limited, and any selectable marker (e.g., auxotrophic marker, antibiotic resistance marker) that functions in the selected recombinant microbial cell can be chosen.

[0076] In certain embodiments, the recombinant fungal strains of the present disclosure have been designed / constructed to express heterologous reporter proteins. In certain embodiments, the heterologous reporter proteins include, but are not limited to, recombinant phytase (reporter) proteins, recombinant lipase (reporter) proteins, recombinant glucoamylase (reporter) proteins, and the like.

[0077] In certain embodiments, exemplary phytase (reporter) proteins include, but are not limited to, native or engineered (variant) phytases. For example, PCT Publication No. WO 2003 / 038111 generally describes purified enzymes having phytase activity derived from various filamentous fungal species (e.g., Penicillium species, Fusarium species, Humicola species, Talaromyces species), as well as suitable methods for constructing recombinant fungal strains that express / produce such phytase proteins and methods for their determination. Similarly, PCT Publication Nos. WO 2008 / 097619, WO 2009 / 129489, and WO 2013 / 119470 (each of which is incorporated herein by reference in its entirety) describe engineered (variant) Buttiauxella phytases, as well as related methods for constructing fungal strains that express / produce such variant phytases and methods for their determination.

[0078] 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 2011 / 020852, and WO 2021 / 212095 generally describe methods for constructing fungal strains that express / produce glucoamylase and methods for their determination.

[0079] In certain other embodiments, exemplary lipase (reporter) proteins include, but are not limited to, native or engineered (variant) lipases. For example, PCT Publication No. WO 2020 / 190782 generally describes methods for constructing fungal strains that express / produce lipase and methods for their determination.

[0080] In certain additional one or more 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 described below in Part IV.

[0081] In certain embodiments, the phytase (reporter) protein, lipase (reporter) protein, and / or glucoamylase (reporter) protein are referred to as exemplary "proteins of interest".

[0082] As used herein, the term "glycoprotein" may refer to a protein of interest that contains one or more oligosaccharide (carbohydrate / glycan) chains that are covalently linked to one or more amino acid (residue) side chains (of the glycoprotein) via one or more glycosidic bonds. In other embodiments, the term "glycoprotein" may refer to a protein that contains an oligosaccharide side chain but later loses the oligosaccharide side chain. For example, as understood by those skilled in the art, the covalently linked oligosaccharide (glycan) chains of a glycoprotein may be partially or completely cleaved by cellular enzymes and / or exogenously introduced enzymes.

[0083] As is generally known for filamentous fungal cells, N-linked glycans are initially derived from a tetradecasaccharide composed of three monosaccharide building blocks (mannose (Man), glucose (Glc), and N-acetylglucosamine (GlcNAc)), which contains the "Glc3Man9GlcNAc2" structure, and the terminal GlcNAc is transferred to an appropriately positioned asparagine residue on the nascent glycoprotein. During passage through the secretory pathway, processing of the glycan generally removes some sugar residues and, in some cases, adds additional sugar residues. For example, partially or fully processed glycans reported for Trichoderma include GlcMan8GlcNAc2, GlcMan7GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2. In some cases, almost the entire glycan chain is removed after secretion, resulting in a protein with only a single GlcNAc residue.

[0084] Similarly, O-linked glycans are linked to the hydroxyl group of L-serine or L-threonine, and this also occurs during the secretory process. The composition of the O-linked glycans of fungi that have been reported is highly variable, and many linear and branched configurations of oligosaccharides have been reported (Goto, 2007). O-linked oligosaccharides may include Glc, Man, and two conformations of galactose (Gal) (designated Galp and Galf), and additionally contain phosphate or sulfate bonds. For example, O-glycans reported for Trichoderma reesei include Man3, phosphorylated-Man2, sulfated-Man2, ManGlc, GlcGalp, and GlcManGalp.

[0085] As used herein, the phrase "high-mannose (Man) structure" refers to an oligosaccharide having at least six (6) mannose residues (Man), such as Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, GlcMan8GlcNAc2.

[0086] As used herein, the phrase "homogeneous N-linked glycan pattern" means that a glycoprotein contains "Man5GlcNAc2" as the major N-linked glycan or consists of "Man5GlcNAc2" as the major N-linked glycan. In certain embodiments of the present disclosure, a glycoprotein comprising a homogeneous N-linked glycan pattern contains at least about 75% Man5GlcNAc2 as the major N-linked glycan pattern. In other embodiments, a glycoprotein comprising a homogeneous N-linked glycan pattern contains at least 70% to 100% of Man5GlcNAc2 as the major N-linked glycan pattern.

[0087] As used herein, the phrase "monoglucosylated structure" specifically refers to the N-linked glycan of a protein, wherein the monoglucosylated structure contains a single (1) glucose (Glc) residue and a variable number of mannose (Man), two GlcNAc, and optionally other residues or consists of a single (1) glucose (Glc) residue and a variable number of mannose (Man), two GlcNAc, and optionally other residues (e.g., GlcMan9GlcNAc2).

[0088] As used herein, the term "glycation" as used in phrases such as "protein glycation", "recombinant protein glycation", "glycation of a protein", and "glycation of a glycoprotein" specifically refers to the "non-enzymatic glycosylation" of a protein (glycoprotein) as understood in the art. As described herein, glycation (non-enzymatic glycosylation) of many target proteins is a particularly undesirable (unwanted) result. For example, as summarized by Vetter and Indurthi (2011), even a moderate protein glycation event can lead to unwanted protein structural changes.

[0089] 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.

[0090] Thus, as used and described herein, "higher levels of reducing sugars" present in fermentation broths, downstream protein recovery processes, protein preparations, etc. are generally associated with higher levels of protein glycation, where this increased level (amount) of protein glycation is particularly undesirable, as exemplified by Vetter and Indurthi (2011).

[0091] As used herein, the terms "mannosidase protein" and "mannosidase" are used interchangeably, and such mannosidases include "α-mannosidases" (e.g., α-mannanase-1; Mds1, α-mannosidase-2; Mds2) and "glucosidases".

[0092] As used herein, phrases such as "a gene encoding a mannosidase," "a mannosidase encoding gene," and "a mannosidase gene" are used interchangeably, wherein these mannosidase encoding genes include genes encoding "α-mannosidase" (e.g., Mds1, Mds2) and "glucosidase."

[0093] As used herein, the term "lack" refers to that when cultured under the same conditions, the modified, mutant or recombinant filamentous fungal cell / strain does not produce detectable activity of one or more (several) mannosidases compared to the parent (control) filamentous fungal cell / strain, or alternatively, refers to that when cultured under the same conditions, the one or more (several) mannosidases produced are preferably at least 25% less, more preferably at least 50% less, even more preferably 75% less, and most preferably 95% less than the parent filamentous fungal cell / strain. The level of the one or more mannosidases produced by the filamentous fungal cells of the present disclosure can be determined using methods described herein or known in the art.

[0094] As described herein and further described below in the Examples section, various methods and techniques are known that can be used to construct one or more modified strains of the present disclosure.

[0095] As used herein, the wild-type Trichoderma reesei "α-mannosidase-1 gene" (hereinafter abbreviated as "mds1") comprises the nucleic acid (DNA) sequence listed in SEQ ID NO: 1, wherein the wild-type mds1 gene encodes the native "α-mannosidase-1 protein" (hereinafter abbreviated as "Mds1") sequence listed in SEQ ID NO: 2.

[0096] As used herein, the wild-type Trichoderma reesei "α-mannosidase-2 gene" (hereinafter abbreviated as "mds2") comprises the DNA sequence listed in SEQ ID NO:3, wherein the wild-type mds2 gene encodes the native "α-mannosidase-2 protein" (hereinafter abbreviated as "Mds2") sequence listed in SEQ ID NO:4.

[0097] As used herein, the wild-type Trichoderma reesei "glucosidase II α subunit" gene (hereinafter abbreviated as "gls2a" or "gls2α") contains the DNA sequence listed in SEQ ID NO:5, wherein the wild-type gls2a gene encodes the native "glucosidase II α subunit" protein (hereinafter abbreviated as "GIIα") sequence listed in SEQ ID NO:6.

[0098] As used herein, the Trichoderma reesei "glucosidase II α subunit" (gls2a) gene containing a frameshift mutation at nucleotide position 1,965 (SEQ ID NO:5) is referred to as "gls2a Stop" (hereinafter abbreviated as "gls2a Stop "), where the DNA sequence of the gls2a Stop allele is as shown in SEQ ID NO:7, thus encoding a truncated GIIα protein. For example, the mutant gls2a Stop gene (nucleotide position 1,965 of SEQ ID NO:7) contains a frameshift mutation that results in a premature stop codon, where the encoded variant GIIα protein (abbreviated as variant "GIIα Stop ") contains the truncated amino acid sequence listed in SEQ ID NO:8. This frameshift mutation is present in the Trichoderma reesei strain NG14, which is the last common ancestor of strains Rut-C30 and RL-P37.

[0099] As used herein, the Trichoderma strain named "Phy" is the parental Trichoderma strain containing an introduced phytase expression cassette and the gls2α Stop allele. The endogenous cellulase genes (i.e., cbh1, cbh2, egl1, egl2) have previously been deleted from the genome.

[0100] As used herein, the modified Trichoderma strain named "Phy-gls2a R " is derived from the Phy parental strain containing a phytase expression cassette, where the Phy-gls2a R strain contains a restored glsa2 (gls2a R ) allele.

[0101] As used herein, the Trichoderma strain named "GA" is the parental Trichoderma strain containing an introduced glucoamylase (GA) expression cassette and the gls2α Stop allele. The endogenous cellulase genes (i.e., cbh1, cbh2, egl1, egl2) have previously been deleted from the genome.

[0102] As used herein, the modified Trichoderma strain named "GA-gls2a R " is derived from the GA parental strain containing a GA expression cassette, where the GA-gls2a R strain contains a restored glsa2 (gls2a R ) allele.

[0103] As used herein, the "restored glsa2 allele" (abbreviated as "gls2a R”) refers to the insertion of a single nucleotide base that restores the wild-type reading frame and removes the premature stop codon present in the gls2α Stop allele (SEQ ID NO:7), wherein the restored gls2a R allele encodes the native GIIα protein of SEQ ID NO:6. For example, in certain embodiments, the parental Trichoderma reesei cell / strain contains a variant gls2a Stop allele encoding a truncated GIIα Stop protein (gls2a R ), and the genetically modified Trichoderma reesei strain derived therefrom or obtained therefrom contains a restored gls2a

[0104] allele encoding the native GIIα protein. As used herein, the modified Trichoderma strain designated “Phy-Δmds2” is derived from a Phy parental strain containing a phytase expression cassette and a gls2α Stop allele, wherein the Phy-Δmds2 strain further contains a deletion (Δ) of mds2 (Δmds2) gene.

[0105] As used herein, the Trichoderma strain designated “Lip” is a parental Trichoderma strain containing an introduced lipase expression cassette and a gls2α Stop allele. The endogenous cellulase genes (i.e., cbh1, cbh2, egl1, egl2) have previously been deleted from the genome.

[0106] As used herein, the modified Trichoderma strain designated “Lip-Δmds1” is derived from a Lip parental strain containing a lipase expression cassette and a gls2α Stop allele, wherein the Lip-Δmds1 strain further contains a deletion (Δ) of mds1 (Δmds1) gene.

[0107] As used herein, the phrase “Endo T gene product” refers to the secreted protein (i.e., endo-N-acetyl-β-D-glucosaminidase; abbreviated as “ENGase”), which is a member of glycoside hydrolase (GH) family 18 of deglycosylating enzymes. For example, Stals 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), wherein glycosylation analysis of cellulases secreted by the Hypocrea jecorina “Endo T knockout strain” demonstrated the in vivo function of ENGase. The glycan cleavage catalyzed by ENGase occurs between two (2) basal GlcNAc residues, leaving the protein with only a single GlcNAc residue.

[0108] As used herein, the phrase "Endo T deletion" allele (abbreviated as "ETD" allele) specifically refers to a filamentous fungal strain having a genetic modification of the Endo T allele, which genetic modifications result in the strain lacking the production of native (functional) ENGase. More particularly, the Endo T alleles of the recombinant (modified) Trichoderma reesei cells / strains described herein and exemplified below have been genetically modified such that these strains are completely (100%) lacking in the production of Endo T protein or Endo T homologs. In particular, by reference to the wild-type Trichoderma reesei (Hypocrea jecorina) Endo T allele (SEQ ID NO: 36), one of ordinary skill in the art can identify the relevant Endo T genes in other filamentous fungal strains of interest. For example, fungal strains containing the ETD allele are unable to cleave between two (2) basal GlcNAc residues and thus retain larger N-linked glycan chains on secreted proteins (Stals et al., 2012).

[0109] As used herein, the Trichoderma strain named "Cel" is a parental Trichoderma strain containing an introduced cellulase expression cassette and the gls2α Stop allele. This strain was constructed from a strain in which the endogenous cellulase genes (i.e., cbh1, cbh2, egl1, egl2) had previously been deleted from the genome. The genes encoding native cellulases (cbh1, cbh2, egl1, egl2) were re-introduced as a single integrated expression cassette.

[0110] As used herein, the modified Trichoderma strain named "Cel-gls2a R " is derived from the Cel parental strain containing a cellulase expression cassette, wherein the Cel-gls2a R strain contains a restored glsa2 (gls2a R ).

[0111] As used herein, the terms "Endo H" and related phrases (e.g., "Endo H treatment", etc.) specifically refer to an enzymatic treatment of a glycoprotein to remove glycans (remove mannose (Man) from a glycoprotein). In particular, an enzyme having Endo H activity (i.e., endoglycosidase activity), such as "endo-β-N-acetylglucosaminidase H" (Endo H, New England BioLabs), is a recombinant glycosidase that cleaves within the high mannose of N-linked glycoproteins and within the chitobiose core of some hybrid oligosaccharides.

[0112] As used herein, the terms "wild-type" and "native" are used interchangeably and refer to a gene, protein, fungal cell or strain as found in nature.

[0113] As used herein, the terms "recombinant" or "non-natural" refer to organisms, microorganisms, cells, nucleic acid molecules, or vectors that have at least one engineered genetic alteration or have been modified by the introduction of a heterologous nucleic acid molecule, or to cells (e.g., microbial cells) that have been altered so that the expression of heterologous or endogenous nucleic acid molecules or genes can be controlled. Recombinant also refers to cells that are derived from non-natural cells, or are the offspring of non-natural cells that have one or more such modifications. Genetic alterations include, for example, modifications that introduce expressible nucleic acid molecules that encode proteins, or additions, deletions, substitutions of other nucleic acid molecules, or other functional changes in the genetic material of the cell. For example, recombinant cells can express genes or other nucleic acid molecules that are not found in the same or homologous form in natural (wild-type) cells, or can provide altered endogenous gene expression patterns, such as overexpression, underexpression, minimal expression, or no expression at all.

[0114] As used herein, the term "gene" is synonymous with the term "allele" and refers to a nucleic acid that encodes and directs the expression of a protein or RNA. Vegetative forms of filamentous fungi are typically haploid, so a single copy of a given gene (i.e., a single allele) is sufficient to confer a specific phenotype.

[0115] As used herein, the term "gene" means 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 region (5'UTR) or "leader" sequence, 3'UTR or "tail" sequence, promoter sequence, terminator sequence, etc.), as well as intervening sequences (introns) between individual coding segments (exons). For example, a gene (DNA) sequence of interest (GOI) may encode a structural protein, a commercially important industrial protein or peptide (e.g., an enzyme (e.g., protease, mannanase, xylanase, amylase, glucoamylase, cellulase, oxidase, phytase, lipase)), etc. The gene of interest may be a naturally occurring gene, a mutant (modified) gene, or a synthetic gene.

[0116] As used herein, a "functional protein" is a protein that has an activity or function (e.g., enzymatic activity, binding function / activity (e.g., DNA binding), surface active properties, etc.) and has not been mutagenized, truncated, or otherwise modified to eliminate or reduce the function or activity. As noted, a functional polypeptide can be thermostable or thermolabile.

[0117] As used herein, a "functional gene" is a gene that can be used by cellular components to produce an active gene product (typically a protein). In contrast, a "non-functional gene" cannot be used by cellular components to produce an active gene product (i.e., a functional protein), or has a reduced ability to be used by cellular components to produce an active gene product (i.e., a functional protein).

[0118] As used herein, the term "promoter" refers to a nucleic acid sequence that directs the transcription of a downstream gene coding sequence (CDS; or open reading frame (ORF)). Generally, the promoter will be suitable for the host cell (e.g., a fungal cell) in which the target gene is being expressed. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also referred to as "control sequences"), is necessary for the expression of a given gene. Generally, transcriptional and translational regulatory sequences include, but are not limited to, promoter and terminator sequences, including core promoters and enhancer or activator or repressor sequences, transcriptional and translational initiation and termination sequences. In certain embodiments, the promoter is an inducible promoter, a constitutive promoter, a tunable promoter, a synthetic promoter, a tandem promoter, and combinations thereof. In certain embodiments, the inducible promoter is an inducible cellulase gene promoter.

[0119] As used herein, the term "promoter activity" is the ability of a nucleic acid to direct the transcription of a downstream (3') polynucleotide in a host cell. To test promoter activity, the (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 the transcription of the polynucleotide can be evaluated. In some cases, the polynucleotide can encode a protein, and the transcription of the polynucleotide can be evaluated by assessing the production of the protein in the cell.

[0120] 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" to another nucleic acid sequence when the nucleic acid sequence is placed in a functional relationship with the other nucleic acid sequence. For example, a promoter sequence or a terminator sequence is operably linked to a CDS if the promoter sequence or the terminator sequence affects the transcription of the gene coding sequence (CDS); a ribosome binding site is operably linked to a coding sequence if the ribosome binding site is positioned to facilitate translation; a nucleic acid sequence encoding a secretory leader sequence (i.e., a signal peptide) is operably linked to a nucleic acid sequence encoding a polypeptide (e.g., an ORF) if the nucleic acid sequence encoding the secretory leader sequence (i.e., the signal peptide) is expressed as a preprotein involved 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 sequence, are contiguous and in reading phase. However, an enhancer need not be contiguous. Linking two or more nucleic acid sequences (i.e., operably linking) is accomplished using any method known to those of skill in the art.

[0121] As used herein, the phrases "lignocellulose-degrading enzyme", "cellulase enzyme", and "cellulase" are used interchangeably and include glycoside hydrolases (GHs), 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.).

[0122] As used herein, the "endoglucanase" protein may be abbreviated as "EG", the "cellobiohydrolase" protein may be abbreviated as "CBH", the "β-glucosidase" protein may be abbreviated as "BG", and the "xylanase" protein may be abbreviated as "XYL". Thus, as used herein, the gene (or ORF) encoding the EG protein may be abbreviated as "eg", the gene (or ORF) encoding the CBH protein may be abbreviated as "cbh", the gene (or ORF) encoding the BG protein may be abbreviated as "bg", and the gene (or ORF) encoding the 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.

[0123] As used herein, "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, xylanase (xyl) gene promoter sequences, and the like.

[0124] As used herein, the terms "modification" and "genetic modification" are used interchangeably and include: (a) introducing, substituting, or removing one or more nucleotides in a gene, or introducing, substituting, or removing 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 and / or upregulation, (f) site-directed mutagenesis of any one or more of the genes / DNA sequences disclosed herein, and / or (g) random mutagenesis.

[0125] As used herein, the phrases "one or more modified filamentous fungal cells", "one or more mutant filamentous fungal cells", "one or more recombinant fungal cells", "one or more modified filamentous fungal strains", etc. are used interchangeably and refer to filamentous fungal cells derived from (i.e., obtained from) a parental or control filamentous fungal cell belonging to the Pezizomycotina subphylum. For example, a "modified" filamentous fungal cell can be derived from (obtained from) a parental or control filamentous fungal cell, wherein the modified cell contains at least one genetic modification not found in the parental or control cell.

[0126] As used herein, "disruption of a gene", "gene disruption", "inactivation of a gene", and "gene inactivation" are used interchangeably and broadly refer 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 part of a gene (including the polypeptide coding sequence, promoter, enhancer, or other regulatory element), or mutagenesis thereof, wherein mutagenesis encompasses substitution, insertion, deletion, inversion, and any combination and variation thereof, which disrupts / inactivates one or more target genes and substantially reduces or prevents the production of a functional gene product (i.e., a functional protein).

[0127] As used herein, "gene deletion" refers to the removal of the gene from the host cell genome. When a gene includes control elements (e.g., enhancer elements) not immediately adjacent to the gene coding sequence, deletion of the gene refers to the deletion of part or all of the coding sequence, and optionally adjacent enhancer elements (including but not limited to, for example, promoter and / or terminator sequences).

[0128] As used herein, a "heterologous gene" refers to a polynucleotide (DNA) sequence having at least a part of the sequence that is not native or does not exist in its native form in the cell into which it is introduced and / or expressed.

[0129] As used herein, a "heterologous nucleic acid construct" or "heterologous DNA sequence" has a part of the sequence that is not native or does not exist in its native form in the cell in which it is expressed.

[0130] As used herein, a "heterologous protein" is encoded by a heterologous gene, a heterologous nucleic acid (polynucleotide) sequence, a heterologous DNA sequence, etc.

[0131] Thus, in certain embodiments, a heterologous gene, a heterologous nucleic acid construct, a 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 performing other genetic modifications described herein.

[0132] With respect to a control sequence, "heterologous" means a control sequence (e.g., a promoter, enhancer, terminator) that does not function in nature to regulate the expression of a gene that is the same as the gene whose expression it is currently regulating. Typically, a heterologous nucleic acid sequence is not endogenous to the cell or portion of the genome in which it is present and has been added to the cell by infection, transfection, transformation, microinjection, electroporation, etc. A "heterologous" nucleic acid construct can contain a combination of control sequences / DNA coding sequences that is the same as or different from the combination found in the native cell.

[0133] As used herein, the term gene "coding sequence" (abbreviated "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 a reading frame (ORF) that typically 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 contains an uninterrupted reading frame consisting of: (i) a start codon, (ii) a series of codons representing the amino acids of the encoded protein product, and (iii) a stop codon, which is read (or translated) in the 5' to 3' direction.

[0134] As used herein, the term "DNA construct" or "expression construct" refers to a nucleic acid sequence that contains at least two DNA polynucleotide fragments. A DNA or expression construct can be used to introduce a nucleic acid sequence into a fungal host cell. DNA can be generated in vitro (e.g., by PCR) or by any other suitable technique. In some embodiments, the DNA construct contains 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 a terminator. In some embodiments, the DNA construct further contains at least one selectable marker. In additional embodiments, the DNA construct contains a sequence homologous to the host cell chromosome. In other embodiments, the DNA construct contains a sequence non-homologous to the host cell chromosome.

[0135] As used herein, " flanking sequence " refers to any sequence upstream or downstream of the sequence in question (for example, for gene ABC, gene B is flanked by A and C gene sequences). In certain embodiments, the flank of the input sequence is a homology box. In another embodiment, the input sequence and the homology box are included in the unit that the flank of each side is a stuffer sequence. In certain embodiments, the flanking sequence is only present in one side (3 ' or 5 '), but in a preferred embodiment, it is on each side of the sequence flanked. The sequence of each homology box is homologous to the sequence in the filamentous fungus chromosome. These sequences guide the integration position of new constructs in the filamentous fungus chromosome and which part of chromosome (if any) will be replaced by the input sequence.

[0136] As used herein, the term "downregulation" of gene expression includes any method that results in a decrease in the expression of a functional gene product (downregulation).

[0137] 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, cassettes, and the like. Expression vectors may include regulatory sequences such as promoters, signal sequences, coding sequences, and transcription terminators.

[0138] As used herein, "expression vector" means a DNA construct comprising a coding sequence operably linked to suitable control sequences capable of achieving protein expression in a suitable host. Such control sequences may include a promoter to achieve transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences that control transcription and translation termination.

[0139] As used herein, the term "secretion signal sequence" refers to a DNA sequence encoding 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 the larger polypeptide is synthesized. The larger polypeptide is typically cleaved during transport through the secretory pathway to remove the secretory peptide.

[0140] As used herein, the terms "isolated" or "purified" refer to filamentous fungal cells, nucleic acids, or polypeptides that are removed from at least one component with which they are naturally associated.

[0141] As used herein, the term "protein of interest" (POI) refers to a polypeptide that is desired to be expressed in a filamentous fungal cell. Such a protein can be an enzyme, a substrate-binding protein, a surfactant protein, a structural protein, etc., can be expressed at a high level, and can be used for commercial purposes. For example, as generally listed below, POIs include, but are not limited to, phytase, glucoamylase, cellulase, hemicellulase, xylanase, peroxidase, protease, lipase, phospholipase, esterase, cutinase, polyesterase, pectinase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, mannanase, α-glucanase, β-glucanase, hyaluronidase, chondroitinase, laccase, amylase, glucoamylase, acetyl esterase, aminopeptidase, arabinanase, arabinosidase, arabinofuranosidase, carboxypeptidase, catalase, nuclease, deoxyribonuclease, ribonuclease, epimerase, α-galactosidase, β-galactosidase, glucan lyase, endo-β-glucanase, glucose oxidase, glucuronidase, invertase, isomerase, etc.

[0142] The protein of interest (POI) can be encoded by an "endogenous" gene. For example, in certain embodiments, the POI is encoded by a gene that is endogenous to the filamentous fungal cell (strain) (e.g., the wild-type gene encoding the native cellulase group (e.g., cellobiohydrolase, xylanase, endoglucanase, and β-glucosidase) as described above).

[0143] As used herein, the term "productivity improvement" and its variants refer to an increase in the protein of interest produced by a modified (mutant) filamentous fungal cell 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%) relative to the parental (control) filamentous fungal cell when cultured under the same conditions (e.g., medium composition, temperature, pH, cell density, dissolved oxygen, time, etc.).

[0144] As used herein, the term "increased amount" and variations thereof, as used in phrases such as "increased amount of a protein of interest produced by a recombinant cell", refer to an increase in the amount of a protein of interest produced by a modified (mutant) filamentous fungal cell, relative to a parental (control) filamentous fungal cell, 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%) when cultured under the same conditions.

[0145] As used herein, the terms "polypeptide" and "protein" (and / or their respective plural forms) are used interchangeably to refer to any length polymer of amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes are used herein for amino acid residues. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses naturally modified or amino acid polymers modified 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, for example, polypeptides containing one or more amino acid analogs (including, e.g., non-natural amino acids, etc.) and other modifications known in the art.

[0146] As used herein, proteins that are functionally and / or structurally similar are considered "related proteins". Such proteins may be derived from organisms of different genera and / or species, or even different classes of organisms (e.g., bacteria and fungi). Related proteins also encompass homologs determined by primary sequence analysis, determined by secondary or tertiary structure analysis, or determined by immunological cross-reactivity.

[0147] As used herein, the phrase "substantially inactive" or similar phrases means that the specified activity is not detectable in the mixture or is present in an amount that does not interfere with the intended purpose of the mixture.

[0148] As used herein, the term "derived polypeptide" refers to a protein derived or derivable from a protein by adding one or more amino acids to one or both of the N-terminus and C-terminus, substituting one or more amino acids at one or more different sites in the amino acid sequence, deleting one or more amino acids at one or both ends of the protein or at one or more sites in the amino acid sequence, and / or inserting one or more amino acids at one or more sites in the amino acid sequence. Preparation of protein derivatives can be achieved by modifying the DNA sequence encoding the native protein, transforming the DNA sequence into a suitable host, and expressing the modified DNA sequence to form the derived protein.

[0149] Related (and derivative) proteins include "variant proteins". Variant proteins differ from a reference / parent protein (e.g., a wild-type protein) by substitution, deletion, and / or insertion at a small number of amino acid residues. The number of different amino acid residues between the variant and the 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. Variant proteins can share at least about 50%, at least about 60%, at least 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%, or even at least about 99%, or more amino acid sequence identity with the reference protein. Variant proteins can also differ from the reference protein in selected motifs, domains, epitopes, conserved regions, etc.

[0150] As used herein, the term "homologous" protein refers to a protein having similar activity, function, and / or structure to a reference protein. This is not intended to mean that homologs are necessarily evolutionarily related. Thus, it is intended that the term encompasses one or more proteins that are the same, similar, or corresponding (i.e., in terms of structure and function) obtained from different organisms. In some embodiments, it is desirable to identify homologs having similar quaternary, tertiary, and / or primary structures to a reference protein. For example, using the methods further outlined in Example 4 below, one or more gls2a, mds1, and / or mds2 genes from filamentous fungal strains (e.g., Aspergillus niger, Aspergillus oryzae, Myceliophthora thermophila (Thermus thermophilus), etc.) encoding protein homologs having significant amino acid sequence identity to the full-length Trichoderma reesei Gls2a, Mds1, and / or Mds2 proteins of the present disclosure can be readily identified in publicly available genomic databases.

[0151] The degree of homology between sequences can be determined using any suitable method known in the art (see, e.g., Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; programs in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI) such as GAP, BESTFIT, FASTA, and TFASTA; and Devereux et al., 1984). For the 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 of the EMBOSS package (Rice et al., 2000) (preferably version 3.0.0 or later). The optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the non-simplified option) is used as the percentage identity and is calculated as follows:

[0152] (Number of identical residues × 100) / (Alignment length - Total number of gaps in the alignment)

[0153] For the purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) as implemented in the Needle program of the EMBOSS package (Rice et al., 2000, ibid.) (preferably version 3.0.0 or later). The optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the non-simplified option) is used as the percentage identity and is calculated as follows:

[0154] (Number of identical deoxyribonucleotides × 100) / (Alignment length - Total number of gaps in the alignment)

[0155] As used herein, in the case of at least two nucleic acids or polypeptides, the phrases "substantially similar" and "substantially identical" 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 even at least about 99% identity, or higher identity, compared to a reference (i.e., wild-type) sequence. Sequence identity can be determined using known programs such as BLAST, ALIGN, and CLUSTAL with standard parameters.

[0156] As used herein, the terms "purified", "isolated", or "enriched" mean that a biomolecule (e.g., a polypeptide or polynucleotide) has been altered from its native state by separating it from some or all of the naturally occurring components with which it is associated in nature. Such separation or purification can be accomplished by separation techniques well known in the art 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, ultrafiltration, gel electrophoresis, or gradient separation to remove unwanted whole cells, cell debris, impurities, foreign proteins, or enzymes from the final composition. Components that provide additional benefits, such as activators, anti-inhibitors, desired ions, pH-controlling compounds, or other enzymes or chemicals, can then be further added to the purified or isolated biomolecule composition.

[0157] As used herein, a "protein preparation" is any material that contains one or more proteins, typically a solution, usually aqueous.

[0158] As used herein, the term "recovery / recovered / recovering" refers to treating or stabilizing a broth, or at least partially separating a protein from one or more soluble and / or insoluble components of a microbial broth, and / or at least partially separating a protein from one or more solvents (e.g., water or ethanol) in the broth. The recovered protein is typically of higher purity than before the recovery process. However, in some embodiments, the recovered protein can have the same or lower purity than before the recovery process.

[0159] As used herein, the terms "broth", "culture broth", "fermentation broth", and / or "whole fermentation broth" are used interchangeably and refer to a preparation produced by cell fermentation that does not undergo a processing step after fermentation is complete. For example, when filamentous fungal cells are grown to saturation and incubated under carbon-limiting conditions to allow protein synthesis (e.g., express a protein and secrete the protein into the cell culture medium), a whole fermentation broth is typically produced. Typically, the whole fermentation broth is unfractionated and contains spent cell culture medium, metabolites, extracellular polypeptides, and microbial cells.

[0160] As used herein, the phrase "processed broth" refers to a broth that has been conditioned by altering 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, one or more salts, one or more flocculants, one or more reducing agents, one or more enzyme activators, one or more enzyme inhibitors, and / or one or more surfactants), mixing, and / or timed holding of the broth (e.g., 0.5 to 200 hours) without further processing.

[0161] As used herein, "aerobic fermentation" refers to growth in the presence of oxygen.

[0162] As used herein, the term "cell mass" refers to the cell components (including intact and lysed cells) present in a liquid / deep culture. The cell mass can be expressed as dry weight or wet weight.

[0163] As used herein, when comparing the expression / production of a protein of interest (POI) in "unmodified" (parent or control) cells to the expression / production of the same POI in "modified" (recombinant) cells, it is understood that the "unmodified" (control) and "modified" (recombinant) cells are grown / cultured / fermented under the same conditions (e.g., the same conditions such as medium, temperature, pH, etc.). Thus, the POI of the present disclosure can be produced within the host cell or secreted (or transported) into the culture medium.

[0164] It should be understood that the methods of the present disclosure are not limited to a particular order of obtaining modified (mutant) filamentous fungal cells (strains). Modification of a gene can be introduced into the parental strain at any step in constructing the strain to produce an endogenous and / or heterologous protein of interest (POI).

[0165] II. Glycoprotein

[0166] As briefly elaborated in the background art, filamentous fungi (such as Trichoderma species, Aspergillus species, Myceliophthora species, etc.) can be used for the production of target proteins and are thus commonly used in the industrial production of recombinant proteins. In certain embodiments, recombinant (modified) fungal strains are particularly suitable for the production of glycoproteins. As is generally understood by those skilled in the art, the 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 can be O-linked (e.g., glycans linked to the hydroxyl (O) of L-serine or L-threonine). In most eukaryotes, N-linked glycans are first synthesized as lipid-linked tetradecasaccharides (14 sugar units, containing the "Glc3Man9GlcNAc2" precursor), which are transferred in the ER lumen to the appropriate L-asparagine (N-linked) residues 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 excised by the action of α-glucosidase II (GII).

[0167] The α-glucosidase II protein (abbreviated as "GII" protein) is generally considered to be an asymmetric non-spherical heterodimer that contains a catalytic α subunit (GIIα) and a β subunit (GIIβ). Geysens et al. (2005) described the isolation and characterization of the gene encoding the α subunit of α-glucosidase II (GIIα) from the Trichoderma strain Rut-C30, where the gls2α gene encoding the GIIα subunit in the highly cellulolytic strain Rut-C30 contains a frameshift mutation that results in a truncated GIIα protein, and a unique monoglucosylated N-glycan pattern was observed on the proteins produced by this strain. In particular, Geysens et al. (2005) concluded that the truncated GIIα protein can still hydrolyze the first α-1,3-linked glucose (Glc) residue in the Glc2Man9GlcNAc2 N-glycan ER structure, but cannot hydrolyze the innermost α-1,3-linked glucose (Glc2) residue. As described by Geysens et al. (2005), transformation of the Rut-C30 strain with a plasmid encoding the wild-type GIIα subunit of Trichoderma reesei significantly altered the glycosylation profile, reducing the number of monoglucosylated structures and increasing the overall heterogeneity and the amount of high-mannose N-glycans. Complete conversion to high-mannose carbohydrates (e.g., Man9GlcNAc2) was not achieved, which was thought to be due to the endogenous mutant (truncated) GIIα StopCompetition between the subunit and the introduced wild-type (native) GIIα subunit. Satoh et al. (2016) described more recent crystallographic features of the fungal GIIα subunit, which indicated that the contact of Trichoderma reesei gls2a frameshift mutant (gls2a STOP ) with the GIIβ subunit would be reduced.

[0168] As described herein and further described in the Examples section below, the Applicant evaluated the specific role of the gls2a frameshift mutation (gls2a STOP ) in the glycosylation profiles of native (endogenous) and heterologously expressed enzymes. More particularly, as described in the Examples, reporter strains were generated that were derived from a common ancestor of the Trichoderma reesei Rut-C30 strain and that expressed either (native) cellulase or a heterologous phytase reporter glycoprotein. For example, instead of providing an ectopic full-length copy of the gls2a allele described by Geysens et al. (2005), the Applicant repaired (i.e., restored) the original (endogenous) frameshift mutation in each of the described reporter strains, thereby restoring the correct reading frame of the gls2a allele (i.e., the allele gls2a R ).

[0169] Specifically, as presented and described in Example 3, for both the cellulase reporter strain and the phytase reporter strain, the N-glycan patterns of these reporter strains became highly homogeneous, consisting mainly of Man5GlcNAc2, with no high-mannose structures detected. This result is very different from the results found previously (Geysens et al., 2005), where a strain containing both gls2a STOP and the wild-type version was studied, rather than replacing gls2a STOP with a revertant allele. When both alleles were present, the N-linked glycans contained a large amount of monoglucosylated and high-mannose chains.

[0170] As presented and described in Example 1, the Applicant has found that replacing gls2a STOP with a revertant allele in the glucoamylase reporter strain results in a significant increase in glucoamylase production. This result indicates that, in some cases, the processing of N-glycans in the secretory pathway may be rate-limiting for protein production.

[0171] Similarly, the post-production release of mannose (Man) from glycoproteins, oligosaccharides, etc. is a potential source of reducing sugars that can chemically react with surface lysine residues, a phenomenon known as glycation, protein glycation, enzyme glycation, etc. For example, protein glycation can negatively affect the storage stability of industrially produced enzymes as described by Sutthirak et al. (2005) and described herein. However, the exact source and mechanism of free mannose production are generally unknown. Therefore, characterization of the mannose production mechanism will further assist in mitigating mannose release, assist in mitigating protein glycation events, mitigate protein product activity loss, enhance protein product storage stability, etc.

[0172] Based on the above, it is highly desirable in the art to understand the source of free mannose and the related production mechanism of free mannose. R Protein secretion with reduced total glycan content observed in the strains (Examples 1 and 3) was used to demonstrate the role of glycoproteins in producing free sugars during post-fermentation recovery and processing (Example 2). In addition, the applicant identified two 1,2-α-D-mannosidases (i.e., Mds1 and Mds2) in the culture medium during Trichoderma reesei fermentation. For example, Maras et al. (2000) have described α-mannosidase-1 (Mds1). Although this enzyme exhibits mannosidase activity when heterologously expressed in yeast (e.g., Pichia), no mannosidase activity was detected in the culture supernatant of the Trichoderma reesei host strain from which the gene was cloned.

[0173] Therefore, to determine the potential role of two 1,2-α-D-mannosidases (Mds1 and / or Mds2) in the release of mannose in soluble extracts, the genes encoding these proteins were deleted, as described below in the Examples. Substrates for Mds1 and Mds2 can be secreted glycoproteins, cell wall oligosaccharides, and / or other substances. Specifically, as described in the Examples, deletion of each mannosidase gene resulted in a decrease in free mannose levels, suggesting that they play a key role in mannose release. More specifically, deletion of the mannosidase genes resulted in a significant reduction in mannose release and, concomitantly, a corresponding decrease in the amount of undesirable protein glycation in the culture medium during fermentation.

[0174] Accordingly, as described herein, certain embodiments of the present disclosure particularly relate to recombinant filamentous fungal cells (strains) for producing a target protein, methods and compositions for designing and constructing recombinant (modified) fungal cells that produce a target protein, methods and compositions for expressing / producing an endogenous target protein in recombinant filamentous fungal cells, methods and compositions for expressing / producing a heterologous target protein in recombinant filamentous fungal cells, methods and compositions for producing a target protein in recombinant filamentous fungi, wherein the protein produced and / or recovered therefrom has a uniform and consistent N-linked glycosylation pattern and / or a consistent monoglucosylated structure, and / or a reduction in (undesired) glycation events of the target protein, etc. Accordingly, certain other embodiments 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 present disclosure provide proteins / glycoproteins with improved or enhanced overall broth processing performance, proteins / glycoproteins with improved or enhanced downstream recovery and purification performance, improved or enhanced protein / glycoprotein product profiles (such as stability, activity, shelf life, etc.), improved quality control (QC), reduced mannose (Man) release, etc.

[0175] III. Recombinant Nucleic Acids, Molecular Biology, and Methods for Constructing Modified Filamentous Fungal Strains

[0176] As described above, certain embodiments relate to recombinant (modified) fungal strains that are derived from a parental strain comprising a native gene encoding a functional Mds1 protein and / or Mds2 protein, and / or are derived from a parental strain comprising a mutant gene encoding a truncated GIIα protein. In certain embodiments, the modified strain comprises a genetic modification such that the modified strain lacks the production of one or more native genes encoding a functional Mds1 and / or Mds2 protein. In other embodiments, the fungal strains of the present disclosure comprising a variant gls2a gene (gls2a Stop encoding a truncated GIIα Stop ) protein are genetically modified herein to replace the variant gls2a R with a restoring copy of the wild-type gls2a gene (gls2a Stop ) encoding the native full-length GIIα protein. As described above, various methods and techniques can be used for constructing, screening, identifying, selecting, etc. one or more modified strains of the present disclosure.

[0177] Thus, in one or more embodiments of the present disclosure, the modified, mutated or recombinant filamentous fungal cells / strains of the present disclosure lack the production of one or more mannosidases as set forth and illustrated herein (i.e., as compared to one or more control / parent cells). In certain embodiments, when cultured under the same conditions, the modified filamentous fungal cells lacking the production of one or more mannosidases do not produce a detectable activity of one or more (several) mannosidases as compared to the parental (control) filamentous fungal cells / strains, or alternatively, when cultured under the same conditions, 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 (several) mannosidases as compared to the parental / control filamentous fungal cells. The level of the one or more mannosidases produced by the filamentous fungal cells of the present disclosure can be determined using the methods described herein and / or methods known in the art.

[0178] Thus, certain embodiments relate to modified fungal strains that comprise one or more genetic modifications, one or more introduced nucleic acids (such as expression cassettes, targeting vectors, etc.), etc. In any of these embodiments, the modified, mutated, parental and / or control strains may comprise additional genetic modifications as 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 (heterologous or endogenous) proteins of interest.

[0179] Thus, in certain embodiments, one or more genetic elements (such as promoter sequences, gene coding sequences (CDS), 5'-UTR sequences, vectors, polynucleotides, etc.) can be genetically modified as is commonly understood by those skilled in the art. In certain embodiments, genetic modifications include, but are not limited to, (a) introducing, substituting or removing one or more nucleotides in a gene, or introducing, substituting or removing one or more nucleotides in regulatory elements required for transcription or translation of a gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) overexpression (OE) of a gene, (g) specific mutagenesis of any one or more of the genes disclosed herein, and / or (h) random mutagenesis.

[0180] As further described herein, the above one or more methods / techniques can be used to construct one or more genetically modified filamentous fungal strains of the present disclosure. Such methods are particularly suitable for constructing modified filamentous fungal cells / strains lacking the production of one or more functional proteins. For example, in one or more embodiments of the present disclosure, the modified filamentous fungal cells are genetically modified to render these cells lacking the production of one or more mannosidases.

[0181] Thus, in certain embodiments, the modified filamentous fungal cells of the present disclosure are constructed by reducing or eliminating the expression of one or more of the above genes 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, the coding region or regulatory elements required for the expression of the coding region. Examples of such regulatory or control sequences can be promoter sequences or functional portions thereof (i.e., portions sufficient to affect the 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.

[0182] In certain other embodiments, the modified filamentous fungal cells can be constructed by gene deletion to eliminate or reduce the expression of at least one of the aforementioned genes of the present disclosure. Gene deletion techniques enable the partial or complete removal of one or more genes, thereby eliminating their expression or expressing non-functional (or reduced-activity) protein products. In such methods, the deletion of one or more genes can be accomplished by homologous recombination using a plasmid that has been constructed to continuously contain the 5′ and 3′ regions flanking the gene. The continuous 5′ and 3′ regions can be introduced into the fungal cells, for example, on a temperature-sensitive plasmid, in combination with a second selectable marker at the permissive temperature to allow the plasmid to establish in the cells. The cells are then transferred to the non-permissive temperature to select for cells that have integrated the plasmid into one of the chromosomal homologous flanking regions. The selection of plasmid integration is effected by selecting the second selectable marker. After integration, the recombination event at the second homologous flanking region is stimulated by growing the cells at the permissive temperature for several generations without selection. The cells are plated to obtain single colonies, and the colonies are examined for the loss of both selectable markers. Thus, a person skilled in the art can readily identify nucleotide regions in the coding sequence of the gene and / or the non-coding sequence of the gene that are suitable for complete or partial deletion, for example, by referring to one or more gene and / or protein sequences of the present disclosure.

[0183] In other embodiments, the modified filamentous fungal cells of the present disclosure are constructed by introducing, substituting, or removing one or more nucleotides in the gene or regulatory elements required for its transcription or translation. For example, nucleotides can be inserted or removed so as to result in the introduction of a stop codon, the removal of a start codon, or a frameshift of the reading frame. Such modifications can be accomplished by site-directed mutagenesis or mutagenesis generated by PCR 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.

[0184] In another embodiment, can make up modified filamentous fungal cell by gene transformation process, wherein in vitro mutagenesis and the nucleotide sequence corresponding to this gene are to produce defective nucleic acid sequence, then this defective nucleic acid sequence is transformed into parental cell to produce defective gene.Through homologous recombination, defective nucleic acid sequence substitutes endogenous gene.It is desirable that defective gene or gene fragment also encode the marker that can be used for selecting the transformant that contains defective gene.For example, defective gene and selectable marker can be associated and introduced on non-replication or temperature-sensitive plasmid.By selecting marker under the condition that does not allow plasmid replication, influence the selection of plasmid integration.By checking whether bacterium colony loses selectable marker and whether obtains the gene of sudden change and influences the selection of the second recombination event that causes gene replacement.Alternatively, defective nucleic acid sequence can contain the insertion, replacement or disappearance of one or more Nucleotide of gene, as described below.

[0185] In certain embodiments, modified filamentous fungal cells can be constructed via CRISPR-Cas9 editing. For example, the target gene can be modified, destroyed, deleted or down-regulated by means of nucleic acid-guided endonucleases that find their target DNA by binding to guide RNA (e.g., Cas9 and Cpf1) or guide DNA (e.g., NgAgo), which recruits the endonuclease to the target sequence on the DNA, where the endonuclease can produce single-strand or double-strand breaks in the DNA. This targeted DNA break becomes a substrate for DNA repair and can be recombined with the provided editing template to cause gene destruction or deletion or modification. For example, a gene encoding a nucleic acid-guided endonuclease (for this purpose, Cas9 from Streptococcus pyogenes (S.pyogenes)) or a codon-optimized gene encoding the Cas9 nuclease is operably linked to a promoter active in fungal cells and a terminator active in fungal cells, thereby producing a fungal Cas9 expression cassette. Similarly, one skilled in the art easily identifies one or more target sites unique to the target gene. For example, in order to construct a DNA construct encoding a gRNA - pointing to a target site within a gene of interest, a variable targeting domain (VT) will comprise nucleotides of the target site that are 5' of the (PAM) protospacer sequence adjacent to the motif (NGG) and are fused to DNA encoding the Cas9 endonuclease recognition domain (CER) of Streptococcus pyogenes Cas9. The DNA encoding the VT domain is combined with the DNA encoding the CER domain to produce DNA encoding the gRNA. Thus, a fungal cell expression cassette for the gRNA is produced by operably linking the DNA encoding the gRNA to an active promoter in the fungal cell and an active terminator in the fungal cell. In other embodiments, purified Cas9 and gRNA are commercially available, assembled in vitro, and introduced alone or with an incoming DNA repair template.

[0186] In certain embodiments, DNA breaks induced by endonucleases are repaired / replaced with an input sequence. For example, to precisely repair DNA breaks generated by the above-described Cas9 expression cassette and gRNA expression cassette, a nucleotide editing template is provided such that the cell's DNA repair machinery can utilize the editing template. For example, approximately 500 bp of the 5′ of the target gene can be fused to approximately 500 bp of the 3′ of the target gene to generate an editing template that is used by the machinery of the fungal host to repair DNA breaks generated by an RNA-guided endonuclease (RGEN). Shorter nucleotide segments in the form of double-stranded or single-stranded DNA can be used as the editing template.

[0187] Many different methods (e.g., PEG-mediated protoplast transformation, protoplast fusion, electroporation, biolistic method) can be used to co-deliver the Cas9 expression cassette, gRNA expression cassette, or in vitro-formed Cas9-gRNA complex (RNP), and the editing template to filamentous fungal cells. Transformed cells are screened by PCR amplification of the target gene with forward and reverse primers. These primers can amplify the wild-type locus or the modified locus that has been edited by RGEN.

[0188] For example, in certain embodiments, a parental Trichoderma reesei strain containing a variant gls2a allele (gls2a Stop that encodes a truncated GIIα Stop ) protein is genetically modified herein by such a CRISPR-Cas9 editing system. In particular, as described in the examples, a parental Trichoderma reesei strain (RLP37) containing the variant gls2a Stop allele is modified herein by CRISPR-Cas9 editing, wherein the resulting modified strain has a restored gls2a that encodes the native GIIα protein. RAlleles. Those skilled in the art are well aware of methods suitable for introducing polynucleotides into filamentous fungal cells (e.g., Aspergillus species, Trichoderma species, etc.), where standard techniques for transforming filamentous fungi and culturing fungi (which are well known to those skilled in the art) are used to transform the fungal host cells of the present disclosure. Thus, introducing a DNA construct or vector into a fungal host cell includes, for example, the following techniques: transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipid-mediated transfection and DEAE-dextran-mediated transfection), incubation with calcium phosphate DNA precipitation, high-velocity bombardment with DNA-coated microparticles, gene gun or biolistic transformation, protoplast fusion, etc. General transformation techniques are known in the art (see, for example, Ausubel et al., 1987; Sambrook et al., 2001). The expression of heterologous proteins in Trichoderma has been described, for example, in U.S. Patent Nos. 6,022,725; 6,268,328. For the transformation of Aspergillus strains, reference is also made to Cao et al. (2000).

[0189] In other embodiments, modified filamentous fungal cells are constructed by established antisense techniques, such as using nucleotide sequences complementary to a nucleic acid sequence of interest. For example, the expression of a functional gene in 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 is capable of hybridizing with the mRNA produced in the cell. Under conditions that allow the complementary antisense nucleotide sequence to hybridize with the mRNA, the amount of the translated protein is thus reduced or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, etc., all of which are well known to those skilled in the art.

[0190] In other embodiments, a variety of techniques can be used to identify any specific point mutations in an organism, essentially using classical mutagenesis and then exhaustive screening. One such technique is called FIND-IT (Knudsen et al., 2022), where genetically variable populations (from natural variation or induced mutations) are pooled together, and then systematic and repetitive screening is performed using highly sensitive PCR methods to identify individuals containing the desired mutations.

[0191] In certain embodiments, the restoration of gls2a without inserting the original deleted nucleotide Stop Additional means of alleles may achieve phenotypic restoration. For example, adding bases several codons upstream or downstream may produce a GIIα protein that differs from the wild type by only a few amino acids and retains wild-type function.

[0192] In certain other 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, als1, amdS, hphB, pyr2, pyr4, pyrG, sucA, trpC, argB, bleomycin resistance marker, blastocidin resistance marker, pyrithiamine resistance marker, neomycin resistance marker, adenine pathway gene, thymidine kinase marker, etc. In particular embodiments, the selectable marker is pyr2, the compositions and methods of use of which are generally described in PCT Publication No. WO 2011 / 153449.

[0193] Typically, Trichoderma species cells are transformed using protoplasts or cells that have been subjected to permeabilization treatment, typically at a density of 10 5 to 10 7 / mL, particularly 2×10 6 / mL. 100 μL volumes of these protoplasts or cells in a suitable solution (e.g., 1.2 M sorbitol and 50 mM CaCl2) are mixed with the desired DNA. Typically, a high concentration of polyethylene glycol (PEG) is added to the uptake solution. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc. can also be added to the uptake solution to facilitate transformation. Similar procedures can be used for other fungal host cells. See, e.g., U.S. Patent Nos. 6,022,725 and 6,268,328, both of which are incorporated herein by reference.

[0194] In certain embodiments, the mutant strain comprises a genetic modification that replaces (substitutes) the native promoter sequence of the endogenous gene encoding the native protein of the present disclosure with a heterologous promoter sequence. For example, in certain embodiments, the mutant strain comprises a knocked-in heterologous promoter sequence that drives the expression of the endogenous gene encoding the native protein. In other embodiments, the mutant strain comprises a knocked-out (or mutated) native promoter sequence of the endogenous gene encoding the functional protein, such that the mutant strain lacks the production of the native protein.

[0195] In other embodiments, the mutant (or modified) strain comprises one or more introduced nucleic acids that express or overexpress one or more target proteins. For example, in certain embodiments, the mutant strain comprises an introduced polynucleotide (expression cassette) comprising a heterologous promoter (pro) sequence upstream (5′) and operably connected to a downstream (3′) nucleic acid encoding the target protein. Heterologous promoter (pro) sequences suitable for driving expression or overexpression of proteins include any promoter sequences known to those skilled in the art, wherein particularly preferred promoters include any promoter sequences capable of increasing expression of proteins in desired fungal cells. In a related embodiment, the cassette may further comprise a downstream (3′) transcription terminator sequence operably linked to the gene CDS.

[0196] As used herein, promoter and / or terminator sequences are not intended to be restrictive, but are selected so as to work in the desired fungal cell / strain. For example, the promoter sequence can be any nucleotide sequence that shows transcriptional activity in the filamentous fungal cell, including mutant / variant promoters, truncated promoters, tandem promoters, hybrid promoters, synthetic promoters, inducible promoters, tuned promoters, conditional expression systems and combinations thereof. Typically, suitable promoters can be obtained from genes encoding extracellular or intracellular polypeptides that are natural or heterologous (exogenous) to the filamentous fungal cell. Examples of promoters suitable for driving expression of one or more regulated genes of the present disclosure include, but are not limited to, the Trichoderma reesei cDNA1 promoter, the eno1 promoter, the pdc1 promoter, the pki1 promoter, the tef1 promoter, the rp2 promoter, the cbh1 promoter, the cbh2 promoter, the egl1 promoter, the egl2 promoter, and other Trichoderma reesei promoters described in Fitz et al. 2018 (incorporated herein by reference in its entirety), the Aspergillus oryzae thiA promoter, the amylase promoter, the Aspergillus nidulans gpdA promoter, the Aspergillus niger glaA promoter, and the like.

[0197] 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. 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.

[0198] In some embodiments, a heterologous gene is cloned into an intermediate vector and then transformed into a filamentous fungal (host) cell for expression. These intermediate vectors can be 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 expressing the heterologous sequence. For example, a typical expression cassette contains a 5′ promoter operably linked to a heterologous nucleic acid sequence encoding a POI and may further contain sequence signals required for efficient polyadenylation of the transcript, ribosome binding, and translation termination. Additional elements of the cassette can include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

[0199] In addition to the promoter sequence, the expression cassette can also contain a transcription termination region downstream of the structural gene to provide efficient termination. This termination region can be obtained from the same gene as the promoter sequence or can be obtained from a different gene. Although any fungal terminator may function in the present invention, preferred terminators include: the terminator from the Trichoderma cbhI gene, the terminator from the Aspergillus nidulans trpC gene, the glucoamylase gene of Aspergillus awamori or Aspergillus niger, and / or the carboxyl protease gene of Mucor miehei.

[0200] The particular expression vector used to deliver genetic information into the cell is not particularly critical. Any of the conventional vectors 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 yeast centromere plasmids. Epitope tags (e.g., c-myc) can also be added to the recombinant protein to provide a convenient method of isolation.

[0201] Elements that can be included in the expression vector can also be a replicon, a gene encoding antibiotic resistance that allows selection of bacteria carrying the recombinant plasmid, or unique restriction sites in the non-essential region of the plasmid that allow insertion of the heterologous sequence. The particular antibiotic resistance gene selected is not decisive, as any of the many resistance genes known in the art can be suitable. Prokaryotic sequences are preferably selected such that they do not interfere with the replication or integration of the DNA in the fungal host.

[0202] The transformation methods of the present disclosure can result in the stable integration of all or part of the transformation vector into the genome of filamentous fungi. However, transformation resulting in episomal transformation vectors that maintain self-replication is also contemplated. Any known procedures for introducing exogenous (heterologous) nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistic methods, liposomes, microinjection, and any other known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other heterologous genetic material into host cells (see, e.g., Sambrook et al., supra). Agrobacterium-mediated transfection methods are also used, such as the transfection methods described in U.S. Patent No. 6,255,115.

[0203] After introducing one or more expression vectors into the cells, the transformed cells are cultured under conditions conducive to the expression of the gene under the control of the particular gene promoter used, which is selected based on optimal activity under the appropriate fermentation conditions chosen. Large batches of transformed cells can be cultured as described herein. Finally, the product is recovered from the culture using standard techniques.

[0204] As further described and illustrated below (Example 4), the applicant screened suitable gene / protein sequence databases and identified several filamentous genes encoding homologs of the GIIα, Mds1, and Mds2 proteins. By way of example, in Figure 5 the Trichoderma reesei GIIα protein (SEQ ID NO:6), the Aspergillus niger GIIα protein homolog (SEQ ID NO:19; strain ATCC No. 1015), and the Thermus thermophilus GIIα protein homolog (SEQ ID NO:25; strain ATCC No. 42464) are presented.

[0205] Similarly, in Figure 7 the Trichoderma reesei Mds1 protein (SEQ ID NO:2), the Aspergillus niger Mds1 protein homolog (SEQ ID NO:21; strain ATCC No. 1015), and the Thermus thermophilus Mds1 protein homolog (SEQ ID NO:27; strain ATCC No. 42464) are presented, and in Figure 9 the Trichoderma reesei Mds2 protein (SEQ ID NO:4), the Aspergillus niger Mds2 protein homolog (SEQ ID NO:23; strain ATCC No. 1015), and the Thermus thermophilus Mds2 protein homolog (SEQ ID NO:29; strain ATCC No. 42464) are presented.

[0206] More particularly, as described in Example 4 (see Figure 6 , Figure 8 andFigure 10 ), the Aspergillus niger and Thermus thermophilus GIIα, Mds1, and Mds2 protein homologs have significant sequence homology with the Trichoderma reesei GIIα, Mds1, and Mds2 proteins, respectively. For example, the Aspergillus niger genes encoding the Mds1 (SEQ ID NO:21) and Mds2 (SEQ ID NO:23) protein homologs have approximately 51% and 60% amino acid sequence identity with the Trichoderma reesei Mds1 (SEQ ID NO:2) and Mds2 (SEQ ID NO:4) proteins, respectively.

[0207] Accordingly, in one or more embodiments of the present disclosure, by referring to the specification, Examples 1 to 4, and Figures 5 to 11 , one of ordinary skill in the art can readily construct the modified filamentous fungal strains lacking one or more mannosidases as described and set forth herein. For example, as Figure 11 generally shown, the Trichoderma reesei Mds1 protein comprises a total of 523 amino acid residues, wherein the amino acid residues from about position 43 to position 511 of SEQ ID NO:2 are represented by bold residues. In particular, the amino acid positions from about position 43 to about position 511 listed in SEQ ID NO:2 ( Figure 11 ) comprise a glycosyl hydrolase family 47 (GH47) sequence domain, wherein members of this family are α-mannosidases that catalyze the hydrolysis of terminal 1,2-linked α-D-mannose residues.

[0208] Similarly, as Figure 11 shown, the Trichoderma reesei Mds2 protein comprises a total of 794 amino acid residues, wherein the amino acid residues from about position 39 to position 286 of SEQ ID NO:4 are represented by underlined residues, and the amino acid residue positions from about position 292 to about position 773 of SEQ ID NO:4 are represented by bold residues. More particularly, as Figure 11 (SEQ ID NO:4) shown, the amino acid positions from about position 39 to about position 286 comprise an N-terminal glycosyl hydrolase family 92 (GH92) sequence domain, and the amino acid positions from about position 292 to about position 773 comprise a glycosyl hydrolase family 92 sequence domain, wherein members of this family are α-1,2-mannosidases, which are enzymes that remove α-1,2-linked mannose residues.

[0209] In certain embodiments, genes or gene homologs encoding mannosidases (which have amino acid sequence homology to the Mds1 protein of SEQ ID NO:2) are modified herein to render the strain lacking the production of the Mds1 protein (or its homolog), and / or genes or gene homologs encoding mannosidases (which have amino acid sequence homology to the Mds2 protein of SEQ ID NO:4) are modified herein to render the strain lacking the production of the Mds2 protein (or its homolog).

[0210] In certain embodiments, one or more genes (or genetic elements, such as promoters, gene coding sequences, 5'-UTRs, terminators, etc.) encoding one or more mannosidases are genetically modified, for example, by (a) introducing, substituting or removing one or more nucleotides in the gene, or introducing, substituting or removing one or more nucleotides in the regulatory elements required for transcription or translation of the gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) site-directed mutagenesis and / or (h) random mutagenesis of any one or several genes disclosed herein, etc. For example, those skilled in the art can mutagenize, disrupt, delete or interfere with (e.g., RNAi) a part of the gene encoding the GH47 (family) sequence domain of the Mds1 protein ( Figure 11 , bold residues; or the entire GH47 domain), thereby reducing or completely eliminating the production of the functional Mds1 protein. Similarly, those skilled in the art can mutagenize, disrupt, delete or interfere with a part of the gene encoding the GH92 (family) N-terminal sequence domain of the Mds2 protein ( Figure 11 , underlined residues; or the entire GH92 N-terminal sequence domain) and / or the GH92 (superfamily) sequence domain ( Figure 11 , bold residues) of the gene, thereby reducing or completely eliminating the production of the functional Mds2 protein. In other embodiments, those skilled in the art can mutagenize, disrupt, delete, etc. a part of the gene encoding the (GH47; glycoside hydrolase) active site of the Mds1 protein and / or the (GH92; glycoside hydrolase) active site of the Mds2 protein, thereby reducing or completely eliminating the production of the functional Mds1 and / or Mds2 proteins.

[0211] IV. Target Protein

[0212] As described above, certain embodiments relate to genetically modified mutant fungal cells and / or modified (recombinant) fungal cells comprising a gene encoding a protein of interest (POI). More particularly, certain embodiments relate to compositions and methods for expressing / producing such a protein of interest in the modified (mutant) fungal cells of the present disclosure. Thus, in certain embodiments, the recombinant fungal cells produce increased amounts of a protein of interest, including but not limited to enzymes, antibodies, receptor proteins, animal feed proteins, human food proteins, protein biologics, and the like.

[0213] In certain embodiments, the protein of interest is encoded / expressed / produced by an endogenous filamentous fungal gene (e.g., for example, 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 comprises a glycosylated protein (glycoprotein).

[0214] In certain embodiments, the protein of interest is an enzyme selected from the group consisting of: cellulase, hemicellulase, xylanase, peroxidase, protease, lipase, phospholipase, esterase, cutinase, polyesterase, phytase, pectinase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, mannanase, α-glucanase, β-glucanase, hyaluronidase, chondroitinase, laccase, amylase, glucoamylase, acetyl esterase, aminopeptidase, arabinanase, arabinosidase, arabinofuranosidase, carboxypeptidase, catalase, nuclease, deoxyribonuclease, ribonuclease, epimerase, α-galactosidase, β-galactosidase, glucan lyase, endo-β-glucanase, glucose oxidase, glucuronidase, invertase, and isomerase.

[0215] In certain embodiments, the POI is selected from Enzyme Commission (EC) numbers, the Enzyme Commission (EC) numbers being selected from the group consisting of: EC 1, EC 2, EC 3, EC 4, EC 5, and EC 6.

[0216] The optimal conditions for producing a protein will vary with the choice of host cell and the choice of one or more proteins to be expressed. Such conditions can be readily determined by those skilled in the art through routine experimentation and / or optimization.

[0217] The target protein can be purified or isolated after expression. The target protein can be isolated or purified in a variety of ways known to those skilled in the art, depending on the other components present in the sample. Standard purification methods include, but are not limited to, electrophoresis, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse phase HPLC chromatography and chromatofocusing. For example, the target protein can be purified using a standard anti-target protein antibody column. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. The degree of purification required will vary depending on the intended use of the target protein. In some cases, protein purification is not required.

[0218] In some other embodiments, various screening methods can be performed to confirm that the genetically modified fungal cells of the present disclosure produce increased levels of the target protein. In some embodiments, the target protein 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 with the target protein that serves as a detectable tag, or the target protein itself can serve as a selectable or screenable marker. The labeled protein can be detected via Western blot, dot blot (a method available from the Cold Spring Harbor Protocols website), ELISA, or (if labeled with GFP) whole cell fluorescence and / or FACS. For example, a 6-histidine tag will be included as a fusion with the target protein and the tag will be detected by Western blot. If the target protein is expressed at a high enough level, SDS-PAGE combined with Coomassie / silver staining can be performed to detect the increased expression of variant host cells compared to parental (control) cells, in which case no labeling is required. Additionally, other methods can be used to confirm the improved level of the target protein, e.g., HPLC methods for protein separation or standard total protein measurements based on Coomassie blue or BCA reagents to detect an increase in the amount of protein per cell or per milliliter of fermentation medium.

[0219] The detection of specific productivity is another method for evaluating protein production. Specific productivity (Qp) can be determined by the following formula:

[0220] Qp = gP / gDCW·hr

[0221] 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 in hours starting from the inoculation time, which includes the production time as well as the growth time. Finally, if the target protein has enzymatic activity, its expression level can be calculated by enzymatic assay.

[0222] In some other embodiments, the modified filamentous fungal cells exhibit an increased total protein yield, where the total protein yield is defined as the amount of protein (g) produced per gram of carbohydrate feed relative to the (unmodified) parental strain. Thus, as used herein, the total protein yield (g / g) can be calculated using the following equation:

[0223] Yf = Tp / Tc

[0224] where "Yf" is the total protein yield (g / g), "Tp" is the total protein produced during fermentation (g), and "Tc" is the total carbohydrate fed during fermentation (bioreactor) operation (g). In some embodiments, the increase in total protein yield of the modified strain (i.e., relative 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 (parental) cells.

[0225] The total protein yield can also be described as carbon conversion efficiency / carbon yield, e.g., in terms of the percentage (%) of the fed carbon incorporated into the total protein. Thus, in some embodiments, the modified filamentous fungal cells have an increased carbon conversion efficiency (e.g., an increase in the percentage (%) of the fed carbon incorporated into the total protein) relative to the (unmodified) parental strain. In some embodiments, the increase in carbon conversion efficiency of the modified strain (i.e., relative 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 (parental) cells.

[0226] V. Fermentation

[0227] Certain embodiments relate to compositions and methods for producing a target protein, the methods including growing, culturing, or fermenting the modified (mutant) filamentous fungal cells of the present disclosure. Generally, fermentation methods well 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 where the composition of the medium is set at the beginning of fermentation and does not change during fermentation. At the start of fermentation, the medium is inoculated with the desired organism. In this method, fermentation can proceed without adding any components to the system. Typically, batch fermentation qualifies as a "batch" with respect to the addition of nutrients while controlling factors such as pH and oxygen concentration. The broth and culture composition of the batch system change continuously until the time of fermentation cessation. In batch culture, cells progress through a static lag phase to a high-growth logarithmic phase and finally into a stationary phase where the growth rate decreases or stops. If not treated, the cells will apoptose and eventually die. Generally, during the batch phase, most of the product is produced during the logarithmic phase.

[0228] A suitable variant of the standard batch system is the "fed-batch fermentation" system. In this variant of the typical batch system, as fermentation progresses, after the logarithmic phase is completed, the substrate is added incrementally. Fed-batch systems are commonly used to avoid catabolite repression. Continuous feeding of the substrate enables the process to keep its concentration below the critical level that might cause inhibition of cell metabolism and protein production. Batch and fed-batch fermentations are commonly used and well known in the art.

[0229] 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 removed simultaneously for processing. Continuous fermentation typically maintains the culture at a constant (high) density where the cells mainly remain in the logarithmic phase of growth. In other systems, many factors affecting growth can be continuously changed while the cell concentration measured by the turbidity of the medium remains constant. Continuous systems strive to maintain steady-state growth conditions. Therefore, the cell loss caused by the withdrawal of the medium should be balanced with the cell growth rate in fermentation. Methods for regulating nutrients and growth factors for continuous fermentation processes and techniques for maximizing the product formation rate are well known in the field of industrial microbiology.

[0230] Certain embodiments of the present disclosure relate to fermentation procedures for culturing fungi. Fermentation procedures for producing cellulase are known in the art. For example, cellulase can be produced by solid-state culture or submerged culture (including batch, fed-batch, and continuous processes). The culture is generally completed in a growth medium that contains an aqueous mineral salt medium, organic growth factors, a carbon source and an energy source material, molecular oxygen, and of course, an initial inoculum of the filamentous fungal host to be used.

[0231] In addition to the carbon and energy sources, oxygen, assimilable nitrogen and the microbial inoculum, it is necessary to supply appropriate amounts of mineral nutrients in the right proportions to ensure proper microbial growth, maximize the assimilation of the carbon and energy sources by the cells during microbial conversion, and obtain maximum cell yield and maximum cell density in the fermentation medium.

[0232] The composition of the aqueous mineral medium can vary within wide limits, depending in part on the microorganisms and substrates used, as is known in the art. In addition to nitrogen, the mineral medium should also include appropriate amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium in suitable soluble, assimilable ionic forms and combinations, and certain trace elements (such as copper, manganese, molybdenum, zinc, iron, boron, and iodine) in suitable soluble, assimilable forms, all as is known in the art.

[0233] The fermentation process can be an aerobic process in which the required molecular oxygen is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, so long as the contents of the fermentation vessel are maintained at a suitable oxygen partial pressure effective to aid the growth of the microbial species in a vigorous manner.

[0234] Fermentation temperatures may vary somewhat, but for filamentous fungi such as Trichoderma reesei, the temperature will generally be in the range of about 20°C to 40°C, with a range of about 25°C to 34°C often being preferred.

[0235] Microorganism also needs assimilable nitrogen source. Assimilable nitrogen source can be any nitrogenous compound or can release the nitrogenous compound of the form that is suitable for microorganism to carry out metabolic utilization. Although can adopt multiple organic nitrogen source compound such as protein hydrolysate, can utilize cheap nitrogenous compound usually, such as ammonia, ammonium hydroxide, urea and multiple ammonium salt (such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride or multiple other ammonia compound). Ammonia itself is convenient to large-scale operation, and can pass through aqueous fermentation product (fermentation medium) and use by bubbling with suitable amount. Simultaneously, can also adopt such ammonia to help carry out pH control.

[0236] The pH range in aqueous microbial fermentations should be within the exemplary range of about 2.0 to 10.0. In the case of filamentous fungi, the pH is typically within the range of about 2.5 to 8.0; in the case of Trichoderma reesei, the pH is typically within the range of about 3.0 to 7.0. The preferred pH range for microorganisms depends to some extent on the culture medium employed and the particular microorganism, and therefore can be adjusted to some extent, as can be readily determined by one skilled in the art.

[0237] Preferably, the fermentation is carried out in such a way that the carbon-containing substrate can be controlled as a limiting factor, thus providing a good conversion of the carbon-containing substrate to the product and avoiding contamination of the cells with large amounts of unconverted substrate. The latter is not a problem for water-soluble substrates, since any remaining trace amounts can be easily washed away. However, in the case of water-insoluble substrates this may be a problem and additional product handling steps such as suitable washing steps are required.

[0238] As mentioned above, the time to reach this level is not critical and can vary with the particular microorganism and the fermentation process carried out. However, it is well known in the art how to determine the concentration of the carbon source in the fermentation medium and whether the desired level of the carbon source has been reached.

[0239] The fermentation can be carried out in batch or continuous operation. For ease of control, production of a uniform amount of product and most economical use of all equipment, fed-batch operation is more preferred.

[0240] If desired, part or all of the carbon source and energy source material and / or part of the assimilable nitrogen source (such as ammonia) can be added to the aqueous mineral medium before feeding the aqueous mineral medium into the fermenter.

[0241] Preferably at a predetermined rate, or in response to requirements that can be determined by monitoring, such as the concentration of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gas from the fermenter, cell density measurable by dry cell weight, light transmittance, etc., each feed stream introduced into the reactor is controlled. The feeding rates of the various materials can be varied to obtain the maximum production rate and / or the maximum yield.

[0242] In batch operation or preferably fed-batch operation, all equipment, reactors or fermentation devices, vessels or containers, pipes, attached circulation or cooling equipment, etc. are sterilized at the beginning, usually by using steam such as at about 121 °C for at least about 15 minutes. Then, in the presence of all required nutrients, including oxygen and the carbon-containing substrate, the sterilized reactor is inoculated with a culture of the selected microorganism. The type of fermenter used is not important.

[0243] VI. Broth conditioning and protein recovery process

[0244] As outlined above, certain embodiments of the present disclosure relate to culturing (fermenting) filamentous fungal cells to express / produce / secrete a protein of interest (or glycoprotein). Accordingly, certain embodiments relate to a fermentation broth obtained by fermenting filamentous fungal cells that express and secrete proteins in a broth. In certain additional one or more embodiments, one or more protein recovery processes are performed on the fermentation broth containing the glycoprotein of interest. In certain embodiments, the present disclosure provides methods for recovering proteins from a fungal cell fermentation broth, such as obtaining and collecting a filamentous fungal cell fermentation broth containing the protein of interest, performing a filtration process to remove the fungal cells, performing a diafiltration process to reduce the free sugar level, subjecting the broth to heat treatment at about 40 °C for a sufficient amount of time, recovering the protein from the broth, etc., wherein the recovered protein contains a reduced level of glycosylation. In related embodiments, the protein expressed, secreted, and recovered from the broth maintains a high level of enzyme activity and / or has enhanced thermal stability during storage at about room temperature (e.g., about 20 °C to 22 °C).

[0245] More particularly, as described herein, the protein is expressed and secreted into the fermentation broth, wherein one or more protein recovery processes (steps), such as a cell separation process, a protein concentration process, a protein purification process, etc., are performed on the end of fermentation (EOF) broth. Proteins can be recovered from the fermentation broth by procedures known to those skilled in the art to obtain the desired protein preparation. For example, the broth typically contains cell debris (including cells, various suspended solids, and other biomass contaminants) as well as the desired target protein. Those skilled in the art know 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 filtrates. The term "cell separation" or "cell separation process" is not intended to be limiting and includes any cell separation and / or broth clarification methods known to those skilled in the art.

[0246] Similarly, the term "concentration" or "concentration process" is not intended to be limiting and includes concentration methods known to those skilled in the art, such as ultrafiltration, evaporation, centrifugation, etc. Techniques such as ultrafiltration, evaporation, or precipitation can be preferably used to further concentrate the fermentation broth or cell-free filtrate before crystallization. Precipitation of the protein component of the supernatant or filtrate can be accomplished by means of a salt (e.g., ammonium sulfate), followed by purification through various chromatographic procedures (e.g., ion exchange chromatography, affinity chromatography, or similar well-recognized procedures in the art).

[0247] Thus, as generally described above, methods known to those of ordinary skill in the art (e.g., well-recognized separation techniques in the art 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 gradient separation) can be used to recover, purify, enrich, etc., protein formulations, glycoprotein formulations, etc. according to the present disclosure to remove unwanted whole cells, cell debris, impurities, foreign proteins, or enzymes in the final composition. Then, ingredients that provide additional benefits, such as activators, anti-inhibitors, desired ions, pH-controlling compounds, or other enzymes or chemicals, can be further added to the purified or isolated biomolecular composition.

[0248] Thus, in one or more preferred embodiments of the present disclosure, a fermentation broth containing one or more target proteins is collected. In related embodiments, the fermentation terminal broth is collected (harvested) and subjected to one or more recovery processes, which include at least one heat treatment step. In certain embodiments, the heat treatment process is carried out for at least about one (1) hour to about five (5) hours. In related embodiments, the heat treatment process is carried out at a temperature between about 38.5°C and about 41.5°C. In certain additional one or more embodiments, the heat treatment process includes heat-treating the broth at 40°C for about four (4) hours, and then collecting the broth. In certain embodiments, the broth can be cooled to about room temperature (20°C) or lower.

[0249] VII. Exemplary Embodiments

[0250] Non-limiting embodiments of the present disclosure include, but are not limited to:

[0251] 1. A modified filamentous fungal cell derived from a parental cell, the parental cell containing an endogenous gene encoding a functional α-mannosidase protein, wherein the modified cell contains a genetic modification that causes the cell to lack the production of the functional α-mannosidase protein.

[0252] 2. A modified filamentous fungal cell derived from a parental cell, the parental cell containing endogenous genes encoding at least two functional α-mannosidase proteins, wherein the modified cell contains a genetic modification that causes the cell to lack the production of the at least two functional α-mannosidase proteins.

[0253] 3. The modified cell as described in Example 1 or Example 2, wherein one or more endogenous genes encode a functional α-mannosidase protein, and the functional α-mannosidase protein has at least about 50% to 100% identity with 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.

[0254] 4. The modified cell as described in any one of Examples 1 to 3, the modified cell comprising one or more endogenous genes encoding one or more target proteins and / or comprising one or more heterologous genes encoding one or more heterologous target proteins.

[0255] 5. The modified cell as described in Example 1 or Example 2, wherein the functional α-mannosidase protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:21, and SEQ ID NO:27 comprises a glycoside hydrolase family 47 (GH47) sequence domain.

[0256] 6. The modified cell as described in Example 1 or Example 2, wherein the functional α-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 glycoside hydrolase family 92 (GH92) sequence domain and a GH92 superfamily sequence domain.

[0257] 7. The modified cell as described in Example 1 or Example 2, wherein the genetic modification that causes the cell to lack the production of a functional α-mannosidase protein is selected from the group consisting of: (a) introducing, substituting, or removing one or more nucleotides in the gene encoding the functional α-mannosidase protein, and / or introducing, substituting, or removing one or more nucleotides in the regulatory elements required for the transcription or translation of the gene encoding the functional α-mannosidase protein, (b) α-mannosidase gene disruption, (c) α-mannosidase gene conversion, (d) α-mannosidase gene deletion, and (e) downregulation of the α-mannosidase gene.

[0258] 8. The modified cell as described in Example 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.

[0259] 9. The modified cell of embodiment 4, which is fermented under suitable conditions to produce one or more proteins and secrete them into the fermentation broth.

[0260] 10. The modified cell of embodiment 9, wherein the end-of-fermentation (EOF) broth is collected and stored at room temperature for at least 4 hours to about 5 days.

[0261] 11. The modified cell of embodiment 10, wherein the amount of mannose (Man) contained in the stored broth is reduced relative to the amount of Man sugar present in the EOF broth of the parental cells when collected and stored under the same conditions.

[0262] 12. The modified cell of embodiment 9, wherein the end-of-fermentation (EOF) broth is subjected to a heat treatment step, wherein the heat-treated broth comprises a reduced amount of mannose (Man) relative to the amount of Man sugar present in the EOF broth of parental cells producing the same one or more proteins when fermented and heat-treated under the same conditions as the modified cells.

[0263] 13. The modified cell of any one of embodiments 9 to 12, wherein the one or more proteins are recovered from the broth.

[0264] 14. The modified cell of any one of embodiments 9 to 12, wherein the modified cell is fermented under the same conditions as the parent cell for at least about 96 to about 300 hours.

[0265] 15. The modified cell of any one of embodiments 1 to 14, selected from the group consisting of: an Aspergillus species cell, a Ciliate Sporangium species cell, a Fusarium species cell, a Humicola species cell, a Mucor species cell, a Myceliophthora species cell, a Neurospora species cell, a Penicillium species cell, a Cylindrotheca species cell, a Thielavia species cell, a Tolypocladium species cell, and a Trichoderma species cell.

[0266] 16. The modified cell of any one of embodiments 1 to 14, wherein the parent cell is a Trichoderma reesei cell comprising a gene encoding a truncated glucosidase II α-subunit (GIIα Stop ) protein mutation glucosidase II α (gls2a Stop ) allele, wherein the modified Trichoderma reesei cell comprises a restored glucosidase IIα (gls2a) encoding a native glucosidase IIα-subunit (GIIα) protein R ) allele.

[0267] 17. The modified cell of embodiment 16, wherein the parent cell is a strain of Trichoderma reesei selected from the group consisting of Rut-C30, RL-P37, NG14, or a strain derived therefrom comprising a mutation in gls2a Stop ancestral strain of the allele.

[0268] 18. The modified cell of embodiment 13, wherein the one or more proteins of interest produced comprise a homogeneous 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, wherein the modified cell is fermented under the same conditions as the parent cell, and the one or more proteins of interest are recovered under the same conditions.

[0269] 19. The modified cell of embodiment 4, wherein the one or more endogenous or heterologous genes encode a protein selected from the group consisting of a lipase, a glucoamylase, and a phytase.

[0270] 20. The modified cell of embodiment 16, wherein the modified cell produces an increased amount of the protein relative to the parent cell when fermented under the same conditions used to produce the protein.

[0271] 21. A modified Trichoderma reesei cell derived from a parent Trichoderma reesei cell, the parent Trichoderma reesei cell comprising a gene encoding a truncated glucosidase II α-subunit (GIIα Stop ) protein mutation glucosidase II α (gls2a Stop ) allele, wherein the modified cell comprises a restored glucosidase IIα (gls2a) encoding a native glucosidase IIα-subunit (GIIα) R ) allele.

[0272] 22. The modified cell of embodiment 21, wherein the modified cell produces one or more endogenous proteins of interest and / or produces one or more heterologous proteins of interest.

[0273] 23. The modified cell of embodiment 22, wherein the one or more proteins of interest are glycoproteins.

[0274] 24. The modified cell of embodiment 23, wherein the one or more proteins of interest comprise a homogeneous 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 a parent cell, wherein the modified cell is fermented under the same conditions as the parent cell and the one or more proteins of interest are recovered under the same conditions.

[0275] 25. A modified cell as described in Example 22, wherein when fermented under the same conditions for producing a target protein, the modified cell produces an increased amount of the target protein relative to the parental cell.

[0276] 26. A modified cell as described in Example 21, wherein the parental cell is a Trichoderma reesei strain selected from the group consisting of Rut-C30, RL-P37, NG14 strain, or an ancestral strain derived therefrom and containing the mutant gls2a Stop allele.

[0277] 27. A modified cell as described in Example 21, wherein the modified cell further comprises a genetic modification that causes the cell to lack the production of a functional α-mannosidase protein, or comprises a genetic modification that causes the cell to lack the production of at least two functional α-mannosidase proteins.

[0278] 28. A modified cell as described in Example 27, wherein the modified cell is fermented under suitable conditions to produce the one or more proteins and secrete them into the fermentation broth.

[0279] 29. A modified cell as described in Example 28, wherein the fermentation end point (EOF) broth is collected and stored at room temperature for at least 4 hours to about 5 days.

[0280] 30. A modified cell as described in Example 29, wherein the amount of mannose (Man) contained in the stored broth is reduced relative to the amount of Man sugar present in the EOF broth of the parental cell when collected and stored under the same conditions.

[0281] 31. A modified cell as described in Example 28, wherein a heat treatment step is performed on the fermentation end point (EOF) broth, and the amount of mannose (Man) contained in the heat-treated broth is reduced relative to the amount of Man sugar present in the EOF broth of the parental cell that produces the same one or more proteins when fermented and heat-treated under the same conditions as the modified cell.

[0282] 32. A modified cell as described in Example 31, wherein the one or more proteins are recovered from the treated broth.

[0283] 33. A modified cell as described in Example 28, wherein the modified cell and the parental cell are fermented under the same conditions for at least about 96 hours to about 300 hours.

[0284] 34. A modified cell as described in Example 22, wherein the one or more target proteins are selected from the group consisting of lipase, glucoamylase, and phytase.

[0285] 35. A method for fermenting filamentous fungal cells for producing and recovering a protein of interest (POI) in a fermentation broth containing a reduced amount of mannose (Man), the method comprising: obtaining a parental cell comprising an endogenous gene encoding a functional α-mannosidase protein, genetically modifying the cell to lack the production of the functional α-mannosidase protein, and fermenting the modified cell under suitable conditions to produce the POI, wherein the amount of Man sugar contained in the fermentation end point (EOF) broth of the modified cell is reduced relative to the EOF broth of the parental cell fermented under the same conditions.

[0286] 36. A method for fermenting filamentous fungal cells for producing and recovering a protein of interest (POI) in a fermentation broth containing a reduced amount of mannose (Man), the method comprising: obtaining a parental cell comprising at least two endogenous genes encoding at least two functional α-mannosidase proteins, genetically modifying the cell to lack the production of the at least two functional α-mannosidase proteins, and fermenting the modified cell under suitable conditions to produce the POI, wherein the amount of Man sugar contained in the fermentation end point (EOF) broth of the modified cell is reduced relative to the EOF broth of the parental cell fermented under the same conditions.

[0287] 37. The method according to embodiment 35, wherein the endogenous gene encodes a functional α-mannosidase protein that has at least about 50% to 100% identity with 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.

[0288] 38. The method according to embodiment 36, wherein the at least two endogenous genes encode functional α-mannosidase proteins that have at least about 50% to 100% identity with 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.

[0289] 39. The method according to embodiment 35 or embodiment 36, wherein the produced POI is secreted into the fermentation broth.

[0290] 40. The method of embodiment 35 or embodiment 36, wherein the filamentous fungal cell is selected from the group consisting of: an Aspergillus species cell, a Ciliate Sporangium species cell, a Fusarium species cell, a Humicola species cell, a Mucor species cell, a Myceliophthora species cell, a Neurospora species cell, a Penicillium species cell, a Cylindrotheca species cell, a Thielavia species cell, a Tolypocladium species cell, and a Trichoderma species cell.

[0291] 41. The method of embodiment 35 or embodiment 36, wherein the modified cells are fermented under the same conditions as the parent cells for at least about 96 hours to about 300 hours.

[0292] 42. The method of embodiment 35 or embodiment 36, wherein the EOF broth is subjected to a heat treatment process (step).

[0293] 43. The method of embodiment 42, wherein the heat-treated EOF broth from the modified cells comprises a reduced amount of mannose relative to EOF broth from the parental cells subjected to the same heat treatment process.

[0294] 44. The method of embodiment 35 or embodiment 36, wherein the parent cell is a Trichoderma reesei cell comprising a gene encoding a truncated glucosidase II α-subunit (GIIα Stop ) protein mutation glucosidase II α (gls2a Stop ) allele, wherein the modified Trichoderma reesei cell comprises a restored glucosidase IIα (gls2a) encoding a native glucosidase IIα-subunit (GIIα) protein R ) allele.

[0295] 45. The method of embodiment 44, wherein the recovered POI comprises a homogeneous N-linked glycan pattern comprising greater than about 75% Man5GlcNAc2 compared to the N-linked glycan pattern of the same POI produced by a parent cell, wherein the modified cell is fermented under the same conditions as the parent cell.

[0296] 46. The method of embodiment 44, wherein the modified cell produces an increased amount of POI relative to the parent cell when fermented under the same conditions used to produce the POI.

[0297] 47. The method of embodiment 44, wherein the parent cell is a strain of Trichoderma reesei selected from the group consisting of Rut-C30, RL-P37, NG14, or a strain derived therefrom comprising a mutation in gls2a Stop ancestral strain of the allele.

[0298] 48. A method for producing a protein of interest (POI) that comprises a homogenous N-linked glycan pattern, the method comprising (a) obtaining Trichoderma reesei cells that comprise a mutant glucosidase II α-subunit (GIIα Stop ) protein-encoding glucosidase II α (gls2a Stop ) allele, and genetically modifying the cells to express and encode a restored glucosidase II α (gls2a R ) allele that encodes the native glucosidase II α-subunit (GIIα), and fermenting the modified cells under suitable conditions to produce and secrete the POI, and recovering the POI from the fermentation broth, wherein the recovered POI comprises a homogenous N-linked glycan pattern that comprises greater than 75% Man5GlcNAc2 as compared to the N-linked glycan pattern of the same POI produced by the parental cells, wherein the modified cells are fermented under the same conditions as the parental cells, and the POI is recovered under the same conditions.

[0299] 49. The method of embodiment 48, wherein when fermented under the same conditions for producing the POI, the modified cells produce an increased amount of the POI relative to the parental cells.

[0300] 50. The method of embodiment 48, wherein the parental cells are Trichoderma reesei strains selected from the group consisting of: Rut-C30, RL-P37, NG14 strain, or an ancestral strain derived therefrom that comprises the mutant gls2a Stop allele.

[0301] 51. The method of embodiment 48, the method further comprising a genetic modification that renders the cells devoid of production of a functional α-mannosidase protein, or comprising a genetic modification that renders the cells devoid of production of at least two functional α-mannosidase proteins.

[0302] 52. The method of embodiment 48, wherein the modified cells secrete the POI into the fermentation broth.

[0303] 53. The method of embodiment 52, wherein the POI is recovered from the broth.

[0304] 54. The method of embodiment 48, wherein the modified cells are fermented for at least about 96 hours to about 300 hours under the same conditions as the parental cells.

[0305] 55. The method of any one of embodiments 35, 36, or 48, wherein the cells comprise one or more introduced expression constructs that encode the one or more proteins of interest.

[0306] 58. The method according to Example 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.

[0307] Example

[0308] Certain embodiments of the present disclosure can be further understood from the following examples, which should not be construed as limiting. Modifications to the 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).

[0309] Example 1

[0310] Genetic modification of recombinant fungal cells expressing heterologous glycoproteins

[0311] As outlined above, the Trichoderma strains described in this example can be derived from publicly available strains (e.g., RutC-30, RLP37, etc.), which are well-suited for expressing / producing endogenous cellulases and heterologous (recombinant) proteins of interest. More particularly, certain Trichoderma reesei strains of this example are derived from the Trichoderma reesei strain RLP37 after deletion of four native cellulase genes (cbh1, cbh2, egl1, egl2) (i.e., containing the mutant (frameshift) glsa2 Stop allele), as outlined in PCT Publication No. WO 2005 / 001036, which is incorporated herein by reference. Thus, in certain embodiments, the applicant designed and constructed recombinant Trichoderma reesei cells / strains (e.g., strain RLP37) to express heterologous glycoproteins, including phytase (glycoprotein) reporter proteins, glucoamylase (glycoprotein) reporter proteins, and lipase (glycoprotein) reporter glycoproteins. More particularly, any suitable protein can be expressed / produced in one or more recombinant Trichoderma reesei cells of the present disclosure and further tested, screened, assayed, etc., as outlined and described herein. Thus, in certain embodiments of the present disclosure, the applicant screened three (3) exemplary glycoproteins in the parental Trichoderma reesei cells.

[0312] In certain embodiments, the applicant used a parental Trichoderma reesei strain (Phy) containing the mutant glsa2 Stop allele and a modified Trichoderma reesei strain (Phy-gls2a) that produces the same heterologous phytase reporter protein and contains the restored glsa2 R allele R ​) Heterologous phytase reporter proteins were screened. In other embodiments, the applicant screened the same heterologous phytase reporter proteins in a modified Trichoderma reesei strain (Phy-Δmds2) containing a deleted mds2 allele. In other embodiments, the applicant screened the same heterologous phytase reporter proteins in a parental Trichoderma reesei strain (GA-glsa2 Stop ) containing a mutant glsa2 Stop allele and a modified Trichoderma reesei strain (GA-gls2a R ) that produces the same heterologous glucoamylase reporter protein and contains a restored glsa2 R allele. In still other embodiments, the applicant screened heterologous lipase reporter proteins in a parental Trichoderma reesei strain (Lip) and a modified Trichoderma reesei strain (Lip-Δmds1) containing a deleted mds1 allele.

[0313] More particularly, such heterologous phytase protein sequences and the genes encoding them are well known in the art, including but not limited to the phytase and phytase variant sequences described in PCT Publication No. WO 2008 / 097619, PCT Publication No. WO 2009 / 129489, and PCT Publication No. WO2013 / 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, including but not limited to the glucoamylase and glucoamylase variant sequences described in PCT Publication No. WO 2021 / 212095 (incorporated herein by reference in its entirety). Similarly, such heterologous lipase reporter protein sequences and the genes encoding them are well known in the art, including but not limited to PCT Publication No. WO 2020 / 190782 (incorporated herein by reference in its entirety).

[0314] As briefly described above, in certain other embodiments, to more directly compare the data with Trichoderma reesei strains that produce endogenous cellulases (cbh1, cbh2, egl1, egl2), a parental cellulase reporter strain named "Cel" was constructed by reintroducing the cbh1, cbh2, egl1, egl2 genes encoding native cellulases as a single integrated expression cassette.

[0315] In the following examples, the parental Trichoderma reesei strain was modified as described herein, and the resulting transformant (modified) strains were screened by microtiter plate (MTP) fermentation to confirm the expression of cellulases or heterologous glycoproteins.

[0316] As described herein, all Trichoderma strains used in the following examples contain a deletion of the Endo T allele (SEQ ID NO: 36), where the Endo T allele encodes a secreted endo-N-acetyl-β-D-glucosaminidase (deglycosylating enzyme). For example, as summarized by Stals et al. (2012), the mannosyl-glycoprotein endo-N-acetyl-β-D-glucosaminidase has been shown to be the cause of the microscopic heterogeneity observed in Hypocrea jecorina cellulases and hemicellulases, where the deletion of the Endo T allele in Hypocrea jecorina eliminates this activity, thereby retaining larger N-linked glycan chains on the secreted proteins.

[0317] A. Gls2a restoration

[0318] As briefly described above, it was determined that the glsa2 allele in certain Trichoderma strains has a frameshift mutation at nucleotide position 1,965, resulting in abnormal processing of N-linked glycans. This phenotype can affect the production of recombinant glycoproteins. Based on the above, the applicant has restored the mutated (frameshift) glsa2 allele (glsa2 Stop ; SEQ ID NO: 7) to the restored glsa2 allele (gls2a R ; SEQ ID NO: 5), where 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 lignocellulose-degrading enzymes Cbh1, Cbh2, Egl1, Egl2, respectively.

[0319] In particular, Cas9 nuclease and custom synthetic guide RNA (named " RHG2 "; SEQ ID NO: 9) were obtained from Synthego (Menlo Park, California). Cas9-sgRNA complexes were assembled in vitro according to the manufacturer's protocol and used to transform Trichoderma strains, as summarized in PCT Publication No. WO 2016 / 100568 (incorporated herein in its entirety by reference). The specific editing of gls2a frameshift is achieved by comprising 200 picomoles of 90 base pair double-stranded donor DNA (SEQ ID NO: 10), which is assembled from two oligonucleotides. Transformed colonies were screened using PCR, and their products were subjected to Sanger sequencing (data not shown). While the donor fragment provides the missing base to repair the frameshift, it also includes single-base changes to further ensure that the donor fragment and the repaired gene obtained therefrom are no longer cut by Cas9. Base changes are performed at the swing position to avoid changing the protein sequence. However, in some cases, this results in transformants that only contain frameshift repair.

[0320] B.Mds2 deleted

[0321] As mentioned above, the mds2 gene was deleted to assess its effect on releasing mannose from glycoproteins, cell wall oligosaccharides and / or other substances during and after fermentation. In this example, the applicant deleted the Trichoderma reesei mds2 gene (SEQ ID NO: 3) in the above-mentioned phytase reporter strain. More particularly, Cas9 nuclease and custom synthetic guide RNA 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. Transformed colonies were screened using PCR, and their products were subjected to Sanger sequencing (data not shown). When co-transformed with selectable markers targeting other unconnected sites, mds2 deletions caused by non-homologous end joining (NHEJ) and oligomer-mediated homologous recombination (HR oligos provided in the form of single-stranded mixtures, LFP013 (SEQ ID NO: 13) and LFP014 (SEQ NO: 14) (each 100 picomoles)) were detected simultaneously.

[0322] C.Mds1 deletion

[0323] As described above, 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, the applicant deleted the Trichoderma reesei mds1 gene (SEQ ID NO:1) in the above lipase reporter strain. More particularly, Cas9 nuclease and custom guide RNAs TCg3 (SEQ ID NO:15) and TCg4 (SEQ ID NO:16) and tracrRNA were obtained from Synthego Corporation (Menlo Park, California). The Cas9-sgRNA complex was formed according to the manufacturer's protocol. Deletion of mds1 caused by non-homologous end joining (NHEJ) as well as oligomer-mediated homologous recombination (HR oligomer TC128 provided as a mixture of single-stranded oligonucleotides; SEQ ID NO:17 (100 picomoles)) was detected simultaneously when co-transformed with a selectable marker targeting other unlinked sites. Transformed colonies were screened using PCR and their products were Sanger sequenced (data not shown).

[0324] The strains constructed herein are summarized in Table 1 below.

[0325] Table 1 Trichoderma Strains Description

[0326]

[0327]

[0328] D. Protein Expression in Microtiter Plates (MTP)

[0329] The transformed and control strains were cultured as generally described in Example 3 of International Application No. PCT / US2022 / 075210 (incorporated herein by reference in its entirety), except that pre-cultivation was carried out in twenty-four (24)-well plates (CytoOne, catalog number CC7672-754) for twenty-four (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 (for five days). Ten (10) microliters of diluted filtrate were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (NuPAGE system, following the manufacturer's protocol). As Figure 3 shown (left combined detection; untreated), compared to the parental Phy strain, the phytase glycoprotein reporter protein produced by the modified Phy-gls2a R strain showed a slight downward shift in the apparent molecular weight (MW). As Figure 3As shown (right panel; Endo H treatment), the Phy parent strain and the modified Phy-gls2a were treated with endoglycosidase H (EndoH, New England Biolabs) to remove N-glycans. R There was no difference in protein band (MW) size between the strains (restored glsa2 allele). Similarly, the modified GA-gls2a was significantly larger than that of the parental GA and Cel strains. R and Cel-gls2a R SDS-PAGE analysis of the strains also showed a slight downward shift (data not shown). The shift in protein mobility indicated that the glycan chains were smaller, which was subsequently confirmed in Example 3 below.

[0330] The enzyme activity and protein titer of the MTP samples were also determined. The phytase reporter was determined using p-nitrophenyl phosphate substrate (Thermo Scientific, catalog number 34045, 10 mM sodium acetate buffer, pH 5.5). The activity was measured spectrophotometrically (absorbance at 405 nm) after the pH value was shifted with 2N NaOH. The glucoamylase (GA) reporter was determined by detecting glucose released from a maltodextrin solution (Sigma-Aldrich, catalog number 419672) in sodium acetate buffer (pH 4.3) over thirty (30) minutes. After heat inactivation, glucose was measured spectrophotometrically using a D-glucose assay kit (K-GLUC, Megazyme, Bray, Ireland). As shown in Table 2 below, compared to the parent strain (GA), the gls2a strain showed a significantly higher activity than the parent strain (GA). R Modified glucoamylase reporter strain (GA-gls2a R ) were observed to have higher activity titers, while the phytase reporter was significantly different between the parental (Phy) strain genotype and the modified (Phy-gls2a R ) strain genotypes showed no significant differences. As described below, these findings were confirmed in bioreactor experiments.

[0331] Table 2 Enzyme activities measured in MTP supernatant samples

[0332] Strain Active signal GA (parent) 0.41+ / -0.02(A510) <![CDATA[GA-gls2a R (modified) * ]]> 0.55+ / -0.09(A510) Phy (parent) 0.69+ / -0.06(A405) <![CDATA[Phy-gls2a R (modified) # > 0.67+ / -0.03(A405)

[0333] *Average of 22 isolates; # The average of 3 isolates

[0334] E. Protein Expression in Large-Scale Fermenters

[0335] As described herein, the reporter proteins are expressed in large-scale (about 14 L) bioreactors. These fermentations are carried out as generally described in PCT Publication No. WO2020 / 028126, which is incorporated herein by reference in its entirety, wherein the glucose-sophorose feed phase is carried out at 25 °C. For the Phy, Lip, and Cel reporters, the modification did not significantly alter the glycoprotein titer or yield. However, as shown below in Table 3, the restoration of gls2a in the GA reporter (GA-gls2a R ) resulted in a significant improvement in most fermentation performance metrics. In particular, as shown in Table 3, protein yield, titer, and specific productivity (Qp) were measured after about one hundred forty (about 140) hours of fermentation, wherein the modified strain (GA-gls2a R ) had an increased protein yield (62%), an increased titer (53%), and an increased Qp compared to the parental (GA) strain, respectively.

[0336] Table 3 Production of glucoamylase reporter proteins in bioreactors

[0337]

[0338] Example 2

[0339] Determination of large-scale fermentation and mannose release during glycoprotein production and recovery

[0340] As described herein, the reporter proteins are expressed in large-scale (about 14 L) bioreactors. At the end of the fermentation period (about 200 to 300 hours), the soluble proteins of the fermentation broth at the end of fermentation were isolated from the cells using standard protein recovery and processing methods known in the art, including phytase and lipase reporters. After cell separation, diafiltration was carried out using standard methods to reduce the concentration of small molecules including sugars. After this recovery process, the diafiltered samples were separated, one part was heated at 40 °C for four (4) hours and then frozen at -20 °C, while the other part of the sample was immediately frozen at -20 °C, wherein the amount of mannose in the samples before and after the final heat treatment step was measured using standard analytical procedures. In particular, the proteins were removed from the samples by precipitation with acetone. The sugars were then separated by high performance liquid chromatography (HPLC) using a Waters Xbridge column, and then quantified using a Thermo ISQ EM single quad mass spectrometer. Since genetic modifications were carried out in different parental strains (i.e., Phy, Lip, and Cel parental strains), the mannose release from each modified strain was compared to its appropriate parental (control) strain.

[0341] For example, the results of mannose release are presented below in Table 4, which shows that mutations in any of three screening genes (i.e., Δmds1, Δmds2, and gls2a R ) are particularly suitable for reducing the amount of mannose released during a recovery process (including a heating step) (e.g., by at least about 7% to 40%). Thus, as shown herein, this reduction in mannose release is particularly suitable for alleviating glycation events during downstream processing, recovery, and storage of such proteins produced in filamentous fungal strains.

[0342] Table 4

[0343] Mannose release by heat treatment after standard fermentation and recovery from parental and modified strains

[0344]

[0345] Example 3

[0346] Determination of N-glycan composition by mass spectrometry

[0347] In this example, proteins produced by Trichoderma reesei phytase and cellulase reporter strains were characterized by protein and peptide mass spectrometry. Enzyme digestion (Part A) allowed the characterization of N-glycans at specific positions in the polypeptide sequence, while intact mass spectrometry (Part B) allowed the quantitative assessment of one or more overall glycosylation states.

[0348] A. Detection of N-glycans on reporter protein peptides after enzyme digestion

[0349] The supernatant samples obtained from fermenter or microtiter plate cultures by centrifugation or filtration were prepared using the following filtration-assisted sample preparation method for mass spectrometry analysis. In a 1.7 ml Eppendorf tube, 200 μg of protein was combined with 5 μl of 0.2 M dithiothreitol and incubated in an Eppendorf thermomixer (50 °C, 300 rpm) for 30 minutes. After adding 10 μl of 440 mM iodoacetamide, the tube was incubated for another 30 minutes at room temperature in the dark. The protein was then precipitated with 0.5 ml of acetone. After centrifugation (14,000 rpm, 10 minutes), the supernatant was removed and the pellet was dried in a chemical hood for 5 minutes. The pellet was resuspended in 150 μl of 8 M urea (37 °C, 5 minutes) and then filtered using a Millipore Microcon-30 filter (Ultracel YM-30 regenerated cellulose 30,000 NMWL) at 14,000 rpm for 15 minutes. Then ammonium bicarbonate (50 mM) was applied three times, 100 μl each time, and centrifugation (14,000 rpm, 10 minutes) was performed. The collection bottle was replaced, and ammonium bicarbonate (50 mM, 40 μl) and trypsin (0.1 mg / ml, 20 μl) were applied to the filter. It was incubated at 37 °C (300 rpm) for 12 to 18 hours. The filter was washed twice more with 50 mM ammonium bicarbonate (14,000 rpm, 10 minutes each time), and the protein was eluted with 10 μl of 0.1% formic acid (14,000 rpm, 10 minutes).

[0350] The protein digest was analyzed by Ultimate 3000 Nano LC / Thermo Q-Exactive HF. The analytical column for NanoLC was a Thermo PepMap RSLC C18 column with a column size of 75 μm x 50 cm, a particle size of 2 μm, and a pore size of 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 minutes, increasing linearly to 45% B from 10 to 70 minutes; from 70.01 to 90 minutes, the B percentage increased linearly to 99% B and remained at 99% B from 90 to 95 minutes. At 95.01 minutes, the percentage returned to 4% B. The total run time was 120 minutes at a flow rate of 0.3 μl / min. MS / MS was performed on a Thermo Q-Exactive HF. Full mass scan resolution was 60,000. The scan range was 380 to 2000 m / z. The AGC target was 1e6, and the maximum IT was 150 ms. MS2 resolution was 15,000. The cycle count was top 20, NCE was 30, and the fixed first mass was 100 m / z (AGC target 1e5 and maximum IT 100 ms).Glycosylation was analyzed using BioPharma Finder 3.5 software (ThermoFisher Scientific).

[0351] The relative abundance percentage (%) data of phytase-derived peptides and cellulase-derived peptides are shown in Table 5 and Table 6, respectively. R ) resulted in nearly uniform Man5GlcNAcN-glycan content at each analyzed position. For comparison, deletion of mds2, which was evaluated for the Phy reporter protein and found to reduce mannose release during downstream processing (Example 2), produced relatively small changes in glycan composition in fermentor and MTP samples.

[0352] Table 5 Detection of post-translational glycan modifications at *ASN residues

[0353]

[0354] *Asparagine (Asn) residues are indicated by position number relative to the secreted protein.

[0355] ≠ Mannose and glucose have the same molecular mass. Glycan structures with a mass higher than Man5GlcNAc in the parental strain may correspond to the monoglucosylated species described by Geysens et al. (2005). Thus, Hex6 = GlcMan5, Hex7 = GlcMan6, and so on.

[0356] Table 6 Detection of post-translational glycan modifications at *ASN residues

[0357]

[0358] * The asparagine (Asn) residues are numbered relative to the position in the secreted protein. ≠ Mannose and glucose have the same molecular mass. Glycan structures with a mass higher than Man5GlcNAc in the parental strain may correspond to the mono-glucosylated species described by Geysens et al. (2005). Thus, Hex6 = GlcMan5, Hex7 = GlcMan6, and so on.

[0359] B. Detection of N-glycans on intact phytase polypeptide

[0360] In this example, the fermentation supernatant sample was diluted in 0.1% formic acid to achieve a concentration of less than 10 g / l. Then the sample was analyzed using a Thermo Scientific TM Vanquish liquid chromatography system equipped with a Waters BEH size exclusion chromatography column, which was connected to a Thermo Scientific TM Q-Exactive Orbitrap HF mass spectrometer equipped with an HESI ion source. Protein separation was performed using an isocratic mobile phase containing 0.1% formic acid (aqueous solution) based on the size of SEC. Mass spectrometry detection was achieved in positive ion ionization mode. The mass-to-charge ratio (m / z) scan range varied according to the charge state distribution of the target protein observed (e.g., m / z 1800 to m / z 7000). The mass spectrometry resolution was set to 30,000 for analyzing the phytase sample. Data analysis was performed using Thermo Scientific TM BioPharma Finder. As Figure 4 shown, compared with the control strain, a decrease in the change of mass distribution caused by mannose / hexose residues was found in the gls2a R fermentation sample.

[0361] Example 4

[0362] Deletion of MDS1 and MDS2 genes in other filamentous fungal cells

[0363] As outlined 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 Aspergillus niger cells and Thermomyces lanuginosus cells. For example, encoding Trichoderma reesei Mds1 protein ( Figure 7 and Figure 8 ; SEQ ID NO:2) and Mds2 protein ( Figure 9 and Figure 10; Aspergillus niger genes encoding proteins having about 51% and 60% amino acid sequence identity were identified in the genomic sequence of the public reference strain Aspergillus niger 1015.

[0364] 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) targeting the Aspergillus niger mds1 homolog (SEQ ID NO:20), and gRNAs designated LFP031 (gRNA; SEQ ID NO:22) and LFP032 (gRNA; SEQ ID NO:34) targeting the Aspergillus niger mds2 homolog (SEQ ID NO:22). For example, the above-mentioned Cas9 nuclease and custom synthetic guide RNAs can be obtained from a suitable supplier (e.g., Synthego Corporation, Menlo Park, California). In certain embodiments, synthetic donor DNA can be included to provide a homologous directed repair template, including DNA sequences LFP030 (SEQ ID NO:32) and LFP033 (SEQ ID NO:35) in single-stranded or double-stranded versions for the mds1 and mds2 homologs, respectively. Transformants are obtained using unlinked selectable markers (e.g., hygromycin resistance, amdS, pyrG, etc.).

[0365] In certain other embodiments, Thermus thermophilus genes encoding proteins having sequence homology to Trichoderma reesei Mds1 protein ( Figure 7 and Figure 8 ; SEQ ID NO:2) and Mds2 protein ( Figure 9 and Figure 10 ; SEQ ID NO:4) were identified in the genomic sequence of the public reference strain Thermus thermophilus 42464. As described above for the Aspergillus niger gene homologs, one or more gRNAs targeting the Thermus thermophilus mds1 and / or mds2 genes can be readily constructed by those skilled in the art in a similar manner.

[0366] In other embodiments, those skilled in the art can readily construct modified filamentous fungal strains lacking one or more mannosidases as described herein. As Figure 11 shown, the Trichoderma reesei Mds1 protein contains 523 amino acid residues, and the amino acid residues from about position 43 to position 511 of SEQ ID NO:2 (bold residues) contain a glycoside hydrolase family 47 (GH47) sequence domain, and members of this family are α-mannosidases that catalyze the hydrolysis of terminal 1,2-linked α-D-mannose residues. Additionally, asFigure 11 As shown, the Trichoderma reesei Mds2 protein contains 794 amino acid residues, wherein the amino acid residues at positions approximately 39 to 286 of SEQ ID NO: 4 (underlined residues) contain an N-terminal glycoside hydrolase family 92 (GH92) sequence domain, and the amino acid residues at positions approximately 292 to approximately 773 of SEQ ID NO: 4 (bold residues) contain a glycoside hydrolase family 92 sequence domain, wherein members of this family are α-1,2-mannosidases, which are enzymes that remove α-1,2-linked mannose residues.

[0367] Based on the above, those skilled in the art can readily design and construct one or more modified filamentous fungal strains that lack the production of Mds1 protein (or its homolog) and / or lack the production of Mds2 protein (or its homolog). In particular, as described above, one or more genes (or genetic elements, such as promoters, gene coding sequences, 5'-UTR, terminators, etc.) encoding one or more mannosidases have been genetically modified herein, including but not limited to mutagenizing, disrupting, deleting, replacing, interfering with, etc. a portion of the gene encoding the GH47 (family) sequence domain of the Mds1 protein, thereby reducing or completely eliminating the production of functional Mds1 protein. In other embodiments, one or more genes (or genetic elements) encoding one or more mannosidases have been genetically modified herein, including but not limited to mutagenizing, disrupting, deleting, replacing, interfering with, etc. a portion of the gene encoding the GH92 (family) N-terminal sequence domain and / or the GH92 superfamily sequence domain of the Mds2 protein, thereby reducing or completely eliminating the production of functional Mds2 protein. In other embodiments, one or more active site (amino acid) residues of the Mds1 and / or Mds2 protein are mutagenized, disrupted, deleted, etc., thereby reducing or completely eliminating the production of functional Mds1 and / or Mds2 protein.

[0368] Example 5

[0369] Assessment of protein glycosylation and activity

[0370] This example describes a method for assessing one or more proteins produced by the filamentous fungal cells of the present disclosure. More particularly, as generally described above, certain embodiments relate to modified filamentous fungal cells that contain genetic modifications that render the cells lacking the production of functional α-mannosidase proteins and / or contain a restored glucosidase IIα (gls2a) encoding the native glucosidase IIα-subunit (GIIα) R) Alleles. For example, as described above in Sections II and VI, modified filamentous fungal cells lacking functional α-mannosidase production and / or having a restored gls2a R allele can be cultured / fermented under suitable conditions for producing one or more proteins, and the protein is 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 (about 200 to 300 hours), the secreted protein is isolated from the cells using standard protein recovery methods known to those skilled in the art. Similarly, after such cell separation process, a standard diafiltration process can be used to reduce the concentration of small molecules (e.g., sugars), and the diafiltered protein sample is then heat-treated for a sufficient amount of time and then frozen at -20°C.

[0371] In one or more 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 or in the examples, the broth heat-treatment process is carried out at a temperature between about 39.5°C and 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 lasts for a period of about 30 minutes to about 4.5 hours. In related embodiments, the heat-treatment process lasts for about 4 hours at a temperature of about 40°C.

[0372] Thus, after the above recovery and heat-treatment processes, the samples are formulated with a composition suitable for the specific protein and stored at 25°C for 6 months, where an aliquot of each sample is taken out of storage once a month, and the amount of free mannose (Man) is measured, and the glycosylation level and / or protein (enzyme) activity and / or thermal stability of the reported protein are analyzed. For example, the Man level can be measured as generally described above in Example 2. Similarly, as described herein, the glycosylation of the target protein (protein) can be measured by one of several well-known techniques in the art, including but not limited to changes in protein size measured by mass spectrometry (Schmitt et al., 2005(a)), blue fluorescence emission upon UV excitation of glycosylated proteins (Schmitt et al., 2005(b)), characterization of lysine and arginine side-chain modifications (Schmitt et al., 2005(b)), etc.

[0373] In related embodiments, methods known to those of ordinary skill in the art can be used to determine the activity of one or more proteins (enzymes). For example, p-nitrophenyl phosphate can be used as a substrate to measure phytase activity (see Example 1, part D), and glucoamylase activity can be measured by the release of glucose from a maltodextrin solution (see Example 1, part D).

[0374] L-α-phosphatidylcholine (Avanti 441601G, Avanti Polar Lipids, USA) can be used as a substrate dissolved in 50 mM HEPES buffer containing 5 mM CaCl2, and Triton-X100 can be used as an emulsifier to measure lipase activity as described in PCT Publication No. WO 2020 / 190782. For example, the amount of free fatty acids released during the enzyme reaction can be measured using a 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 cellulose as described in Australian Patent Publication No. 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 extent of remaining enzyme activity (such as phytase, glucoamylase, lipase, cellulase, etc.) after heat-treating the sample at temperatures of 40°C, 50°C, 60°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, and 98°C for 10 minutes.

[0375] For example, one or more of the above assays can be performed by those of ordinary skill in the art to evaluate the level of free Man sugar in a heat-treated fermentation broth sample obtained from modified (recombinant) filamentous fungal cells that lack the production of a functional α-mannosidase protein and / or contain gls2a RModification of alleles. Thus, any protein of interest can be expressed in one or more modified filamentous fungal cells of the present disclosure and evaluated as described herein. More particularly, one or more modified filamentous fungal cells / strains can be readily constructed by those skilled in the art for the production of other proteins of interest by reference to one or more of the modified strains and parental strains illustrated in Table 7 below. For example, as contemplated and described herein, proteins of interest (such as phytase, glucoamylase, lipase, cellulase, etc.) produced by one or more modified cells of the present disclosure contain reduced levels of glycosylation (i.e., compared to their respective parents), maintain a high level of protein (enzyme) activity during long-term storage at room temperature, and have improved thermal stability.

[0376] Table 7 Measurement of glycosylation and enzyme stability of parental and modified strains

[0377]

[0378] References

[0379] Australian Patent Publication No. AU2016 / 200955

[0380] PCT Publication No. WO 2003 / 038111

[0381] PCT Publication No. WO 2006 / 040358

[0382] PCT Publication No. WO 2008 / 097619

[0383] PCT Publication No. WO 2009 / 129489

[0384] PCT Publication No. WO 2011 / 153449

[0385] PCT Publication No. WO 2013 / 119470

[0386] PCT Publication No. WO 2020 / 028126

[0387] PCT Publication No. WO 2020 / 190782

[0388] PCT Publication No. WO 2021 / 212095

[0389] Ausubel et al., “Current Protocols in Molecular Biology”, Green Publishing Associates / Wiley Interscience, New York, Vol. 1 and Vol. 2, (1987; 1988, 1989).

[0390] 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.

[0391] Goto, “Protein O-Glycosylation in Fungi: Diverse Structures and Multiple Functions”, Biosci. Biotechnol. Biochem., 71, pages 1415-1427, 2007.

[0392] Knudsen et al., “FIND-IT: Accelerated trait development for a green evolution”, Sci. Adv. 8, eabq2266, 2022.

[0393] Kornfeld and Kornfeld, “Assembly of Asparagine-linked Oligosaccharides”, Ann. Rev. Biochem., 54, pages 631-664, 1985.

[0394] 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

[0395] Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring, New York, 1989.

[0396] Sambrook et al., Molecular Cloning, A Laboratory Manual, 4 th Edition, Cold Spring Harbor Laboratory Press, Cold Spring, New York, 2012.

[0397] 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.

[0398] Schmitt et al., Analytical Biochemistry Volume 338, Issue 2, pages 201 - 215, 2005(b).

[0399] Schmitt et al., Analytical Biochemistry, Volume 346, Issue 1, pages 101 - 106, 2005(a)

[0400] 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.

[0401] 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.

[0402] 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.

[0403] 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 parental cell, the parental cell comprising an endogenous gene encoding a functional α-mannosidase protein and having an introduced expression cassette encoding a protein of interest (POI), wherein the modified cell comprises a genetic modification that results in insufficient production of the functional α-mannosidase protein in the cell.

2. The modified cell of claim 1, wherein the endogenous gene encodes a functional α-mannosidase protein that has at least about 50% to 100% identity with 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, wherein the modified cell is fermented under suitable conditions to produce the POI and secrete the POI into the fermentation broth.

4. The modified cell of claim 1, wherein the end-of-fermentation (EOF) broth is collected and stored at room temperature for at least 4 hours to about 5 days, and the amount of mannose (Man) contained in the collected and stored broth is reduced relative to the amount of Man sugar present in the EOF broth of the parental cell when collected and stored under the same conditions.

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

6. The modified cell according to claim 5, wherein the parental cell is a Trichoderma reesei cell, and the Trichoderma reesei cell contains a mutant glucosidase II α (gls2a Stop ) allele encoding a truncated glucosidase II α-subunit (GIIα Stop ) protein, and the modified Trichoderma reesei cell contains a restored glucosidase II α (gls2a R ) allele encoding a native glucosidase II α-subunit (GIIα) protein.

7. The modified cell of claim 6, wherein when fermented under the same conditions used to produce the POI, the modified cell produces an increased amount of the POI relative to the parental cell.

8. A modified Trichoderma reesei cell derived from a parental Trichoderma reesei cell, wherein the parental Trichoderma reesei cell comprises a mutant glucosidase II α (gls2a Stop ) allele encoding a truncated glucosidase II α - subunit (GIIα Stop ) protein, and wherein the modified cell comprises a restored glucosidase II α (gls2a R ) allele encoding a native glucosidase II α - subunit (GIIα).

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

10. The modified cell of claim 9, wherein when fermented under the same conditions used to produce the one or more proteins of interest, the modified cell produces an increased amount of the one or more proteins of interest relative to the parental cell.

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

12. The modified cell according to claim 8, wherein the modified cell comprises a genetic modification that renders the cell deficient in the production of a functional α-mannosidase protein, or comprises a genetic modification that renders the cell deficient in the production of at least two functional α-mannosidase proteins.

13. A method for fermenting filamentous fungal cells for the production and recovery of a target protein (POI) in a fermentation broth comprising a reduced amount of mannose (Man), the method comprising: (a) obtaining a parental filamentous fungal cell comprising an endogenous gene encoding a functional α-mannosidase protein, and introducing an expression cassette encoding the target protein (POI) into the cell, (b) genetically modifying the cell to render the cell deficient in the production of the functional α-mannosidase protein, and (b) fermenting the modified cell under conditions suitable for producing the POI, wherein the EOF broth from the modified cell comprises a reduced amount of Man sugar relative to the EOF broth obtained from the parental cell fermented under the same conditions.

14. The method according to claim 13, wherein the endogenous gene encodes a functional α-mannosidase protein that has at least about 50% to 100% identity with 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. The method according to claim 13, wherein the filamentous fungal cells are selected from the group consisting of cells of Aspergillus species, cells of Emericella species, cells of Fusarium species, cells of Humicola species, cells of Mucor species, cells of Myceliophthora species, cells of Neurospora species, cells of Penicillium species, cells of Cylindrocarpon species, cells of Thielavia species, cells of Tolypocladium species, and cells of Trichoderma species.

16. The method according to embodiment 15, wherein the parental cell is a Trichoderma reesei cell, and the Trichoderma reesei cell comprises a mutant glucosidase II α (gls2a Stop ) allele encoding a truncated glucosidase II α - subunit (GIIα Stop ) protein, wherein the modified Trichoderma reesei cell comprises a restored glucosidase II α (gls2a R ) allele encoding a native glucosidase II α - subunit (GIIα) protein.

17. A method for producing a target protein (POI) comprising a homogeneous N-linked glycan pattern, the method comprising: (a) Obtain Trichoderma reesei cells containing a mutant glucosidase II α-subunit (GIIα Stop ) protein-encoding glucosidase II α (gls2a Stop ) allele, and genetically modify the cells to express and encode a restored glucosidase II α (gls2a R ) allele, the restored glucosidase II α (gls2a R ) allele encoding a native glucosidase II α-subunit (GIIα), ferment the modified cells under suitable conditions to produce and secrete the POI, and (b) recovering the POI from the fermentation broth, wherein the recovered POI comprises a homogeneous N-linked glycan pattern comprising greater than 75% Man5GlcNAc2 as compared to the N-linked glycan pattern of the same POI produced by the parental cell, wherein the modified cell and the parental cell are fermented under the same conditions and the POI is recovered under the same conditions.

18. The method according to claim 17, wherein the modified cell produces an increased amount of the POI relative to the parental cell when fermented under the same conditions for producing the POI.

19. The method according to claim 17, wherein the method comprises a genetic modification that causes the cell to lack the production of a functional α-mannosidase protein, or comprises a genetic modification that causes the cell to lack the production of at least two functional α-mannosidase proteins.

20. The method according to claim 17, wherein the POI is a glycoprotein.

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