Compositions and methods for enhancing protein production in filamentous fungal cells

Abstract

The present disclosure relates generally to mutant and genetically modified filamentous fungal cells and methods for use in the production of proteins of interest. More particularly, as described herein, the mutant and / or modified fungal cells (strains) of the present disclosure are well suited for use in industrial-scale fermentation processes for enhanced expression / production of proteins of interest in the absence and / or presence of an inducing substrate.

Description

[Technical Field]

[0001] The present disclosure relates generally to the fields of molecular biology, biochemistry, industrial fermentation, protein production, filamentous fungi, etc. Accordingly, certain embodiments of the present disclosure relate to mutant and / or genetically modified filamentous fungal cells and methods thereof for use in producing proteins of interest.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 013,741, filed April 22, 2020, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing The sequence listing text file submitted herewith includes the file "NB41754WOPCT_SequenceListing.txt," created on March 22, 2021, and having a size of 136 kilobytes. This sequence listing complies with 37 C.FR § 1.52(e) and is hereby incorporated by reference in its entirety. [Background technology]

[0004] Cellulose, a component of lignocellulosic plant material, is the most abundant polysaccharide found in nature. Similarly, filamentous fungi are known in the art to be efficient decomposers of plant biomass and, in fact, are a major source of industrially relevant lignocellulolytic enzymes (hereinafter collectively referred to as "cellulase" enzymes). For example, filamentous fungi are known to produce extracellular cellulase enzymes (e.g., cellobiohydrolases, endoglucanases, β-glucosidases) that hydrolyze the β-(1,4)-linked glycosidic bonds of cellulose to produce glucose (i.e., thereby endowing these fungi with the ability to utilize cellulose for growth). In particular, the filamentous fungus Trichoderma reesei (T. reesei; an anamorph of the fungus Hypocrea jecorina) is known to be an efficient producer of cellulase enzymes (see, e.g., WO 1998 / 15619, WO 2005 / 028636, WO 2006 / 074005, WO 1992 / 06221, WO 1992 / 06209, WO 1992 / 06183, WO 2002 / 12465, etc.), and as such, filamentous fungi have been exploited for their ability to produce enzymes useful in the production of commodities such as cellulose-derived ethanol, textiles and clothing, detergents, fibers, food and feed additives, and other industrial uses.

[0005] The expression / production of these industrially relevant enzymes in Trichoderma fungal cells is known to depend on the carbon source available for growth. The expression / production of cellulase enzymes by filamentous fungi is an energy-consuming process; therefore, both induction and repression mechanisms have evolved to ensure efficient production of these enzymes. For example, various genes encoding enzymes required for the degradation of plant cell wall materials (i.e., lignocellulolytic enzymes, e.g., cellulases / hemicellulases) are "activated" in the presence of an "inducing" substrate and "repressed" in the presence of easily metabolized carbon sources (e.g., D-glucose) that are more favorable than plant biomass, through a mechanism known as "carbon catabolite repression" ("CCR"). Thus, cellulase genes are strongly repressed by glucose and induced thousands of times by cellulose and certain disaccharides (e.g., sophorose, lactose, and gentibiose). For example, the expression level of the major cellobiohydrolase 1 (cbh1) is "upregulated" several thousand-fold in media containing inducing carbon sources such as cellulose or sophorose compared to glucose-containing media (Ilmen et al., 1997).

[0006] Generally, commercial-scale production of enzymes / polypeptides by filamentous fungi such as Trichoderma is generally by solid-state or submerged culture, including batch, fed-batch, and continuous-flow processes. For example, one of the most problematic and expensive aspects of industrial cellulase production in Trichoderma is providing a suitable inducer (i.e., induction substrate) to Trichoderma host cells. For example, as in laboratory-scale experiments, commercial-scale cellulase (enzyme) production is "induced" by growing fungal cells on solid cellulose (i.e., induction substrate) or by culturing the cells in the presence of a disaccharide inducer (i.e., induction substrate) such as "lactose."

[0007] Unfortunately, on an industrial scale, both methods of "induction" have drawbacks that result in the high costs associated with cellulase production, and thus, there remains a continuing and unmet need in the art for cost-effective, commercial-scale production of enzymes / polypeptides by filamentous fungi without the need or requirement to provide expensive induction substrates (e.g., sophorose, lactose, etc.) for such production. As described and exemplified herein, the mutant and / or engineered fungal cells (strains) of the present disclosure are well suited for use in industrial-scale fermentation processes for enhanced expression / production of proteins of interest in the presence or absence of an induction substrate. Summary of the Invention [Means for solving the problem]

[0008] Certain embodiments of the present disclosure relate to mutant and genetically modified Trichoderma fungal cells for use in commercial-scale production of polypeptides. More particularly, certain embodiments of the present disclosure relate to novel variant Ace3 transcription factor (TF) proteins capable of upregulating expression of one or more genes encoding one or more lignocellulolytic enzymes. Accordingly, certain other embodiments relate to polynucleotides (nucleic acid sequences) encoding such novel (i.e., variant) Ace3 transcription factor (TF) proteins disclosed herein. Certain other embodiments relate to compositions and methods for constructing, modifying, testing, screening, isolating, etc., Trichoderma fungal cells (strains) containing and expressing the variant Ace3 transcription factor (TF) proteins disclosed herein.

[0009] Accordingly, certain embodiments of the present disclosure are directed to an isolated polynucleotide encoding a variant Ace3TF protein, wherein the variant Ace3TF comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO: 2 and comprises a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO: 2. In a related embodiment, the polynucleotide encodes a variant Ace3TF comprising a functional N-terminal zinc binuclear cluster (Zn2Cys6) DNA-binding domain comprising at least 90% sequence identity to SEQ ID NO: 29. In other embodiments, the genetic modification of one or more C-terminal amino acid residues selected from amino acid positions 673-689 of SEQ ID NO: 2 comprises an amino acid deletion, an amino acid insertion, an amino acid substitution, or a combination thereof.

[0010] In certain other embodiments of the polynucleotide, the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2, a deletion of at least the last eight C-terminal amino acid positions 682-689 of SEQ ID NO:2, a deletion of at least the last nine C-terminal amino acid positions 681-689 of SEQ ID NO:2, a deletion of at least the last ten C-terminal amino acid positions 680-689 of SEQ ID NO:2, a deletion of at least the last eleven C-terminal amino acid positions 679-689 of SEQ ID NO:2, This includes a deletion of at least the last 12 C-terminal amino acid positions 678-689 of SEQ ID NO:2, a deletion of at least the last 13 C-terminal amino acid positions 677-689 of SEQ ID NO:2, a deletion of at least the last 14 C-terminal amino acid positions 676-689 of SEQ ID NO:2, a deletion of at least the last 15 C-terminal amino acid positions 675-689 of SEQ ID NO:2, a deletion of at least the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2, or a deletion of at least the last 17 C-terminal amino acid positions 673-689 of SEQ ID NO:2.

[0011] In certain embodiments, an isolated polynucleotide of the present disclosure encodes a variant Ace3 TF that comprises at least 90% sequence identity to an amino acid sequence selected from any one of SEQ ID NO:9 to SEQ ID NO:18.

[0012] Certain other embodiments of the present disclosure relate to an isolated Trichoderma mutant cell comprising a mutant ace3 gene encoding a variant Ace3 transcription factor (TF) protein, wherein the variant Ace3 TF protein comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO: 2 and comprises one or more mutated C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO: 2. In certain embodiments, when the mutant cell is fermented under conditions suitable for the production of lignocellulolytic enzymes, the variant Ace3 TF protein of the mutant cell upregulates expression of genes encoding lignocellulolytic enzymes in the absence of an inducing substrate.

[0013] Certain other embodiments of the present disclosure relate to genetically modified Trichoderma fungal cells derived from a parent Trichoderma fungal cell that contains an ace3 gene encoding an Ace3 transcription factor (TF) protein that comprises at least 90% sequence identity to SEQ ID NO:2, wherein the genetically modified cell comprises a modified ace3 gene that encodes a variant Ace3 TF protein that comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2 and that comprises a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2.

[0014] Certain other embodiments of the present disclosure relate to a genetically modified Trichoderma fungal cell derived from a parent Trichoderma fungal cell that contains a mutant ace3 gene encoding a variant Ace3 transcription factor (TF) protein comprising at least 90% sequence identity to SEQ ID NO:4, wherein the N-terminus of SEQ ID NO:4 does not comprise an intact zinc binuclear (Zn2Cys6) DNA binding sequence set forth in SEQ ID NO:29, wherein the genetically modified cell comprises a modified ace3 gene encoding a variant Ace3 TF protein comprising at least 90% sequence identity to positions 1-672 of SEQ ID NO:2, wherein the N-terminus of SEQ ID NO:2 comprises an intact zinc binuclear (Zn2Cys6) DNA binding sequence set forth in SEQ ID NO:29, and wherein the genetically modified Trichoderma fungal cell comprises a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2.

[0015] In certain other embodiments, the present disclosure relates to methods for screening for variant Ace3 TF proteins that induce expression of a gene encoding a lignocellulolytic enzyme in the absence of an inducing substrate. In other embodiments, the present disclosure provides methods for screening for variant Ace3 TF proteins that induce expression of a gene encoding a reporter protein in the absence of an inducing substrate. In other embodiments, the present disclosure is directed to methods for producing a lignocellulolytic enzyme in a Trichoderma fungal cell in the absence of an inducing substrate. In certain other embodiments, the present disclosure relates to methods for producing a protein of interest (POI) in a Trichoderma fungal cell in the absence of an inducing substrate. [Brief explanation of the drawings]

[0016] [Figure 1]Figure 1 shows an amino acid sequence alignment of the Ace3-S protein (SEQ ID NO: 4), the Ace3-L protein (SEQ ID NO: 6), and the Ace3-LC protein (SEQ ID NO: 2). As shown in Figure 1A, the six N-terminal cysteine ​​amino acids of the zinc binuclear cluster (Zn2Cys6) DNA-binding domain are indicated by gray-shaded cysteines (C). As shown in Figure 1A, the Ace3-S protein (SEQ ID NO: 4) contains a truncated N-terminus, in which two of the six cysteine ​​residues of the zinc (Zn2Cys6) DNA-binding domain are missing (deleted) relative to the N-termini of the Ace3-L and Ace3-LC proteins. Similarly, as shown in Figure 1B, the C-terminus of the Ace3-L protein is truncated relative to the Ace3-S and Ace3-LC proteins, which contain a wild-type C-terminus ending in glycine (G), as opposed to the truncated C-terminus of the Ace3-L protein, which ends in aspartic acid (D).

[0017] [Figure 2]Figure 2 shows the amino acid sequence of the Ace3-LC protein, which contains 689 amino acid residues and a wild-type C-terminus ending with glycine (G) at position 689 (Figure 2A, SEQ ID NO: 2). Additionally, Figure 2 shows the amino acid sequences of 16 different Ace3 proteins containing incremental C-terminal truncations (i.e., relative to the C-terminus of the wild-type Ace3-LC protein; SEQ ID NO: 2). As shown in Figure 2, the Ace3 C-term-5 protein (Figure 2A; SEQ ID NO: 7) contains 684 amino acid residues and a 5 amino acid truncation at the C-terminus ending with serine (S) at position 684 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2), the Ace3 C-term-6 protein (Figure 2A; SEQ ID NO: 8) contains 683 amino acid residues and a 6 amino acid truncation at the C-terminus ending with threonine (T) at position 683 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2), the Ace3 C-term-7 protein (Figure 2B; SEQ ID NO: 9) contains 682 amino acid residues with a 7 amino acid truncation at the C-terminus ending with asparagine (N) at position 682 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2), and the Ace3 C-term-8 protein (Figure 2B; SEQ ID NO: 10) contains an 8 amino acid truncation at the C-terminus ending with arginine (R) at position 681 (relative to the WT Ace3 C-terminus). The Ace3 C-term-9 protein (Figure 2B; SEQ ID NO: 11) contains 680 amino acid residues with a 9 amino acid truncation at the C-terminus ending with a leucine (L) at position 680 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2). The Ace3 C-term-10 protein (Figure 2C; SEQ ID NO: 12) contains 679 amino acid residues with a 10 amino acid truncation at the C-terminus ending with a glutamine (Q) at position 679 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2). The Ace3 C-term-12 protein (Figure 2C; SEQ ID NO: 13) contains 677 amino acid residues with a 12 amino acid truncation at the C-terminus ending with a serine (S) at position 677 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2).The Ace3 C-term-14 protein (FIG. 2D; SEQ ID NO: 15) contains 675 amino acid residues with a 14 amino acid truncation at the C-terminus ending at alanine (A) at position 675 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2). The Ace3 C-term-15 protein (FIG. 2D; SEQ ID NO: 16) contains 674 amino acid residues with a 15 amino acid truncation at the C-terminus ending at serine (S) at position 674 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2). The Ace3 C-term-16 protein (FIG. 2D; SEQ ID NO: 17) contains 673 amino acid residues with a 16 amino acid truncation at the C-terminus ending at aspartic acid (D) at position 673 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2). The C-term-17 protein (Figure 2E; SEQ ID NO: 18) contains 672 amino acid residues with a 17 amino acid truncation at the C-terminus ending with a leucine (L) at position 672 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2). The Ace3 C-term-18 protein (Figure 2E; SEQ ID NO: 19) contains 671 amino acid residues with an 18 amino acid truncation at the C-terminus ending with an arginine (R) at position 671 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2). The Ace3 C-term-19 protein (Figure 2E; SEQ ID NO: 20) contains 670 amino acid residues with a 19 amino acid truncation at the C-terminus ending with a threonine (T) at position 670 (relative to the WT Ace3 C-terminus; SEQ ID NO: 2). The Ace3 C-term-20 protein (Figure 2F; SEQ ID NO: 21) contains 670 amino acid residues with a 20 amino acid truncation at the C-terminus ending with a leucine (L) at position 669 (relative to the WT Ace3 C-terminus). The Ace3 C-term-25 protein (Figure 2F; SEQ ID NO: 22) contains 664 amino acid residues with a 25 amino acid truncation at the C-terminus (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with a valine (V) at position 664;

[0018] [Figure 3] Figure 3 shows the amino acid sequence of the Ace3 zinc binuclear cluster (Zn2Cys6) DNA-binding domain (SEQ ID NO: 29), which contains six cysteine ​​(C) residues (indicated by bold underlined C residues), and the DNA-binding domain is located within amino acid positions 73 to 109 of the Ace3-LC protein (SEQ ID NO: 2) and Ace3-L (SEQ ID NO: 6) proteins.

[0019] [Figure 4] Figure 4 shows the wild-type C-terminus of the Ace3-LC protein (SEQ ID NO: 2), ending with glycine (G) at amino acid position 689. As shown in Figure 4, C-terminal amino acids indicated in bold text are assigned negative numbers, with the most C-terminal amino acid position (i.e., glycine (G); position 689) assigned a negative one (-1), the threonine (T) at position 685 assigned a negative five (-5), the leucine (L) at position 680 assigned a negative ten (-10), and so on. As generally described in the Examples and detailed description herein, the C-terminal amino acid region of the Ace3 protein shown in Figure 4, with the bolded C-terminal amino acids and gray shading (i.e., from -7 (T) threonine to -17 (D) aspartic acid), is a particularly suitable region for genetic modification (e.g., truncation, internal deletion, insertion, substitution, and combinations thereof).

[0020] [Figure 5] Figure 5 shows an alignment of Ace3-LC with the wild-type C-terminus (i.e., amino acid positions 641-689; SEQ ID NO: 2) with the C-terminus of the Ace3-LC C-term-7 protein (amino acid positions 641-682; SEQ ID NO: 9) and the C-terminus of the Ace3-LC C-term-17 protein (amino acid positions 641-672; SEQ ID NO: 18). As shown in Figure 5, the bold and underlined C-terminal amino acid residues (DSKASDQLRN) of the Ace3 protein are a particularly suitable region for genetic modification (e.g., truncation, internal deletion, insertion, substitution, and combinations thereof).

[0021] [Figure 6] FIG. 6 is a schematic map of the plasmid pYL72, which contains a polynucleotide sequence encoding an Ace3 protein (eg, Ace3-LC protein; SEQ ID NO: 2) containing a wild-type (ie, full-length) C-terminus ending in glycine.

[0022] [Figure 7]7 shows the average total secreted protein titers of T. reesei strains fermented under inducing (lactose; Lac) and non-inducing (glucose; Glu) conditions. As shown in FIG. 7, the T. reesei strains screened included a control strain (RL-P37), a "C-term-WT" strain expressing an Ace3-LC protein (SEQ ID NO: 2) containing a wild-type C-terminus, a "C-term-5" strain expressing a variant Ace3-LC protein (SEQ ID NO: 7) containing a C-terminal truncation of 5 amino acids, a "C-term-10" strain expressing a variant Ace3-LC protein (SEQ ID NO: 12) containing a C-terminal truncation of 10 amino acids, and a "C-term-11" strain expressing a variant Ace3-LC protein (SEQ ID NO: 13) containing a C-terminal truncation of 11 amino acids. The strains included the "C-term-11" strain expressing a variant Ace3-LC protein containing a C-terminal truncation of 15 amino acids (SEQ ID NO: 6), the "C-term-15" strain expressing a variant Ace3-LC protein containing a C-terminal truncation of 15 amino acids (SEQ ID NO: 16), the "C-term-20" strain expressing a variant Ace3-LC protein containing a C-terminal truncation of 20 amino acids (SEQ ID NO: 21), and the "C-term-25" strain expressing a variant Ace3-LC protein containing a C-terminal truncation of 25 amino acids (SEQ ID NO: 22). As shown in Figure 7, the parental control strain RL-P37 was only able to produce large amounts of protein on lactose (i.e., under inducing conditions) and produced minimal basal levels of protein on glucose (i.e., under non-inducing conditions). In contrast, daughter strains (cells) expressing Ace3-LC variants containing truncations of 10, 11, or 15 amino acids at the C-terminus (i.e., SEQ ID NO: 12, SEQ ID NO: 6, and SEQ ID NO: 16, respectively) showed increased protein production under both lactose (inducing) and glucose (non-inducing) conditions.

[0023] [Figure 8]FIG. 8 shows the mean total secreted protein titers and standard deviations from 2-4 biological replicates fermented under inducing (lactose; Lac) and non-inducing (glucose; Glu) conditions. As shown in Figure 8, the T. reesei strains screened included a control strain (RL-P37), a "C-term-WT" strain expressing an Ace3-LC protein (SEQ ID NO: 2) containing a wild-type C-terminus, a "C-term-5" strain expressing a variant Ace3-LC protein (SEQ ID NO: 7) containing a C-terminal truncation of 5 amino acids, a "C-term-6" strain expressing a variant Ace3-LC protein (SEQ ID NO: 8) containing a C-terminal truncation of 6 amino acids, a "C-term-7" strain expressing a variant Ace3-LC protein (SEQ ID NO: 9) containing a C-terminal truncation of 7 amino acids, a "C-term-8" strain expressing a variant Ace3-LC protein (SEQ ID NO: 10) containing a C-terminal truncation of 8 amino acids, a "C-term-9" strain expressing a variant Ace3-LC protein (SEQ ID NO: 11) containing a C-terminal truncation of 9 amino acids, and a "C-term-10" strain expressing a variant Ace3-LC protein (SEQ ID NO: 12) containing a C-terminal truncation of 10 amino acids. a "C-term-10" strain expressing a variant Ace3-LC protein (SEQ ID NO: 12), a "C-term-11" strain expressing a variant Ace3-LC protein with a C-terminal truncation of 11 amino acids (SEQ ID NO: 6), a "C-term-12" strain expressing a variant Ace3-LC protein with a C-terminal truncation of 12 amino acids (SEQ ID NO: 13), a "C-term-13" strain expressing a variant Ace3-LC protein with a C-terminal truncation of 13 amino acids (SEQ ID NO: 14), a "C-term-14" strain expressing a variant Ace3-LC protein with a C-terminal truncation of 14 amino acids (SEQ ID NO: 15), a "C-term-15" strain expressing a variant Ace3-LC protein with a C-terminal truncation of 15 amino acids (SEQ ID NO: 16), and a "C-term-16" strain expressing a variant Ace3-LC protein with a C-terminal truncation of 16 amino acids (SEQ ID NO: 17).The strains included a "C-term-17" strain expressing a variant Ace3-LC protein containing a 17 amino acid C-terminal truncation (SEQ ID NO: 18), a "C-term-18" strain expressing a variant Ace3-LC protein containing an 18 amino acid C-terminal truncation (SEQ ID NO: 19), a "C-term-19" strain expressing a variant Ace3-LC protein containing a 19 amino acid C-terminal truncation (SEQ ID NO: 20), a "C-term-20" strain expressing a variant Ace3-LC protein containing a 20 amino acid C-terminal truncation (SEQ ID NO: 21), and a "C-term-25" strain expressing a variant Ace3-LC protein containing a 25 amino acid C-terminal truncation (SEQ ID NO: 22).

[0024] [Figure 9] Figure 9 shows the total secreted protein titers of strain T4abc (containing an ace3 truncation allele encoding the Ace3 protein of SEQ ID NO: 6), strain T4abc Ace3_rev (containing a reverted Ace3 allele encoding a wild-type ace3 protein (full-length C-terminus; SEQ ID NO: 2)), and strain T4abc del-cbh1 (containing a deletion of the cbh1 (cellobiohydrolase) gene), where each strain was evaluated in two independent shake flasks (e.g., Flask A and Flask B). As shown in Figure 9, when the ace3 mutation was reverted in the T4abc background to encode full-length Ace3, the total secreted protein titer was reduced by 73% compared to the T4abc parent strain encoding the truncated form of Ace3.

[0025] [Figure 10] FIG. 10 is a schematic map of the ace3 reversion cassette plasmid designated pRATT346.

[0026] [Figure 11]FIG. 11 is a schematic map of plasmid pA3L02 containing a polynucleotide sequence encoding a variant Ace3-LC protein having a mutation at amino acid position 673 (SEQ ID NO: 32).

[0027] [Figure 12] Figure 12 shows an amino acid sequence alignment of the native Ace3-LC C-terminus (Figure 12; Ace3-LC amino acid residue positions 641-689; SEQ ID NO: 2) with the C-terminus of Ace3-L (Figure 12; Ace3-L positions 641-678; SEQ ID NO: 6), Ace3-LC-V5 (Figure 12; Ace3-LC-V5 positions 641-692; SEQ ID NO: 67), and Ace3-LC-V5(6xHis) (Figure 12; Ace3-LC-V5(6xHis) positions 642-701; SEQ ID NO: 69). As shown in Figure 12, the V5 tag is indicated by bolded amino acid residues, and the six histidine (His) tags are indicated by underlined H amino acid residues, separated by a three-amino acid "RTG" linker sequence.

[0028] [Figure 13] FIG. 13 is a schematic map of plasmid pYL18 containing a polynucleotide sequence encoding a variant Ace3-LC protein having a C-terminal amino acid substitution with a V5 epitope tag (SEQ ID NO: 67).

[0029] [Figure 14] FIG. 14 shows the total secreted protein titers of T. reesei strains fermented under inducing (Glu / Sop) and non-inducing (Glu) conditions.

[0030] [Figure 15] Figure 15 shows a partial sequence of the wild-type Ace3-LC protein (SEQ ID NO: 2) containing the full-length (wild-type) Ace3-LC C-terminal amino acid positions 641 to 689 of SEQ ID NO: 30. As shown in Figure 15 (SEQ ID NO: 30), amino acid positions are shown as S641 to G689, with capital letters indicating the amino acid residue and the subsequent subscripted number indicating its position. DETAILED DESCRIPTION OF THE INVENTION

[0031] Biological sequence description SEQ ID NO: 1 is the wild-type Trichoderma reesei polynucleotide sequence containing the gene encoding the Ace3 protein (SEQ ID NO: 2), designated "Ace3-LC."

[0032] SEQ ID NO:2 is the amino acid sequence of the Ace3-LC protein encoded by SEQ ID NO:1.

[0033] SEQ ID NO:3 is a T. reesei polynucleotide sequence containing the gene encoding the Ace3 protein designated "Ace3-S" (SEQ ID NO:4).

[0034] SEQ ID NO:4 is the amino acid sequence of the Ace3-S protein encoded by SEQ ID NO:3.

[0035] SEQ ID NO: 5 is a T. reesei polynucleotide sequence containing the gene encoding the Ace3 protein designated "Ace3-L" (SEQ ID NO: 6).

[0036] SEQ ID NO: 6 is the amino acid sequence of the Ace3-L protein encoded by SEQ ID NO: 5.

[0037] SEQ ID NO: 7 is the amino acid sequence of a variant Ace3 protein designated "C-term-5."

[0038] SEQ ID NO: 8 is the amino acid sequence of a variant Ace3 protein designated "C-term-6."

[0039] SEQ ID NO: 9 is the amino acid sequence of a variant Ace3 protein designated "C-term-7."

[0040] SEQ ID NO: 10 is the amino acid sequence of a variant Ace3 protein designated "C-term-8."

[0041] SEQ ID NO: 11 is the amino acid sequence of a variant Ace3 protein designated "C-term-9."

[0042] SEQ ID NO: 12 is the amino acid sequence of a variant Ace3 protein designated "C-term-10."

[0043] SEQ ID NO: 13 is the amino acid sequence of a variant Ace3 protein designated "C-term-12."

[0044] SEQ ID NO: 14 is the amino acid sequence of a variant Ace3 protein designated "C-term-13."

[0045] SEQ ID NO: 15 is the amino acid sequence of a variant Ace3 protein designated "C-term-14."

[0046] SEQ ID NO: 16 is the amino acid sequence of a variant Ace3 protein designated "C-term-15."

[0047] SEQ ID NO: 17 is the amino acid sequence of a variant Ace3 protein designated "C-term-16."

[0048] SEQ ID NO: 18 is the amino acid sequence of a variant Ace3 protein designated "C-term-17."

[0049] SEQ ID NO: 19 is the amino acid sequence of a variant Ace3 protein designated "C-term-18."

[0050] SEQ ID NO: 20 is the amino acid sequence of a variant Ace3 protein designated "C-term-19."

[0051] SEQ ID NO: 21 is the amino acid sequence of a variant Ace3 protein designated "C-term-20."

[0052] SEQ ID NO: 22 is the amino acid sequence of a variant Ace3 protein designated "C-term-25."

[0053] SEQ ID NO: 23 is the nucleic acid primer sequence (TP218).

[0054] SEQ ID NO:24 is the nucleic acid primer sequence (TP220).

[0055] SEQ ID NO: 25 is the nucleic acid primer sequence (TP125).

[0056] SEQ ID NO: 26 is the nucleic acid primer sequence (TP123).

[0057] SEQ ID NO: 27 is the nucleic acid primer sequence (TP221).

[0058] SEQ ID NO: 28 is the nucleic acid primer sequence (TP222).

[0059] SEQ ID NO: 29 is the amino acid sequence of the Ace3 binuclear (Zn2Cys6) zinc DNA binding domain.

[0060] SEQ ID NO: 30 comprises amino acid residue positions 641-689, which define the "Ace3-LC C-terminus."

[0061] SEQ ID NO:31 is a polynucleotide sequence encoding the Ace3-LC C-terminus of SEQ ID NO:30.

[0062] SEQ ID NO: 32 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_673."

[0063] SEQ ID NO: 33 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_674."

[0064] SEQ ID NO: 34 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_675."

[0065] SEQ ID NO: 35 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_676."

[0066] SEQ ID NO: 36 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_677."

[0067] SEQ ID NO: 37 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_678."

[0068] SEQ ID NO: 38 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_679."

[0069] SEQ ID NO: 39 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_680."

[0070] SEQ ID NO: 40 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_681."

[0071] SEQ ID NO: 41 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_682."

[0072] SEQ ID NO: 42 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus mut_683."

[0073] SEQ ID NO: 43 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_672."

[0074] SEQ ID NO: 44 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_673."

[0075] SEQ ID NO: 45 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_674."

[0076] SEQ ID NO: 46 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_675."

[0077] SEQ ID NO: 47 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_676."

[0078] SEQ ID NO: 48 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_677."

[0079] SEQ ID NO: 49 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_678."

[0080] SEQ ID NO: 50 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_679."

[0081] SEQ ID NO: 51 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_680."

[0082] SEQ ID NO: 52 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_681."

[0083] SEQ ID NO: 53 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_682."

[0084] SEQ ID NO: 54 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus ins_683."

[0085] SEQ ID NO: 55 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_673-675."

[0086] SEQ ID NO: 56 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_674-676."

[0087] SEQ ID NO: 57 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_675-677."

[0088] SEQ ID NO: 58 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_676-678."

[0089] SEQ ID NO: 59 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_677-679."

[0090] SEQ ID NO: 60 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus del_678-680."

[0091] SEQ ID NO: 61 is a polynucleotide sequence encoding an Ace3-LC C-terminus variant designated "Ace3-LC C-terminus del_679-681."

[0092] SEQ ID NO: 62 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_680-682."

[0093] SEQ ID NO: 63 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_681-683."

[0094] SEQ ID NO: 64 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_682-684."

[0095] SEQ ID NO: 65 is a polynucleotide sequence encoding an Ace3-LC C-terminal variant designated "Ace3-LC C-terminal del_683-685."

[0096] SEQ ID NO:66 is a polynucleotide sequence encoding the Ace3-LC C-terminal V5 substitution comprising the amino acid sequence of SEQ ID NO:67.

[0097] SEQ ID NO: 67 is the amino acid sequence of the "Ace3-LC C-terminal V5 substitution" variant.

[0098] SEQ ID NO:68 is a polynucleotide sequence encoding an Ace3-LC C-terminal V5-(6xHis) substitution comprising the amino acid sequence of SEQ ID NO:69.

[0099] SEQ ID NO: 69 is the amino acid sequence of the "Ace3-LC C-terminal V5-(6xHis) substitution" variant.

[0100] SEQ ID NO: 70 is an sgRNA targeting the gla1 locus.

[0101] Detailed Description I. Overview As described herein, certain embodiments of the present disclosure relate to mutant and / or genetically modified Trichoderma fungal cells for use in commercial-scale production of a protein of interest. More particularly, certain embodiments of the present disclosure relate to novel Ace3 (variant) transcription factor (TF) proteins capable of upregulating expression of one or more genes encoding lignocellulolytic enzymes. Accordingly, certain embodiments are directed to polynucleotides (nucleic acid sequences) encoding the novel (variant) Ace3 TF proteins disclosed herein. For example, certain embodiments of the present disclosure are directed to isolated polynucleotides encoding variant Ace3 TF proteins, wherein the variant Ace3 TF comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2 and comprises a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2.

[0102] Accordingly, certain embodiments relate to genetically modified Trichoderma fungal cells comprising an ace3 gene encoding a variant Ace3TF protein of the present disclosure, wherein the modified cells are capable of producing a protein of interest in the absence of an inducing substrate. Certain other embodiments relate to genetically modified Trichoderma fungal cells comprising an ace3 gene encoding a variant Ace3TF protein of the present disclosure, wherein the modified cells are capable of increasing the amount of the protein of interest produced in the presence of an inducing substrate (i.e., compared to the amount of the same protein of interest produced by a control Trichoderma fungal cell comprising a wild-type ace3 gene encoding a wild-type Ace3 protein (e.g., SEQ ID NO: 2) when the modified and control Trichoderma fungal cells are cultured / fermented under the same conditions in the presence of an inducing substrate for the production of a POI). Accordingly, certain other embodiments of the present disclosure relate to compositions and methods for constructing, modifying, testing, screening, isolating, etc., modified Trichoderma fungal cells (strains) that contain and express the variant Ace3 transcription factor (TF) proteins of the present disclosure.

[0103] II. Definition Before describing the present compositions and methods in further detail, the following terms and phrases are defined. Terms not defined should be accorded the ordinary meaning used and known to those skilled in the art.

[0104] All publications and patents cited herein are hereby incorporated by reference.

[0105] Where a range of values ​​is presented, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within that range, is encompassed within the compositions and methods. The upper and lower limits of these smaller ranges are independently included in the smaller ranges and are also encompassed within the compositions and methods of the invention, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the compositions and methods of the invention.

[0106] In this specification, some ranges are expressed by numerical values ​​preceded by the term "about." In this specification, the term "about" is used to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically recited number, the unrecited close or approximate number may be a number that, in the context in which it is presented, provides a substantially equivalent number to the specifically recited number. For example, the term "about" in connection with a numerical value means the numerical value of that number unless the term is clearly defined otherwise in the context. - 10%~ + As another example, the phrase "a pH value of about 6" refers to a pH value of 5.4 to 6.6, unless the pH value is specifically defined otherwise.

[0107] The headings provided herein are not limitations of the various aspects or embodiments of the present compositions and methods, which can be had by reference to the specification as a whole, and accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0108] In accordance with this detailed description, 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 dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, reference to "the dosage" includes one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0109] It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a prelude to the use of exclusive terminology such as "only," "only," "except," "not including," or the use of any such "negative" limitation or "condition" in connection with the recitation of claim elements.

[0110] Additionally, it should be noted that the term "comprising," as used herein, means "including, but not limited to" the component following the term "comprising." The component following the term "comprising" is required or essential, but a composition containing this component may further include other non-essential or optional components.

[0111] It should also be noted that the term "consisting of," as used herein, means "including and limited to" the component(s) following the term "consisting of." Thus, the component(s) following the term "consisting of" are required or essential, and no other components are present in the composition.

[0112] It will be apparent to those skilled in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the compositions and methods described herein. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0113] As used herein, the term "ascomycota fungal cell" refers to any organism of the phylum Ascomycota in the kingdom Fungi. Examples of ascomycota fungal cells include, but are not limited to, filamentous fungi within the subdivision Pezizomycotina, such as Trichoderma species, Aspergillus species, Myceliophthora species, and Penicillium species.

[0114] As used herein, the term "filamentous fungi" refers to all filamentous fungal forms of the subkingdoms Eumycota and Oomycota. For example, filamentous fungi include, but are not limited to, species of the genera Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma.

[0115] In some embodiments, the filamentous fungus is a Trichoderma fungal cell (strain), such as, but not limited to, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0116] In some embodiments, the filamentous fungus can be an Aspergillus fungal cell (strain), such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae.

[0117] In some embodiments, the filamentous fungus is a T. reesei cell from T. reesei strain "Rut-C30," available from the American Type Culture Collection under Trichoderma reesei ATCC accession number 56765. In other embodiments, the filamentous fungus is a T. reesei cell from T. reesei strain "RL-P37," available from the U.S. Department of Agriculture, Northern Regional Research Laboratory culture collection under NRRL number 15709.

[0118] As used herein, the term "mutant cell," when used in phrases such as "mutant Trichoderma fungal cell," refers specifically to any naturally occurring Trichoderma fungal (mutant) cell comprising a mutant ace3 gene encoding a mutant Ace3 TF of the present disclosure. For example, a population of Trichoderma fungal cells can be screened according to the methods described herein to identify naturally occurring mutant Trichoderma fungal cells capable of producing a protein of interest in the absence of an inducing substrate. For example, in certain embodiments, the mutant Trichoderma fungal cells identified above are isolated from a population of Trichoderma fungal cells and cultured / fermented under conditions suitable for producing a protein of interest in the absence of an inducing substrate.

[0119] As used herein, the phrases "variant filamentous fungal cell," "modified filamentous fungal cell," "variant fungal cell," "modified fungal cell," and the like refer to filamentous fungal cells derived from (i.e., derived from) a parent filamentous fungal cell belonging to the subphylum Pezizomycotina. Thus, as used herein, an "modified" or "variant" filamentous fungal cell is derived from a "parent" filamentous fungal cell, and a "mutant" cell contains at least one genetic modification not present in the "parent" cell. For example, when comparing a "modified cell" to a "parent cell" of the present disclosure, the "parent" cell serves as an unmodified "control" cell for the "modified" cell containing at least one genetic modification.

[0120] As used herein, the terms "modification" and "genetic modification" are used interchangeably and include: (a) the introduction, substitution, or removal of one or more nucleotides in a gene (or its ORF), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of a gene or its ORF; (b) gene disruption; (c) gene conversion; (d) gene deletion; (e) gene downregulation; (f) directed mutagenesis; and / or (g) random mutagenesis of any one or more genes disclosed herein.

[0121] As used herein, "gene disruption," "gene disruption," "gene inactivation," and "gene inactivation" are used interchangeably and refer broadly to any genetic modification that substantially prevents the production of a functional gene product (e.g., a protein) in a host cell. Exemplary gene disruption methods include complete or partial elimination of or mutagenesis of any portion of a gene, including a polypeptide-encoding sequence, promoter, enhancer, or other regulatory element (wherein mutagenesis encompasses substitutions, insertions, deletions, inversions, and any combinations and variations thereof that disrupt / inactivate the target gene and substantially reduce or prevent the production of a functional gene product (i.e., a protein)).

[0122] As used herein, the terms "downregulation" of gene expression and "upregulation" of gene expression include any method that results in a decrease (downregulation) or increase (upregulation) of gene expression. For example, gene downregulation can be achieved by RNA-induced gene silencing, genetic modification of regulatory elements such as promoters, ribosome binding sites (RBS) / Shine-Dalgarno sequences, untranslated regions (UTRs), codon alterations, etc.

[0123] As used herein, a "targeting vector" is a vector that contains a polynucleotide sequence homologous to a region in a host cell chromosome into which the targeting vector is transformed and that can drive homologous recombination at that region. For example, targeting vectors are used to introduce mutations into a host cell chromosome by homologous recombination. In some embodiments, the targeting vector contains other non-homologous sequences (i.e., stuffer or flanking sequences), e.g., added to the ends. The ends can be closed, e.g., by insertion into a vector, to cause the targeting vector to form a closed circle. For example, in certain embodiments, the parent Trichoderma fungal cell contains ace3 encoding a variant Ace3 protein (e.g., Ace3-S; SEQ ID NO: 4) containing a truncated (non-functional) N-terminal zinc binuclear cluster DNA-binding domain.

[0124] Thus, as described and contemplated herein, in certain embodiments, the ace3 gene encoding a variant Ace3 protein (e.g., Ace3-S; SEQ ID NO:4) is genetically modified (e.g., transformed) by introducing into a parent cell one or more "targeting vectors" designed to restore the native DNA-binding domain of the Ace3 protein. For example, the native Ace3 DNA-binding domain comprises the amino acid sequence set forth in SEQ ID NO:29. The selection and / or construction of an appropriate vector (e.g., to restore native Ace3 DNA binding within the ace3 gene) is clearly within the knowledge of one of ordinary skill in the art. Similarly, in other embodiments, one or more targeting vectors are designed / constructed to delete, introduce (insert), and / or substitute one or more nucleotides into the ace3 gene, as described herein, such that the encoded Ace3 protein comprises a modified C-terminus.

[0125] As used herein, "flanking sequence" refers to any sequence upstream or downstream of the sequence under consideration (e.g., in gene ABC, gene B is flanked by gene sequences A and C). In certain embodiments, the incoming sequence is flanked on both sides by homology boxes. In other embodiments, the incoming sequence and homology box comprise a unit flanked on both sides by stuffer sequences. In some embodiments, flanking sequences are present on only one side (3' or 5'), but in preferred embodiments, they are present on both sides of the flanking sequence. The sequence of each homology box is homologous to a sequence in the chromosome of Trichoderma fungus. These sequences direct where in the chromosome of Trichoderma fungus the novel construct will be integrated and which portion of the chromosome of Trichoderma fungus will be replaced by the incoming sequence. In other embodiments, the 5' and 3' ends of the selectable marker are flanked by polynucleotide sequences comprising a portion of an inactivated chromosomal segment.

[0126] As used herein, the term "gene" is synonymous with the term "allele" in that it refers to a nucleic acid that encodes and directs the expression of a protein or RNA. Vegetative forms of filamentous fungi are generally haploid, so a single copy of a particular gene (i.e., a single allele) is sufficient to confer a particular phenotype.

[0127] 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.

[0128] As used herein, Trichoderma reesei "strain QM6a" contains the Ace3 gene encoding the Ace3 protein designated "Ace3-S" (SEQ ID NO: 4). See reference 4.

[0129] As used herein, Trichoderma reesei "strain RL-P37" contains the Ace3 gene encoding the Ace3 protein designated "Ace3-L" (SEQ ID NO: 6).

[0130] As used herein, the "Ace3-LC" protein comprises the amino acid sequence of SEQ ID NO: 2. As shown in FIG. 1, the Ace3-LC protein comprises 689 amino acid residues, with an N-terminal amino acid sequence including an intact zinc binuclear cluster (Zn2Cys6) DNA-binding domain (FIG. 1A; indicated by six bolded and grayed cysteine ​​(C) residues) and a full-length (wild-type; non-truncated) C-terminus (FIG. 1B) ending with a glycine (G) residue at position 689. As used herein, the last 49 amino acid residue positions of the native Ace3-LC protein (SEQ ID NO: 2; i.e., residue positions 641-689) are set forth as SEQ ID NO: 30 (see, e.g., FIG. 15). Thus, in certain embodiments, a variant Ace3 protein comprises a modified C-terminus, which is compared to the native Ace3-LC C-terminal amino acid positions 641-689 of SEQ ID NO: 30.

[0131] As used herein, the "Ace3-LC" protein comprises the amino acid sequence of SEQ ID NO: 6. As shown in Figure 1, the Ace3-L protein comprises an N-terminal amino acid sequence that includes an intact zinc binuclear cluster (Zn2Cys6) DNA-binding domain (indicated by six bolded and grayed cysteine ​​(C) residues; Figure 1A), and a truncated C-terminus that ends in an aspartic acid (D) residue (Figure 1B, as compared to, e.g., the Ace3-LC protein C-terminus; SEQ ID NO: 2).

[0132] As used herein, the "Ace3-S" protein comprises the amino acid sequence of SEQ ID NO: 4. As shown in FIG. 1A, the Ace3-S protein comprises a shorter (truncated) N-terminal amino acid sequence compared to Ace3-LC (FIG. 1A; SEQ ID NO: 2) and / or Ace3-L (FIG. 1A; SEQ ID NO: 6). Thus, as shown in FIG. 1A, the Ace3-S protein does not contain an intact zinc binuclear cluster (Zn2Cys6) DNA-binding domain (i.e., compared to the N-terminal sequences of Ace3-LC and Ace3-L).

[0133] As used herein, the phrases "Ace3 DNA-binding domain" and "Ace3 zinc binuclear cluster (Zn2Cys6) DNA domain" may be used interchangeably and refer to an amino acid sequence that contains homology to the Ace3 DNA-binding domain of SEQ ID NO: 29. For example, the Ace3 DNA-binding domain of SEQ ID NO: 29 (FIG. 3) is located at amino acid positions 73-109 of the Ace3-LC (SEQ ID NO: 2) and Ace3-L (SEQ ID NO: 6) proteins.

[0134] As used herein, the term "upregulate," when used in phrases such as "the Ace3 TF protein 'upregulates' the expression of a gene encoding a lignocellulolytic enzyme," specifically refers to an Ace3 TF protein that can upregulate the expression of a gene encoding a lignocellulolytic enzyme in the absence of an inducing substrate. For example, a modified filamentous fungal cell of the present disclosure that contains an Ace3 TF protein that upregulates the expression of a gene encoding a lignocellulolytic enzyme can produce the lignocellulolytic enzyme in the absence of an inducing substrate. In contrast, a filamentous fungal (control) cell that contains a wild-type ace3 gene encoding a wild-type Ace3 TF protein does not upregulate the expression of a gene encoding a lignocellulolytic enzyme in the absence of an inducing substrate.

[0135] As used herein, the term "lignocellulolytic enzyme" includes, but is not limited to, cellobiohydrolases, endoglucanases, and β-glucosidases. As used herein, the term "increased amount," when used in phrases such as "the modified cells produce an 'increased amount' of a lignocellulolytic enzyme in the presence of an inducing substrate," the 'increased amount' of lignocellulolytic enzyme produced is compared to the amount of the same lignocellulolytic enzyme produced by the parent cell when the modified cell and the parent cell are cultured / fermented in the presence of the same inducing substrate under the same conditions for the production of a POI.

[0136] As described in the Examples section below and explained below, certain embodiments of the present disclosure are directed to genetically modified Trichoderma fungal cells (strains) derived from a parent Trichoderma fungal cell (strain) that contains an ace3 gene encoding an Ace3 transcription factor (TF) protein that contains at least 90% sequence identity to SEQ ID NO: 2, wherein the genetically modified cells contain a variant Ace3 gene that encodes a variant Ace3 TF protein that contains at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO: 2 and that contains a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO: 2. For example, in certain embodiments, the modified Trichoderma fungal cells of the present disclosure contain a variant Ace3 TF protein that is capable of upregulating expression of an open reading frame (ORF) encoding a protein of interest (POI) when the ORF is operably linked to an upstream (5') promoter sequence derived from a gene encoding a lignocellulolytic enzyme.

[0137] Thus, as used herein, the term "upregulate," when stated in phrases such as "a variant Ace3 TF protein capable of upregulating expression of an ORF encoding a protein of interest (POI), wherein the ORF is operably linked to an upstream (5') promoter sequence derived from a gene encoding a lignocellulolytic enzyme," specifically refers to an Ace3 TF protein capable of upregulating expression of an ORF encoding a POI in the absence of an inducing substrate.

[0138] As used herein, "upstream (5') promoter sequences derived from genes encoding lignocellulolytic enzymes" include, but are not limited to, cellobiohydrolase promoter sequences, endoglucanase promoter sequences, β-glucosidase promoter sequences, and xylanase promoter sequences.

[0139] As used herein, the term "increased amount" when used in phrases such as "the engineered cells produce an 'increased amount' of a protein of interest (POI) in the presence of an inducing substrate," refers to the "increased amount" of POI produced compared to the amount of the same POI produced by a parent or control cell. For example, an engineered filamentous fungal cell of the present disclosure comprising an Ace3 TF protein capable of upregulating expression of an ORF encoding a POI (i.e., where the ORF is operably linked to a 5' promoter sequence derived from a gene encoding a lignocellulolytic enzyme) produces an increased amount of POI when cultured / fermented in the presence of an inducing substrate compared to the amount of the same POI produced by a control cell comprising a wild-type ace3 gene encoding a wild-type Ace3 TF protein (e.g., SEQ ID NO: 2) when cultured / fermented in the presence of the same inducing substrate.

[0140] As used herein, specific amino acid deletions (truncations) of the wild-type Ace3 C-terminus are abbreviated as "C-term," followed by a numerical designation (e.g., "C-term-5" through "C-term-20" and "C-term-25"), where the numerical designation represents the number of amino acid residues deleted from the Ace3 C-terminus (i.e., relative to the wild-type Ace3 C-terminus ending at glycine (G) at position 689; SEQ ID NO: 2). For example, with reference to the wild-type Ace3 C-terminus shown in FIG. 4 (i.e., which ends at glycine (G) at position 689), a "C-term-7" protein contains a 7-amino acid deletion (truncation) of the last 7 C-terminal amino acids (i.e., "TSTTVVG"), and a "C-term-17" protein contains a 17-amino acid deletion (truncation) of the last 17 C-terminal amino acids (i.e., "DSKASDQLRNTSTTVVG").

[0141] In certain embodiments, reference is made to the wild-type (Ace3) C-terminal amino acid residues and negative numbers are assigned for visual clarity, e.g., as shown in Figure 4. The most C-terminal amino acid position of the Ace3-LC protein (i.e., glycine (G) at position 689) is assigned a negative one (-1), the threonine (T) at position 685 is assigned a negative five (-5), the leucine (L) at position 680 is assigned a negative ten (-10), the lysine (K) at position 675 is assigned a negative fifteen (-15), etc.

[0142] As used herein, the Ace3 "C-term-5" protein (Figure 2A; SEQ ID NO: 7) comprises 684 amino acid residues with a C-terminal five amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with serine (S) at position 684.

[0143] As used herein, the Ace3 "C-term-6" protein (Figure 2A; SEQ ID NO: 8) contains 683 amino acid residues, terminating with a threonine (T) at position 683, and a C-terminal 6 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2).

[0144] As used herein, the Ace3 "C-term-7" protein (Figure 2B; SEQ ID NO: 9) comprises 682 amino acid residues with a C-terminal 7 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with asparagine (N) at position 682.

[0145] As used herein, the Ace3 "C-term-8" protein (Figure 2B; SEQ ID NO: 10) comprises 681 amino acid residues with a C-terminal 8 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with arginine (R) at position 681.

[0146] As used herein, the Ace3 "C-term-9" protein (Figure 2B; SEQ ID NO: 11) comprises 680 amino acid residues with a C-terminal 9 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with leucine (L) at position 680.

[0147] As used herein, the Ace3 "C-term-10" protein (Figure 2C; SEQ ID NO: 12) comprises 679 amino acid residues with a C-terminal 10 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with glutamine (Q) at position 679.

[0148] As used herein, the Ace3 "C-term-12" protein (Figure 2C; SEQ ID NO: 13) contains 677 amino acid residues with a C-terminal 12 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with serine (S) at position 677.

[0149] As used herein, the Ace3 "C-term-13" protein (Figure 2C; SEQ ID NO: 14) contains 676 amino acid residues with a C-terminal 13 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with an alanine (A) at position 676.

[0150] As used herein, the Ace3 "C-term-14" protein (Figure 2D; SEQ ID NO: 15) comprises 675 amino acid residues with a C-terminal 14 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with a lysine (K) at position 675.

[0151] As used herein, the Ace3 "C-term-15" protein (Figure 2D; SEQ ID NO: 16) contains 674 amino acid residues with a C-terminal 15 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with serine (S) at position 674.

[0152] As used herein, the Ace3 "C-term-6" protein (Figure 2D; SEQ ID NO: 17) contains 673 amino acid residues with a C-terminal 16 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with an aspartic acid (D) at position 673.

[0153] As used herein, the Ace3 "C-term-17" protein (Figure 2E; SEQ ID NO: 18) contains 672 amino acid residues with a 17 amino acid truncation at the C-terminus (relative to the WT Ace3 C-terminus; SEQ ID NO: 2), ending with a leucine (L) at position 672.

[0154] As used herein, the Ace3 "C-term-18" protein (Figure 2E; SEQ ID NO: 19) contains 671 amino acid residues with an 18 amino acid truncation at the C-terminus (relative to the WT Ace3 C-terminus; SEQ ID NO: 2), ending with arginine (R) at position 671.

[0155] As used herein, the Ace3 "C-term-19" protein (Figure 2E; SEQ ID NO: 20) comprises 670 amino acid residues with a 19 amino acid truncation at the C-terminus (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with a threonine (T) at position 670.

[0156] As used herein, the Ace3 "C-term-20" protein (Figure 2F; SEQ ID NO: 21) comprises 669 amino acid residues with a C-terminal 20 amino acid truncation (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with leucine (L) at position 669.

[0157] As used herein, the Ace3 "C-term-25" protein (Figure 2F; SEQ ID NO: 22) comprises 664 amino acid residues with a 25 amino acid truncation at the C-terminus (relative to the WT Ace3 C-terminus; SEQ ID NO: 2) ending with a valine (V) at position 664.

[0158] As used herein, the phrase "modified or variant cells comprising a genetic modification to express a gene encoding an Ace3 "C-term" protein" includes, but is not limited to, the introduction of at least one copy of a gene (or ORF) encoding an Ace3 "C-term" protein of the present disclosure (e.g., C-term-7; SEQ ID NO:9 through C-term-17; SEQ ID NO:18). In certain embodiments, the gene (or ORF) encoding the Ace3 C-term protein is operably linked to an upstream (5') heterologous promoter sequence (i.e., the ace3 gene is not linked to its native ace3 promoter sequence) and / or the gene (or ORF) encoding the Ace3 C-term protein is operably linked to a downstream (3') heterologous terminator sequence.

[0159] In certain embodiments, modified cells comprising a genetic modification to express a gene encoding the Ace3 "C-term" protein of the present disclosure (e.g., C-term-7; SEQ ID NO: 9 to C-term-17; SEQ ID NO: 18) comprise a modification of the endogenous ace3 gene by CRISPR / Cas9 editing, wherein the CRISPR / Cas9-modified ace3 gene encodes the Ace3 "C-term" protein of the present disclosure.

[0160] In certain embodiments, fungal cells of the present disclosure can be screened for the presence of an endogenous ace3 gene encoding a truncated N-terminal zinc binuclear cluster DNA-binding domain. For example, Figure 3 (SEQ ID NO: 29) shows the intact DNA-binding domain as found in the Ace3-L (SEQ ID NO: 6) and Ace3-LC (SEQ ID NO: 2) proteins. Thus, in certain embodiments, parental fungal cells screened and confirmed as containing the (endogenous) truncated N-terminus are genetically modified herein to restore the full-length (wild-type) N-terminus (SEQ ID NO: 29).

[0161] Similarly, fungal cells of the present disclosure can be screened for the presence of an endogenous ace3 gene encoding a full-length (wild-type) Ace3 C-terminus (e.g., Ace3-LC; SEQ ID NO:2). For example, FIG. 4 shows the full-length (wild-type) C-terminus of the Ace3-LC protein (SEQ ID NO:2). Accordingly, in certain embodiments, parental fungal cells screened and confirmed as containing an endogenous ace3 gene encoding a full-length (wild-type) C-terminus are genetically modified herein to delete (truncate) at least 7, but not more than 17, amino acids from the full-length (wild-type) C-terminus. Thus, as shown in FIG. 4, the Ace3 C-terminal amino acid residue positions indicated by gray shading (i.e., from position -7 (T) to position -17 (D)) indicate the minimum (at least 7) ​​and maximum (not more than 17) number of amino acid deletions (truncations) permitted at the Ace3 C-terminus, as further described below.

[0162] In other embodiments, the modified filamentous fungal cells of the present disclosure will comprise additional genetic modifications. For example, in certain embodiments, such variant filamentous fungal cells may further comprise a genetic modification that reduces the expression and / or activity of a gene encoding the carbon catabolite repressor protein "Cre1" or the "Ace1" repressor protein. In other embodiments, such modified filamentous fungal cells further comprise a genetic modification that introduces at least one copy of xylanase regulator 1 (Xyr1).

[0163] As used herein, the term "host cell" refers to a filamentous fungal cell capable of acting as a host and expression vehicle for an incoming sequence (i.e., a polynucleotide sequence that is introduced into the cell), as described herein.

[0164] A "heterologous" nucleic acid construct or sequence has a portion of a sequence that is not native to the cell in which it is expressed or that does not exist in its natural form. With respect to regulatory sequences, heterologous refers to regulatory sequences (e.g., promoters, enhancers) that do not naturally exert a regulatory effect on the same gene that they currently regulate expression. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, but rather have been added to the cell by infection, transfection, transformation, microinjection, electroporation, etc. A "heterologous" nucleic acid construct can contain a regulatory sequence / DNA coding sequence combination that is identical to or different from the regulatory sequence / DNA coding sequence combination found in the native cell. Similarly, a heterologous protein often refers to two or more subsequences (e.g., a fusion protein) that are not found in the same relationship to each other in nature.

[0165] As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene or its open reading frame (ORF). A promoter is generally appropriate for the host cell in which the target gene is expressed. A promoter, along with other transcriptional and translational regulatory nucleic acid sequences (also called "control sequences"), is necessary to express a given gene. Generally, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In certain embodiments, the promoter is an inducible promoter. In other embodiments, the promoter is a constitutive promoter.

[0166] As used herein, a "promoter sequence" is a DNA sequence recognized by a particular filamentous fungus for expression purposes. A "promoter" is defined as an array of nucleic acid control sequences that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the transcription start site, e.g., a TATA element, in the case of a polymerase II type promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under particular environmental or developmental regulation.

[0167] As used herein, the term "operably linked" refers to the functional linkage of a nucleic acid expression control sequence (e.g., a promoter or an array of transcription factor binding sites) with a second nucleic acid sequence, such that the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. Thus, a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader (i.e., a signal peptide) is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. In other embodiments, the ligation is performed by seamless cloning, which joins DNA in a sequence-independent, scarless manner, typically performed using commercially available systems such as, but not limited to, Gibson Assembly (NEB), NEBuilder HiFi DNA Assembly (NEB), Golden Gate Assembly (NEB), and the GeneArt Seamless cloning and Assembly system (ThermoFisher Scientific).

[0168] As used herein, the term "heterologous promoter" or "heterologous promoter sequence," when used in phrases such as "an ace3 gene (or its ORF) operably linked to an upstream (5') heterologous promoter sequence," is meant to distinguish it from the native ace3 promoter sequence, and the heterologous promoter sequence operably linked to the ace3 gene can be any promoter sequence that functions in the filamentous fungal cells of the present disclosure (i.e., excluding the native ace3 gene promoter).

[0169] Similarly, when the term "heterologous promoter sequence" is used in phrases such as "a gene of interest (GOI) encoding a protein of interest (POI) is operably linked to an upstream (5') heterologous promoter sequence," the term "heterologous promoter" is meant to distinguish it from the native promoter sequence of the gene of interest; the heterologous promoter sequence operably linked to the GOI encoding the POI can be any promoter sequence that functions in the filamentous fungal cell of the present disclosure (i.e., excluding the native promoter of the GOI).

[0170] As used herein, the term "DNA construct" or "expression construct" refers to a nucleic acid sequence comprising 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. The DNA can be generated in vitro (e.g., by PCR) or by any other suitable technique. In some preferred embodiments, the DNA construct comprises a sequence of interest (e.g., encoding an Ace3-L protein). In certain embodiments, the polynucleotide sequence of interest is operably linked to a promoter. In some embodiments, the DNA construct further comprises at least one selectable marker. In further embodiments, the DNA construct comprises a sequence homologous to a host cell chromosome. In other embodiments, the DNA construct comprises a sequence heterologous to a host cell chromosome.

[0171] As used herein, the phrase "lignocellulolytic" enzymes includes cellobiohydrolases, endoglucanases and β-glucosidases.

[0172] As used herein, the terms "cellulase," "cellulolytic enzyme," or "cellulase enzyme" refer to bacterial or fungal enzymes, such as exoglucanases, exocellobiohydrolases, endoglucanases, and / or β-glucosidases. These different types of cellulase enzymes work synergistically to convert cellulose and its derivatives to glucose. For example, many microorganisms, such as the wood-rotting fungus Trichoderma, the composting bacteria Thermomonospora (now Thermobifida), Bacillus, and Cellulomonas; Streptomyces; and the fungi Humicola, Aspergillus, and Fusarium, produce enzymes that hydrolyze cellulose. The enzymes produced by these microorganisms are a mixture of three active proteins useful in the conversion of cellulose to glucose: endoglucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (BG). As defined herein, the terms "endoglucanase" (EG), "cellobiohydrolase" (CBH), and "β-glucosidase" (BG) are used interchangeably with their abbreviations "EG," "CBH," and "BG," respectively.

[0173] As used herein, the term "coding sequence" refers to a nucleotide sequence that directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0174] As defined herein, "open reading frame" (hereinafter "ORF") means a nucleic acid or nucleic acid sequence (whether naturally occurring, non-naturally occurring, or synthetic) that comprises a continuous reading frame consisting of (i) an initiation codon, (ii) a series of codons that designate amino acids, and (iii) a stop codon, and the ORF is read (or translated) in the 5' to 3' direction.

[0175] As used herein, the term "gene" refers to a segment of DNA involved in producing a polypeptide chain, which may or may not include regions preceding and following the coding region (e.g., 5' untranslated (5' UTR) or "leader" sequence, and 3' UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons). A gene may encode commercially important industrial proteins or peptides, such as enzymes (e.g., proteases, mannanases, xylanases, amylases, glucoamylases, cellulases, oxidases, lipases, etc.). A gene of interest may be a naturally occurring gene, a mutated gene, or a synthetic gene.

[0176] The term "recombinant," as used herein with respect to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by the modification of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses naturally occurring genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0177] The term "vector" is defined herein as a polynucleotide designed to carry a nucleic acid sequence for introduction into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage or viral particles, DNA constructs, cassettes, etc. Expression vectors can include regulatory sequences such as a promoter, signal sequence, coding sequence, and transcription terminator.

[0178] As used herein, "expression vector" refers to a DNA construct containing a coding sequence operably linked to suitable control sequences capable of directing the expression of a protein in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0179] As used herein, the term "secretory signal sequence" refers to a DNA sequence that encodes a polypeptide (i.e., a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through the secretory pathway of the cell in which it is synthesized. The larger polypeptide is typically cleaved to remove the secretory peptide during passage through the secretory pathway.

[0180] As used herein, the term "induction" refers to an increase in transcription of a gene that results in the synthesis of a protein of interest (POI) in a filamentous fungal cell at a significantly greater rate in response to the presence of an "inducer" (i.e., an inducing substrate). To measure "induction" of a "gene of interest" (GOI) or "ORF of interest" encoding a POI, variant filamentous fungal (host) cells are treated with a candidate inducing substrate (inducer) and compared to parental filamentous fungal (control, unmodified) cells that have not been treated with the inducing substrate (inducer). Thus, if the (untreated) parental (control) cells are assigned a relative protein activity value of 100%, induction of the GOI encoding the POI in the variant (modified) host cells is achieved when the activity value (i.e., relative to the control cells) is greater than 100%, greater than 105%, greater than 110%, greater than 150%, greater than 200-500% (i.e., relative to the control), or even higher.

[0181] As used herein, "inducer," "inducers," "inducing substrate," or "inducing substrates" are used interchangeably and refer to any compound that causes a filamentous fungal cell to produce "increased amounts" of a polypeptide (e.g., an enzyme, receptor, antibody, etc.) or to produce a compound / substance other than that which would be produced in the absence of the inducing substrate. Examples of inducing substrates include, but are not limited to, sophorose, lactose, gentiobiose, and cellulose.

[0182] As used herein, the term "inducing feed material" refers to a composition comprising at least an "inducing substrate" (sophorose, lactose, gentiobiose, cellulose) that is fed to a filamentous fungal cell.

[0183] As used herein, the terms "isolated" or "purified" refer to a filamentous fungal cell, nucleic acid, or polypeptide that has been removed from at least one component with which it is naturally associated.

[0184] As defined herein, the term "protein of interest" or "POI" refers to a polypeptide that is desired to be expressed in a filamentous fungal cell. Such proteins may be enzymes, substrate-binding proteins, surface-active proteins, structural proteins, etc., and may be expressed at high levels and intended for commercialization. Proteins of interest may be encoded by endogenous genes or heterologous genes. Proteins of interest may be expressed intracellularly or as secreted (extracellular) proteins.

[0185] In certain embodiments, the gene (or ORF) encoding the POI to be expressed / produced in a filamentous fungal cell of the present disclosure comprises an upstream (5') cellulase gene promoter (e.g., cbh1) operably linked to the POI-encoding gene or ORF. Thus, expression of the POI-encoding gene or ORF under the control of the cellulase promoter (e.g., cbh1) is upregulated in the presence of one or more of the modified Ace3 TF proteins described herein.

[0186] As used herein, the terms "polypeptide" and "protein" (and / or their respective plurals) are used interchangeably and refer to polymers of any length comprising amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified, either naturally or by intervention (e.g., by 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 analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0187] As used herein, functionally and / or structurally similar proteins are considered to be "related proteins." Such proteins may be derived from organisms of different genera and / or species, or from organisms of different taxa (e.g., bacteria and fungi). Related proteins also encompass homologs as determined by primary sequence analysis, by secondary or tertiary structure analysis, or by immunological cross-reactivity.

[0188] As used herein, the phrase "substantially free of activity," or similar phrases, means that the particular activity cannot be detected in the mixture or is present in an amount that does not interfere with the intended purpose of the mixture.

[0189] As used herein, the term "derived polypeptide" refers to a protein obtained or obtainable from a protein by the addition of one or more amino acids to one or both of the N- and C-termini, the substitution of one or more amino acids at one or more different sites in the amino acid sequence, the deletion of one or more amino acids at one or both termini of the protein or at one or more sites in the amino acid sequence, and / or the insertion of one or more amino acids at one or more sites in the amino acid sequence. Preparation of a protein derivative can be accomplished by modifying a DNA sequence encoding the native protein, transforming the DNA sequence into a suitable host, and expressing the modified DNA sequence to form a derived protein.

[0190] Related (and derived) proteins include "variant proteins." Variant proteins differ from a reference / parent protein (e.g., a wild-type protein) by substitutions, deletions, and / or insertions of a small number of amino acid residues. The number of different amino acid residues between a variant protein and a parent protein can be one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more amino acid residues. A variant protein can share at least about 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 at least about 99% or more amino acid sequence identity with the reference protein. A variant protein can also differ from a reference protein in selected motifs, domains, epitopes, conserved regions, etc.

[0191] As used herein, the term "homologous protein" refers to a protein that has a similar activity and / or structure to a reference protein. Homologues are not necessarily evolutionarily related. Thus, the term is intended to encompass identical, similar, or corresponding proteins (i.e., with respect to structure and function) obtained from different organisms. In some embodiments, it is desirable to identify homologues that have a similar quaternary, tertiary, and / or primary structure to the reference protein. In some embodiments, the homologous protein induces a similar immune response as the reference protein. In some embodiments, the homologous protein is genetically engineered to produce an enzyme with a desired activity.

[0192] The degree of homology between sequences may be determined using any suitable method known in the art (see, for example, Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., 1984).

[0193] As used herein, the phrases "substantially similar" and "substantially identical," in the context of at least two nucleic acids or polypeptides, typically mean that the polynucleotide or polypeptide comprises a sequence having at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% or more identity compared to a reference (i.e., wild-type) sequence. Sequence identity can be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters.

[0194] As used herein, "gene deletion" refers to the removal of a gene from the genome of a host cell. When a gene contains regulatory elements (e.g., enhancer elements) that are not located immediately adjacent to the coding sequence of the gene, gene deletion refers to the deletion of the coding sequence and, optionally, the deletion of adjacent enhancer elements, including, but not limited to, for example, promoter and / or terminator sequences.

[0195] As used herein, "gene disruption" broadly refers to any genetic or chemical manipulation, i.e., mutation, that substantially prevents a cell from producing a functional gene product, e.g., a protein, within a host cell. Exemplary disruption methods include complete or partial deletion or mutagenesis of any portion of a gene, including a polypeptide coding sequence, promoter, enhancer, or other regulatory element, including substitutions, insertions, deletions, inversions, and combinations and variations thereof, all of which mutations substantially prevent the production of a functional gene product. Gene expression can also be disrupted using RNAi, CRISPR / Cas9, or any other method that eliminates, reduces, or diminishes gene expression.

[0196] As used herein, "aerobic fermentation" refers to growth in the presence of oxygen.

[0197] As used herein, the term "cell broth" refers collectively to the medium and cells in a liquid / submerged culture.

[0198] As used herein, the term "cell mass" refers to the cellular components present in a liquid / submerged culture, including intact cells and lysed cells. Cell masses can be expressed as dry weight or wet weight.

[0199] As used herein, a "functional protein" is a protein that has an activity, such as an enzymatic activity, a binding activity, a surfactant property, etc., and that has not been mutated, truncated, or otherwise modified to eliminate or reduce that activity.

[0200] As used herein, a "functional gene" is a gene that can be used by cellular constituents to produce an active gene product, typically a protein. A functional gene is the opposite of a disrupted gene that cannot be used by cellular constituents to produce an active gene product, or that has been modified to reduce the ability of cellular constituents to use it to produce an active gene product.

[0201] As used herein, a "protein of interest" is a protein whose production is desired in a submerged culture of filamentous fungal cells. Generally, proteins of interest are commercially important in industrial, pharmaceutical, animal health, and food and beverage applications, and it is desirable to produce them in large quantities. A protein of interest is to be distinguished from the countless other proteins expressed by filamentous fungal cells, which are generally not of interest as products and are primarily considered background protein contaminants.

[0202] III. Activator of cellulase expression 3 (Ace3) As reviewed by Hakkinen et al. (2014), a T. reesei gene designated "activator of cellulase expression 3" (ace3) encodes a transcription factor (TF) protein designated "Ace3," and the Ace3 TF regulates cellulase and hemicellulase gene expression. Furthermore, applicant's International Publication No. WO 2018 / 067599 identifies specific variant forms of the Ace3 TF protein that can upregulate cellulase / hemicellulase expression / production in T. reesei strains in the absence of inducing substrates (e.g., lactose, sophorops). For example, WO 2018 / 067599 describes surprising and unexpected results when evaluating the cloned ace3 ORF described in Hakkinen et al. (2014) (i.e., based on the ace3 T. reesei "strain QM6a" (annotated)) in comparison with the ace3 ORF based on the Treesei "strain RUT-C30" (annotated).

[0203] More specifically, WO 2018 / 067599 identified mutant ace3 genes encoding variant Ace3 TFs capable of upregulating cellulase / hemicellulase gene expression in T. reesei strains in the absence of an inducing substrate. As described in WO 2018 / 067599, the mutant ace3 genes in T. reesei strains such as RL-P37 and RUT-C30 contained a premature stop codon at the C-terminus of the variant Ace3 TF (i.e., compared to the C-terminus of the encoded wild-type Ace3 TF protein), resulting in a truncation of 11 amino acids. For example, as shown in Figure 1A of the present disclosure. The variant Ace3 protein, designated "Ace3-S" (SEQ ID NO: 4), has a shorter (truncated) N-terminal sequence in which two of the six cysteine ​​residues of the zinc binuclear cluster (Zn2Cys6) DNA-binding domain (i.e., the wild-type Ace3 TF zinc binuclear cluster (Zn2Cys6) DNA-binding domain; as seen in SEQ ID NO: 29) are deleted, and as shown in Figure 1B, the variant Ace3-S TF protein (SEQ ID NO: 4) contains a full-length (wild-type) C-terminus ending in glycine (G). Furthermore, as described in WO 2018 / 067599 and shown in FIG. 1A of the present disclosure, the wild-type Ace3 protein designated "Ace3-LC" (SEQ ID NO: 2) contains a full-length N-terminus with an intact zinc binuclear cluster (Zn2Cys6) DNA-binding domain (SEQ ID NO: 29), and as shown in FIG. 1B, the wild-type Ace3-LC protein (SEQ ID NO: 2) contains a full-length (wild-type) C-terminus ending in glycine (G). In contrast, as shown in FIG. 1A of the present disclosure, the variant Ace3 protein designated "Ace3-L" (SEQ ID NO: 6) contains a full-length (wild-type) N-terminus with an intact zinc binuclear cluster (Zn2Cys6) DNA-binding domain (SEQ ID NO: 29), and as shown in FIG. 1B, the Ace3-L protein (SEQ ID NO: 6) contains an 11-amino acid truncated C-terminus ending in aspartic acid (D).A recent publication by Zhang et al. (2019) further substantiated the applicant's experimental observation that an Ace3 variant with a full-length zinc cluster (Zn2Cys6) DNA-binding domain can bind to DNA.

[0204] Thus, as outlined in WO 2018 / 067599, Trichoderma cells containing an ace3 gene (or open reading frame thereof) encoding a variant Ace3-L protein (i.e., containing an 11 amino acid truncation at the C-terminus; SEQ ID NO: 6) were able to produce lignocellulolytic enzymes in the absence of an inducing substrate (i.e., when fermented / cultured under identical conditions) compared to Trichoderma cells containing an ace3 gene (or open reading frame thereof) encoding a variant Ace3-S protein (SEQ ID NO: 4) or a wild-type Ace3-LC protein (SEQ ID NO: 2). More recently, a publication by Chen et al. (2020) further substantiated the applicant's experimental observation that Trichoderma cells containing a mutant ace3 gene encoding a variant Ace3 TF protein having a truncation (deletion) of the last 11 C-terminal amino acid residues at the native ace3 locus (i.e., compared to the full-length (wild-type) Ace3 C-terminus) enable such Trichoderma cells to produce increased amounts of lignocellulolytic enzymes (e.g., cellulases / hemicellulases) under inducing conditions compared to Trichoderma cells containing a wild-type ace3 gene encoding the wild-type Ace3 protein.

[0205] Therefore, when comparing the results of the present disclosure with the results / observations of other authors' publications (e.g., Zhang et al. 2019; Chen et al. 2020), the applicants emphasize and note the following differences between these observations. For example, as generally described herein and shown in the Examples section below, the applicants use a strain with an ace3 gene encoding a truncated Ace3 TF protein at its native locus as the (unmodified) parent strain, and overexpress a variant ace3 gene encoding a variant Ace3 TF protein at an ectopic locus in the (modified) daughter strain. Thus, the (modified) daughter cells of the present disclosure produce a mixture of overexpressed variant Ace3 TF proteins in addition to the native Ace3 TF protein with a truncated C-terminus. In contrast, the Chen et al. (2020) publication only introduces ace3 truncation at its native locus. Similarly, Zhang et al. (2019) described a promoter swap strategy to overexpress two variant Ace3 TF proteins (wherein the first variant Ace3 TF contains an incomplete zinc cluster (Zn2Cys6) DNA-binding domain at the N-terminus and a wild-type C-terminus, and the second variant Ace3 TF contains a complete / intact zinc cluster (Zn2Cys6) DNA at the N-terminus and a wild-type C-terminus, similar but not identical to the wild-type Ace3 TF) at the ace3 locus of T. reesei strain QM6a in the absence of its native Ace3 TF. Thus, while the two studies by Zang et al. (2019) and Chen et al. (2020) mentioned above generally demonstrate specific improvements in protein production under inducing conditions (i.e., when cultured / fermented in the presence of an inducing substrate), neither publication demonstrates or describes improvements in protein production under non-inducing conditions (i.e., when cultured / fermented in the absence of an inducing substrate).

[0206] In the present disclosure, applicants further evaluated and analyzed the wild-type C-terminus of the Ace3-LC TF protein (SEQ ID NO: 2; this wild-type Ace3-LC protein requires an induction substrate to express / produce lignocellulolytic enzymes) compared with the truncated C-terminus of the Ace3-L TF protein (SEQ ID NO: 6; this Ace3-L protein does not require an induction substrate to express / produce lignocellulolytic enzymes). More specifically, as described in Example 1 below, applicants constructed multiple ace3 expression vectors encoding variant Ace3 proteins containing consecutive amino acid deletions (truncations) at the C-terminus.

[0207] For example, a first set of six vectors containing C-terminal deletions ranging from 0 to 25 amino acids, using five amino acid deletion increments, was constructed (see, e.g., Table 1; Set 1). Similarly, a second set of 12 vectors containing C-terminal deletions ranging from 6 to 19 amino acids, using one amino acid deletion increments, was constructed (Table 1; Set 2). Thus, the 18 Ace3 C-terminal variant proteins constructed in Example 1 (listed in Table 3) are based on the Ace3-LC protein sequence (SEQ ID NO: 2), which contains the full-length (wild-type) Ace3 C-terminus ending at glycine (G) at position 689.

[0208] Applicant subsequently tested / screened the constructed T. reesei strains (Table 3) for protein production under "non-induction" conditions (glucose) and "induction" conditions (glucose / sophorose, or lactose). For example, as shown in Figure 7. Applicant evaluated total protein production using a first set of six strains expressing the (Ace3-LC) C-terminal variants. The parental control strain (RL-P37) was only able to produce large amounts of protein on lactose (i.e., under "induction" conditions) and produced minimal basal levels of protein on glucose (i.e., under "non-induction" conditions). Similarly, as shown in Figure 7, daughter cells expressing the (Ace3-LC) C-terminal variants with the 10 amino acid deletion (Cterm-10), 11 amino acid deletion (Cterm-11), or 15 amino acid deletion (Cterm-15) showed increased protein production under both lactose ("induction" conditions) and glucose ("non-induction" conditions). For example, as shown in Figure 7. The increase in fold change is approximately 1.8-fold on glucose compared to the protein levels produced by the parent strain (RL-P37) on lactose, and approximately 3-fold on lactose compared to the protein levels produced by the parent strain (RL-P37) on lactose.

[0209] In contrast, as shown in Figure 7, the wild-type (Ace3-LC) C-terminus (i.e., containing a 0 amino acid deletion; SEQ ID NO: 2) and the (Ace3-LC) C-terminal variants containing 20 amino acid deletions (SEQ ID NO: 21) or 25 amino acid deletions (SEQ ID NO: 22) significantly reduced total protein production on both lactose and glucose. These results indicate that truncation of the wild-type (Ace3-LC) C-terminus is essential for the function of upregulating protein production and that there are upper and lower limits to the number of amino acids and truncations tolerated at the Ace3 C-terminus.

[0210] To further explore the upper and lower limits of tolerated amino acid deletions at the wild-type (Ace3-LC) C-terminus, Applicant screened T. reesei strains containing C-terminal deletions ranging from 6 to 19 amino acids using one-amino acid deletion increments (see, e.g., Table 1, Set 2; and Table 3). For example, as shown in Figure 8, truncation of 5 or 6 C-terminal amino acids showed results similar to the parent strain (i.e., basal protein production on glucose and higher production on lactose). In contrast, truncation of at least the last 7 amino acids at the C-terminus (Figure 5) and truncation of up to and including 17 amino acids (Figure 5) showed improved production under both glucose (non-inducing) and lactose (inducing) conditions, as shown in Figure 8.

[0211] For example, T. reesei strains expressing the (Ace3-LC) C-terminal variants C-term-7 (SEQ ID NO: 9), C-term-8 (SEQ ID NO: 10), C-term-9 (SEQ ID NO: 11), C-term-10 (SEQ ID NO: 12), C-term-12 (SEQ ID NO: 13), C-term-13 (SEQ ID NO: 14), C-term-14 (SEQ ID NO: 15), C-term-15 (SEQ ID NO: 16), C-term-16 (SEQ ID NO: 17), and C-term-17 (SEQ ID NO: 18) showed an approximately two-fold increase in total protein production under glucose (non-inducing) conditions compared to the parent strain RL-P37 on lactose, and an approximately three-fold increase in total protein production under lactose (inducing) conditions compared to the parent strain RL-P37 on lactose. In contrast, T. reesei strains expressing the wild-type Ace3-LC protein (i.e., containing the wild-type Ace3 C-terminus; SEQ ID NO: 2) produced reduced amounts of protein on lactose compared to the parent strain RL-P37 on lactose, with production on glucose being minimal (Figure 8). T. reesei strains expressing the (Ace3-LC) C-terminal variant with fewer than seven amino acid deletions at the C-terminus produced amounts of protein on glucose and lactose comparable to the parent strain RL-P37 under the same conditions. T. reesei strains expressing the (Ace3-LC) C-terminal variant with more than 17 amino acid deletions at the C-terminus produced minimal amounts of protein under either glucose or lactose conditions (Figure 8).

[0212] As described in Example 2 below, Applicants molecularly reverted a C-terminal truncation point mutation in the ace3 locus (i.e., encoding Ace3-L; SEQ ID NO: 6) to the wild-type (Ace3) C-terminal sequence of QM6a (i.e., encoding Ace3-LC) ending in (G)glycine (FIG. 1B; SEQ ID NO: 2). More specifically, as described in Example 2 and shown in FIG. 9, Applicants evaluated the protein production of three strains in shake flask fermentation. The three strains included the "T4abc" strain (containing an ace3 truncation allele encoding Ace3-L; SEQ ID NO: 6), the "T4abc ace3_rev" strain (containing an ace3 full-length allele encoding Ace3-LC; SEQ ID NO: 2), and the "T4abc del-cbh1" strain (containing a deletion of its endogenous cellobiohydrolase (cbh1) gene). As shown in Figure 9, when the ace3 mutation (Ace3-L; SEQ ID NO: 6) was reverted to encode the wild-type (full-length) Ace3 C-terminus (Ace3-LC; SEQ ID NO: 2) in a T4abc background, the total secreted protein titer was reduced by approximately 73%. For comparison, deletion of the gene encoding the most prominent secreted protein (Cbh1) reduced the total secreted protein titer by only 47%.

[0213] In addition to the native Ace3 C-terminal truncations (deletions) described above, Applicants have contemplated, constructed, tested, and validated certain other Ace3 C-terminal modifications described herein. More specifically, without wishing to be bound by any particular mechanism or method of manipulation, Applicants believe that other genetic modifications of the Ace3 C-terminus (e.g., substitutions, insertions, internal (C-terminal) deletions, and combinations thereof) are equally suitable genetic modifications for improving protein production, as described herein. For example, as shown in Example 3 below, Applicants describe compositions and methods for constructing an Ace3 TF C-terminal variant library and screening such a library to improve protein productivity in Trichoderma fungal cells. The Ace3 C-terminal variant library is generated by modifying the coding sequence of the full-length (wild-type) Ace3-LC C-terminus, which encodes amino acid positions 641-689 of SEQ ID NO:2. For example, Figure 15 (SEQ ID NO: 30) shows the full-length (wild-type) Ace3-LC C-terminus encoding amino acid positions S641 to G689. More specifically, Applicants describe three different Ace3 C-terminus libraries, including a site-substitution library (Library 1), a scanning insertion library (Library 2), and a scanning deletion library (Library 3).

[0214] Example 4 of the present disclosure describes the molecular replacement of the last 11 amino acid residues at the C-terminus of the Ace3-LC protein with a V5 epitope tag or a V5-(6xHis) tandem tag. Trichoderma strains expressing either an Ace3-LC protein with a C-terminal V5 tag substitution (i.e., Ace3-LC-V5) or an Ace3-LC protein with a C-terminal V5-(6xHis) tandem tag substitution (i.e., Ace3 LC-V5-(6xHis)) were tested (fermented) under both "uninduced" (Glu) and "induced" (Glu / Sop) conditions.

[0215] For example, modified (daughter) T. reesei cells containing and expressing a variant Ace3-L TF protein (SEQ ID NO: 6) containing an 11-amino acid truncation produced large amounts of secreted protein under both induced (Glu / Sop) and uninduced (Glu) conditions. Furthermore, modified (daughter) T. reesei cells containing and expressing Ace3-LC-V5 (i.e., containing 14 amino acid substitutions in the last 11 amino acids) and variant (daughter) T. reesei cells containing and expressing Ace3-LC-V5-(6xHis) (i.e., containing 23 amino acid substitutions in the last 11 amino acids) also exhibited high protein productivity, although approximately 10% lower than that of the Ace3-L variant, under both induced (Glu / Sop) and uninduced (Glu) conditions (see Figure 14). Thus, these results indicate that genetic modifications of the Ace3 C-terminus, including but not limited to substitutions, insertions, internal (C-terminal) deletions, and combinations thereof, are equally suitable genetic modifications for improving protein productivity, as described herein.

[0216] IV. Recombinant Nucleic Acids and Molecular Biology In certain embodiments, the present disclosure is directed to an isolated polynucleotide (nucleic acid sequence) encoding a variant Ace3 TF protein. In certain embodiments, the variant Ace3 TF comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2 and comprises a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2. Accordingly, certain other embodiments relate to genetically modified Trichoderma fungal cells comprising an ace3 gene encoding a variant Ace3 TF protein of the present disclosure, wherein the modified cell is capable of producing a protein of interest (POI) in the absence of an inducing substrate and / or the modified cell is capable of producing increased amounts of the POI in the presence of an inducing substrate. In certain embodiments, "modification" or "genetic modification" includes: (a) the introduction, substitution, or removal of one or more nucleotides in a gene (or its ORF), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of a gene or its ORF; (b) gene disruption; (c) gene conversion; (d) gene deletion; (e) gene downregulation; (f) directed mutagenesis; and / or (g) random mutagenesis of any one or more genes disclosed herein.

[0217] Thus, in certain embodiments, the present disclosure is directed to a recombinant nucleic acid comprising a gene or ORF encoding an Ace3 protein. In certain embodiments, the recombinant nucleic acid comprises a polynucleotide expression cassette for producing an Ace3 variant protein in a filamentous fungal host cell. In other embodiments, the polynucleotide expression cassette is comprised within an expression vector. In certain embodiments, the expression vector is a plasmid.

[0218] In other specific embodiments, the recombinant nucleic acid (or its polynucleotide expression cassette or its expression vector) further comprises one or more selectable markers. Selectable markers for use in filamentous fungi include, but are not limited to, alsl, amdS, hygR, pyr2, pyr4, pyrG, sucA, bleomycin resistance markers, blasticidin resistance markers, pyrithiamine resistance markers, chlorimuron ethyl resistance markers, neomycin resistance markers, adenine pathway genes, tryptophan pathway genes, thymidine kinase markers, and the like. In specific embodiments, the selectable marker is pyr2, the components and methods of use of which are generally described in WO 2011 / 153449. Thus, in certain embodiments, a polynucleotide construct encoding an Ace3 protein of the present disclosure comprises, operably linked thereto, a nucleic acid sequence encoding a selectable marker.

[0219] In another embodiment, the recombinant nucleic acid, polynucleotide construct, polynucleotide expression cassette, or expression vector thereof comprises a heterologous promoter driving expression of a gene (or ORF) encoding a variant Ace3 protein. More particularly, in certain embodiments, the heterologous promoter is a constitutive promoter or an inducible promoter. In certain embodiments, the heterologous promoter is selected from the group consisting of rev3 promoter, bxl promoter, tkl1 promoter, PID104295 promoter, dld1 promoter, xyn4 promoter, PID72526 promoter, axe1 promoter, hxk1 promoter, dic1 promoter, opt promoter, gut1 promoter, and pki1 promoter. Thus, in certain embodiments, the recombinant nucleic acid (or polynucleotide construct, polynucleotide expression cassette, or expression vector thereof) comprises a promoter sequence operably linked 5' to the nucleic acid sequence encoding the variant Ace3 protein.

[0220] In another embodiment, the recombinant nucleic acid (or polynucleotide construct, polynucleotide expression cassette, or expression vector thereof) further comprises a nucleic acid sequence encoding a native ace3 terminator sequence. Thus, in certain embodiments, the recombinant nucleic acid (or polynucleotide construct, polynucleotide expression cassette, or expression vector thereof) comprises a heterologous promoter operably linked 5' to the nucleic acid sequence encoding the Ace3 protein, and also comprises a native ace3 terminator sequence operably linked 3' to the nucleic acid sequence encoding the variant Ace3 protein (e.g., 5'-Pro-ORF-Term-3', where "Pro" is the constitutive promoter, "ORF" encodes Ace3, and "Term" is the native ace3 terminator sequence).

[0221] An example of a regulatory or control sequence may be a promoter sequence or a functional portion thereof (i.e., a portion 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.

[0222] In certain other embodiments, a gene of interest (or ORF) encoding a protein of interest is placed under the control of a promoter sequence derived from a gene encoding a lignocellulolytic enzyme. For example, in certain embodiments, the gene of interest (or ORF) encoding a protein of interest is placed under the control of the cbh1 promoter, such that expression of the GOI is upregulated in the presence of a variant Ace3 TF protein of the present disclosure. Thus, in certain embodiments, the promoter sequence derived from a gene encoding a lignocellulolytic enzyme is a cellobiohydrolase promoter sequence, an endoglucanase promoter sequence, a β-glucosidase promoter sequence, or a xylanase promoter sequence.

[0223] In certain embodiments, modified Trichoderma fungal cells of the present disclosure are constructed by reducing or eliminating expression of a gene of interest (GOI) using methods well known in the art, such as insertion, disruption, substitution, or deletion. The portion of the gene to be modified or inactivated can be, for example, a coding region or a regulatory element required for expression of the coding region.

[0224] In certain other embodiments, modified Trichoderma fungal cells are constructed by gene deletion to eliminate or reduce expression of at least one GOI. Gene deletion techniques allow for the partial or complete removal of genes, thereby eliminating their expression or resulting in the expression of a non-functional (or reduced activity) protein product. In such methods, gene deletion can be accomplished by homologous recombination using a plasmid constructed to contain adjacent 5' and 3' regions flanking the gene.

[0225] In other embodiments, modified Trichoderma fungal cells of the present disclosure are constructed by introducing, substituting, or removing one or more nucleotides within a gene or regulatory element required for its transcription or translation. For example, nucleotides may be inserted or removed to introduce a stop codon, remove a start codon, or cause a frameshift in the open reading frame. Such modifications may be accomplished by site-directed mutagenesis or PCR-generated mutagenesis, according to methods known in the art.

[0226] In another embodiment, modified Trichoderma fungal cells are constructed by a gene transformation process. For example, in gene transformation, a nucleic acid sequence corresponding to a gene is mutated in vitro to generate a defective nucleic acid sequence, which is then transformed into a parent Trichoderma fungal cell to generate the defective gene. The defective nucleic acid sequence replaces the endogenous gene through homologous recombination. It may also be desirable for the defective gene or gene fragment to encode a marker that can be used to select for transformants containing the defective gene. For example, the defective gene can be introduced into a non-replicating or temperature-sensitive plasmid associated with a selectable marker. Selection for integration of the plasmid is carried out by selecting for that marker under conditions that do not permit plasmid replication. Selection for a second recombination event resulting in gene replacement is carried out by examining colonies for loss of the selectable marker and acquisition of the mutated gene (Perego, 1993). Alternatively, the defective nucleic acid sequence can contain an insertion, substitution, or deletion of one or more nucleotides of the gene, as described below.

[0227] In other embodiments, modified Trichoderma fungal cells are constructed using established antisense technology, using a nucleotide sequence complementary to the nucleic acid sequence of a gene (Parish and Stoker, 1997). More specifically, gene expression by Trichoderma fungal cells can be reduced (downregulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which can be transcribed within the cell and hybridize to mRNA produced within the cell. Thus, under conditions in which the complementary antisense nucleotide sequence can hybridize to the mRNA, the amount of translated protein is reduced or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, and the like, all of which are well known to those skilled in the art.

[0228] In other embodiments, modified Trichoderma fungal cells are produced / constructed by CRISPR-Cas9 editing. For example, a gene of interest can be disrupted (or deleted or downregulated) by a nucleic acid-guided endonuclease that finds its target DNA by binding to a guide RNA (e.g., Cas9) and Cpf1 or a guide DNA (e.g., NgAgo), which recruits the endonuclease to a target sequence on the DNA, where the endonuclease can generate single- or double-strand breaks in the DNA. This targeted DNA break can become a substrate for DNA repair and recombine with the provided editing template to disrupt or delete the gene. For example, a gene encoding a nucleic acid-guided endonuclease (for this purpose, Cas9 from S. pyogenes) or a codon-optimized gene encoding a Cas9 nuclease is operably linked to a promoter active in Trichoderma cells and a terminator active in Trichoderma cells, thereby generating a Trichoderma Cas9 expression cassette. Similarly, one or more target sites unique to a gene of interest are readily identified by one of skill in the art. For example, to construct a DNA construct encoding a gRNA directed to a target site within a gene of interest, the variable targeting domain (VT) would include the nucleotides of the target site 5' to a (PAM) protospacer adjacent motif (TGG), the nucleotides of which are fused to DNA encoding the Cas9 endonuclease recognition domain for S. pyogenes Cas9 (CER). Combination of the DNA encoding the VT domain with the DNA encoding the CER domain thereby generates DNA encoding the gRNA. Thus, a Trichoderma expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in Trichoderma cells and a terminator active in Trichoderma cells.

[0229] In certain embodiments, the DNA break induced by endonuclease is repaired / replaced using an incoming sequence.For example, to precisely repair the DNA break generated by the above-mentioned Cas9 expression cassette and gRNA expression cassette, a nucleotide editing template is provided so that the DNA repair mechanism of the cell can use the editing template.For example, about 500 bp of the 5' of the targeting gene can be fused to about 500 bp of the 3' of the targeting gene to generate an editing template, and this template is used by the mechanism of the Trichoderma host to repair the DNA break generated by RNA-guided endonuclease (RGEN).

[0230] The Cas9 expression cassette, gRNA expression cassette, and editing template can be co-delivered into filamentous fungal cells using a number of different methods (e.g., protoplast fusion, electroporation, natural competence, or induced competence). Transformed cells are screened by PCR amplification of the target locus by amplifying the locus using forward and reverse primers. These primers can amplify the wild-type locus or the modified locus edited by RGEN.

[0231] In yet other embodiments, modified Trichoderma fungal cells are constructed by random or directed mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis and translocation. Genetic modification can be performed by subjecting parent cells to mutagenesis and screening for mutant cells in which gene expression is reduced or eliminated. Mutagenesis can be directed or random, and can be performed, for example, by using suitable physical or chemical mutagenizing agents, by using suitable oligonucleotides, or by subjecting DNA sequences to PCR-generated mutagenesis. Furthermore, mutagenesis can be performed by any combination of these mutagenesis methods. Examples of physical or chemical mutagenizing agents suitable for purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When such agents are used, mutagenesis is generally carried out by incubating the parent cells to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutant cells that exhibit reduced or no gene expression.

[0232] Therefore, in certain embodiments, 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, methods for introducing a DNA construct or vector into a fungal host cell include transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection-mediated and DEAE-dextrin-mediated transfection), incubation with calcium phosphate DNA precipitates, high-velocity guns using DNA-coated microparticles, gene guns or biolistic transformation, protoplast fusion, and the like. Common transformation techniques are known in the art (see, for example, Ausubel et al., 1987; Sambrook et al., 2001 and 2012; and Campbell et al., 1989). Expression of heterologous proteins in Trichoderma has been described, for example, in U.S. Patent Nos. 6,022,725; 6,268,328; Harkki et al., 1991 and Harkki et al., 1989. For transformation of Aspergillus strains, see also Cao et al. (2000).

[0233] Generally, transformation of Trichoderma fungi is usually performed within 10 5 ~10 7 / mL, especially 2 × 10 6Permeabilized protoplasts or cells are used at a density of 100 μL / mL. These protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCl2) are mixed with the desired DNA in a volume of 100 μL. Generally, a high concentration of polyethylene glycol (PEG) is added to the uptake solution. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc. may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Pat. Nos. 6,022,725 and 6,268,328 (both of which are incorporated by reference).

[0234] In certain embodiments, the present disclosure is directed to the expression and production of one or more proteins of interest that are endogenous to the filamentous fungal host cell (i.e., the endogenous proteins are produced by a variant fungal host cell of the present disclosure that includes a genetic modification to express a variant Ace3 protein). In other embodiments, the present disclosure is directed to the expression and production of one or more proteins of interest that are heterologous to the filamentous fungal host cell. Thus, the present disclosure generally relies on routine techniques in the field of recombinant genetics. Essential texts disclosing the general methods used in the present disclosure include Sambrook et al., (2002) and others. nd Edition, 1989); Kriegler (1990) and Ausubel et al., (1994).

[0235] Thus, in certain embodiments, a heterologous gene or ORF encoding a protein of interest is introduced into a filamentous fungal (host) cell. In certain embodiments, the heterologous gene or ORF is typically cloned into an intermediate vector before being transformed into the filamentous fungal (host) cell for replication and / or expression. These intermediate vectors may be, for example, prokaryotic vectors such as plasmids or shuttle vectors. In certain embodiments, expression of the heterologous gene or ORF is under the control of its native promoter. In other embodiments, expression of the heterologous gene or ORF is placed under the control of a heterologous promoter, which may be a heterologous constitutive promoter or a heterologous inducible promoter.

[0236] Those skilled in the art know that a native (natural) promoter can be modified by replacing, substituting, adding, or deleting one or more nucleotides without altering its function, and the practice of the present invention encompasses, but does not mandate, such modifications to the promoter.

[0237] An expression vector / construct typically contains a transcription unit or expression cassette that contains all additional elements required for expression of a heterologous sequence. For example, a typical expression cassette contains a 5' promoter operably linked to a heterologous nucleic acid sequence encoding a protein of interest and may further contain sequence signals required for efficient polyadenylation of the transcript, a ribosome binding site, and translation termination. Additional elements of the cassette may include an enhancer and, if genomic DNA is used as the structural gene, an intron with functional splice donor and acceptor sites.

[0238] In addition to a promoter sequence, the expression cassette may also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene. While any fungal terminator is likely to be functional in the present invention, preferred terminators include the terminator from the Trichoderma cbhI gene, the terminator from the Aspergillus nidulans trpC gene (Yelton et al., 1984; Mullaney et al., 1985), the Aspergillus awamori or Aspergillus niger glucoamylase gene (Nunberg et al., 1984; Boel et al., 1984), and / or the Mucor miehei carboxyl protease gene (EP 0 215 594).

[0239] The particular expression vector used to deliver genetic information into cells is not particularly critical. Any conventional vector used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include bacteriophages λ and M13, as well as plasmids such as pBR322-based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags, such as c-myc, can also be added to recombinant proteins to provide convenient isolation methods.

[0240] Elements that can be included in an expression vector also include a replicon, a gene encoding antibiotic resistance to allow for selection of bacteria harboring the recombinant plasmid, or a unique restriction site in a non-essential region of the plasmid to allow for insertion of heterologous sequences. The particular antibiotic resistance gene selected is not critical, as any of a number of resistance genes known in the art may be suitable. Prokaryotic sequences are preferably selected so as not to interfere with DNA replication or integration in Trichoderma reesei.

[0241] The transformation methods of the present invention may result in stable integration of all or part of the transformation vector into the filamentous fungal genome. However, transformation that results in the maintenance of an autonomously replicating extrachromosomal transformation vector is also contemplated.

[0242] Any known procedure for introducing foreign nucleotide sequences into host cells can be used. These include calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors, and any other known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells (see, e.g., Sambrook et al., supra). Agrobacterium-mediated transfection, such as that described in U.S. Patent No. 6,255,115, is also useful. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one heterologous gene into the host cell that is capable of expressing that gene.

[0243] After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the gene under the control of the cellulase gene promoter sequence. Large batches of transformed cells can be cultured as described herein. Finally, the product is recovered from the culture using standard techniques.

[0244] Thus, the disclosure herein provides for the expression and enhanced secretion of desired polypeptides whose expression is under the control of cellulase gene promoter sequences, including naturally occurring cellulase genes, fusion DNA sequences, and various heterologous constructs. The invention also provides methods for the high level expression and secretion of such desired products.

[0245] V. Target Protein As noted above, certain embodiments of the present disclosure relate to genetically modified filamentous fungal cells comprising a genetic modification that expresses a gene or ORF encoding a variant Ace3 protein described herein. More particularly, certain embodiments relate to compositions and methods for expressing / producing a protein of interest (POI) in such modified fungal cells encoding the variant Ace3 protein in the absence of an inducing substrate. Certain other embodiments relate to compositions and methods for increasing production of a POI in such modified fungal cells in the presence of an inducing substrate.

[0246] As described herein, proteins of interest (POI) include hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, mannanases, β-glucanases, hyaluronidases, chondroitinases, laccases, amylases, and the like. Examples of enzymes that can be used include, but are not limited to, enzymes such as α-galactosidase, β-galactosidase, α-glucanase, glucanlysase, endo-β-glucanase, glucose oxidase, glucuronidase, invertase, and isomerase.

[0247] Thus, in certain embodiments, the POI is selected from an Enzyme Commission (EC) number selected from the group consisting of EC1, EC2, EC3, EC4, EC5 or EC6.

[0248] For example, in certain embodiments, the POI is selected from the group consisting of EC 1.10.3.2 (e.g., laccase), EC 1.10.3.3 (e.g., L-ascorbic acid oxidase), EC 1.1.1.1 (e.g., alcohol dehydrogenase), EC 1.11.1.10 (e.g., chloride peroxidase), EC 1.11.1.17 (e.g., peroxidase), EC 1.1.1.27 (e.g., L-lactate dehydrogenase), EC 1.1.1.47 (e.g., glucose 1-dehydrogenase), EC 1.1.3.X (e.g., glucose oxidase), EC 1.1.3.10 (e.g., pyranose oxidase), EC 1.13.11.X (e.g., dioxygenase), EC 1.13.11.12 (e.g., linoleic acid l3S-lipoxygenase), EC 1.1.3.13 (e.g., alcohol oxidase), EC 1.14. and EC 1.1.99.18 (e.g., cellobiose dehydrogenase), EC 1.1.99.29 (e.g., pyranose dehydrogenase), EC 1.2.1.X (e.g., fatty acid reductase), EC 1.2.1.10 (e.g., acetaldehyde dehydrogenase), EC 1.5.3.X (e.g., fructosylamine reductase), EC 1.8.1.X (e.g., disulfide reductase), and EC 1.8.3.2 (e.g., thiol oxidase).

[0249] In certain embodiments, the POI is selected from the group consisting of EC 2.3.2.13 (e.g., transglutaminase), EC 2.4.1.X (e.g., hexosyltransferase), EC 2.4.1.40 (e.g., alternasucrase), EC 2.4.1.18 (e.g., 1,4 α-glucan branching enzyme), EC 2.4.1.19 (e.g., cyclomaltodextrin glucanotransferase), EC 2.4.1.2 (e.g., dextrin dextranase), EC 2.4.1.20 (e.g., cellobiose phosphorylase), EC 2.4.1.25 (e.g., 4-α-glucanotransferase), EC 2.4.1.333 (e.g., 1,2-β-oligoglucan phosphotransferase), E Transferase enzymes include, but are not limited to, EC2 (transferase) enzymes selected from EC2.4.1.4 (e.g., amylosucrase), EC2.4.1.5 (e.g., dextransucrase), EC2.4.1.69 (e.g., galactoside 2-α-L-fucosyltransferase), EC2.4.1.9 (e.g., inulosucrase), EC2.7.1.17 (e.g., xylulokinase), EC2.7.7.89 (formerly EC3.1.4.15, e.g., [glutamine synthetase]-adenylyl-L-tyrosine phosphorylase), EC2.7.9.4 (e.g., α-glucan kinase), and EC2.7.9.5 (e.g., phosphoglucan kinase).

[0250] In other embodiments, the POI is selected from the group consisting of EC 3.1.XX (e.g., esterases), EC 3.1.1.1 (e.g., pectinases), EC 3.1.1.14 (e.g., chlorophyllases), EC 3.1.1.20 (e.g., tannases), EC 3.1.1.23 (e.g., glycerol-ester acylhydrolases), EC 3.1.1.26 (e.g., galactolipases), EC 3.1.1.32 (e.g., phospholipase A1), EC 3.1.1.4 (e.g., phospholipase A2), EC 3.1.1.6 (e.g., acetylesterases), EC 3.1.1.72 (e.g., acetylxylases), and the like. lan esterase), EC 3.1.1.73 (e.g., feruloyl esterase), EC 3.1.1.74 (e.g., cutinase), EC 3.1.1.86 (e.g., rhamnogalacturonan acetyl esterase), EC 3.1.1.87 (e.g., fumosin B1 esterase), EC 3.1.26.5 (e.g., ribonuclease P), EC 3.1.3.X (e.g., phosphate ester hydrolase), EC 3.1.30.1 (e.g., Aspergillus nuclease S1), EC 3.1.30.2 (e.g., Serratia marcescens nuclease), marcescens nuclease), EC 3.1.3.1 (e.g., alkaline phosphatase), EC 3.1.3.2 (e.g., acid phosphatase), EC 3.1.3.8 (e.g., 3-phytase), EC 3.1.4.1 (e.g., phosphodiesterase I), EC 3.1.4.11 (e.g., phosphoinositide phospholipase C), EC 3.1.4.3 (e.g., phospholipase C), EC 3.1.4.4 (e.g., phospholipase D), EC 3.1.6.1 (e.g., arylsulfatase), E C3.1.8.2 (e.g., diisopropyl-fluorophosphatase), EC3.2.1.10 (e.g., oligo-1,6-glucosidase), EC3.2.1.101 (e.g., mannan endo-1,6-α-mannosidase), EC3.2.1.11 (e.g., α-1,6-glucan-6-glucanohydrolase), EC3.2.1.131 (e.g., xylan α-1,2-glucuronosidase), EC3.2.1.132 (e.g., chitosan N-acetylglucosaminohydrolase), EC3.2.1.139 (e.g., α-glucuronidase), EC 3.2.1.14 (e.g., chitinase), EC 3.2.1.151 (e.g., xyloglucan-specific endo-β-1,4-glucanase), EC 3.2.1.155 (e.g., xyloglucan-specific exo-β-1,4-glucanase), EC 3.2.1.164 (e.g., galactan endo-1,6-β-galactosidase), EC 3.2.1.17 (e.g., lysozyme), EC 3.2.1.171 (e.g., rhamnogalacturonan hydrolase), EC 3.2.1.174 (e.g., , rhamnogalacturonan rhamnohydrolase), EC 3.2.1.2 (e.g., β-amylase), EC 3.2.1.20 (e.g., α-glucosidase), EC 3.2.1.22 (e.g., α-galactosidase), EC 3.2.1.25 (e.g., β-mannosidase), EC 3.2.1.26 (e.g., β-fructofuranosidase), EC 3.2.1.37 (e.g., xylan 1,4-β-xylosidase), EC 3.2.1.39 (e.g., glucan endo-1,3-β-D-glucosidase), EC 3.2.1.40 (e.g., α -L-rhamnosidase), EC 3.2.1.51 (e.g., α-L-fucosidase), EC 3.2.1.52 (e.g., β-N-acetylhexosaminidase), EC 3.2.1.55 (e.g., α-N-arabinofuranosidase), EC 3.2.1.58 (e.g., glucan 1,3-β-glucosidase), EC 3.2.1.59 (e.g., glucan endo-1,3-α-glucosidase), EC 3.2.1.67 (e.g., galacturan 1,4-α-galacturonidase), EC 3.2.1.68 (e.g., isoamylase), EC 3.2 .1.7 (e.g., 1-β-D-fructan fructanohydrolase), EC 3.2.1.74 (e.g., glucan 1,4-glucosidase), EC 3.2.1.75 (e.g., glucan endo-1,6-β-glucosidase), EC 3.2.1.77 (e.g., mannan 1,2-(1,3)-α-mannosidase), EC 3.2.1.80 (e.g., fructan β-fructosidase), EC 3.2.1.82 (e.g., exo-poly-α-galacturonosidase), EC 3.2.1.83 (e.g., κ-carrageenase), EC 3.2.1.89 (e.g., arabinogalactan endo-1,4-β-galactosidase), EC 3.2.1.91 (e.g., cellulose 1,4-β-cellobiosidase), EC 3.2.1.96 (e.g., mannosyl-glycoprotein endo-β-N-acetylglucosaminidase), EC 3.2.1.99 (e.g., arabinan endo-1,5-α-L-arabinanase), EC 3.4.XX (e.g., peptidase), EC 3.4.11.X (e.g., aminopeptidase), EC 3.4.11.1 (e.g., leucyl aminopeptidase), EC 3.4.11 .18 (e.g., methionyl aminopeptidase), EC 3.4.13.9 (e.g., Xaa-Pro dipeptidase), EC 3.4.14.5 (e.g., dipeptidyl-peptidase IV), EC 3.4.16.X (e.g., serine-type carboxypeptidase), EC 3.4.16.5 (e.g., carboxypeptidase C), EC 3.4.19.3 (e.g., pyroglutamyl-peptidase I), EC 3.4.21.X (e.g., serine endopeptidase), EC 3.4.21.1 (e.g., chymotrypsin), EC 3.4.21.19 (e.g., , glutamyl endopeptidase), EC 3.4.21.26 (e.g., prolyl oligopeptidase), EC 3.4.21.4 (e.g., trypsin), EC 3.4.21.5 (e.g., thrombin), EC 3.4.21.63 (e.g., oryzen), EC 3.4.21.65 (e.g., thermomycolin), EC 3.4.21.80 (e.g., streptoglycin A), EC 3.4.22.X (e.g., cysteine ​​endopeptidase), EC 3.4.22.14 (e.g., actinidin), EC 3.4.22.2 (e.g., papain), EC 3.4.2 2.3 (e.g., ficain), EC 3.4.22.32 (e.g., stem bromelain), EC 3.4.22.33 (e.g., fruit bromelain), EC 3.4.22.6 (e.g., chymopapain), EC 3.4.23.1 (e.g., pepsin A), EC 3.4.23.2 (e.g., pepsin B), EC 3.4.23.22 (e.g., endothiapepsin), EC 3.4.23.23 (e.g., Mucor pepsin), EC 3.4.23.3 (e.g., gastricin), EC 3.4.24.X (e.g., metalloendopeptidase), EC 3.4.24.39 (e.g., deuterolysin), EC 3.4.24.40 (e.g., serralysin), EC 3.5.1.1 (e.g., asparaginase), EC 3.5.1.11 (e.g., penicillin amidase), EC 3.5.1.14 (e.g., N-acyl-fatty-L-amino acid amidohydrolase), EC 3.5.1.2 (e.g., L-glutamine amidohydrolase), EC 3.5.1.28 (e.g., N-acetylmuramoyl-L-alanine amidase), EC 3.5.1.4 (e.g., amidase), EC 3.5.1.4 and hydrolase enzymes, including, but not limited to, EC 3 (hydrolase) enzymes selected from EC 3.5.4 (e.g., protein-L-glutamine amidohydrolase), EC 3.5.1.5 (e.g., urease), EC 3.5.1.52 (e.g., peptide-N(4)-(N-acetyl-β-glucosaminyl)asparagine amidase), EC 3.5.1.81 (e.g., N-acyl-D-amino acid deacylases), EC 3.5.4.6 (e.g., AMP deaminase), and EC 3.5.5.1 (e.g., nitrilases).

[0251] In other embodiments, the POI is a lyase enzyme, including but not limited to, an EC4 (lyase) enzyme selected from EC 4.1.2.10 (e.g., mandelonitrile lyase), EC 4.1.3.3 (e.g., N-acetylneuraminic acid lyase), EC 4.2.1.1 (e.g., carbonic anhydrase), EC 4.2.2.- (e.g., rhamnogalacturonan lyase), EC 4.2.2.10 (e.g., pectin lyase), EC 4.2.2.22 (e.g., pectate trisaccharide lyase), EC 4.2.2.23 (e.g., rhamnogalacturonan endolyase), and EC 4.2.2.3 (e.g., mannuronic acid-specific alginate lyase).

[0252] In certain other embodiments, the POI is an isomerase enzyme, including, but not limited to, an EC5 (isomerase) enzyme selected from EC 5.1.3.3 (e.g., aldose 1-epimerase), EC 5.1.3.30 (e.g., D-psicose 3-epimerase), EC 5.4.99.11 (e.g., isomaltulose synthase), and EC 5.4.99.15 (e.g., (1 / 4)-α-D-glucan 1-α-D-glucosylmutase).

[0253] In yet other embodiments, the POI is a ligase enzyme, including but not limited to, an EC6 (ligase) enzyme selected from EC 6.2.1.12 (e.g., 4-coumarinate:coenzyme A ligase) and EC 6.3.2.28 (e.g., L-amino acid α-ligase).

[0254] Optimal conditions for protein production will vary with the choice of host cell and the protein to be expressed, and such conditions can be readily ascertained by one skilled in the art by routine experimentation and / or optimization.

[0255] The target protein can be purified or isolated after expression. The target protein can be isolated or purified by various methods known to those skilled in the art, depending on what other components are present in the sample. Standard purification methods include electrophoretic techniques, molecular techniques, immunological techniques, and chromatographic techniques, such as ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, as well as chromatofocusing. For example, the target protein can be purified using a standard anti-target protein antibody column. In relation to protein concentration, ultrafiltration and diafiltration techniques are also useful. The degree of purification required depends on the intended use of the target protein. In some cases, protein purification is not necessary.

[0256] In certain other embodiments, various screening methods can be performed to confirm that the genetically modified fungal cells of the present disclosure produce increased levels of a protein of interest. The expression vector can encode a polypeptide fusion to the target protein that functions as a detectable label, or the target protein itself can function as a selectable or screenable marker. The labeled protein can be detected by Western blotting, dot blotting (methods available on the Cold Spring Harbor Protocols website), ELISA, or, if the label is GFP, whole-cell fluorescence or FACS. For example, a 6-histidine tag can be included as a fusion to the target protein, and this tag is detected by Western blotting. If the target protein is expressed at sufficiently high levels, SDS-PAGE combined with Coomassie / silver staining can be performed to detect increased expression in the variant host cells over the parental (control) cells, in which case no labeling is required. Additionally, other methods, such as detecting increased protein activity or amount per cell or per milliliter of medium, can be used to confirm improved levels of the protein of interest, which, or a combination of these methods, allows for efficient continuation of the culture or fermentation for longer periods of time.

[0257] Determining specific productivity is another way to assess protein production. Specific productivity (Qp) can be calculated by the following formula: Qp=gP / gDCW·hr

[0258] (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) from the time of inoculation, including production time and growth time).

[0259] VI. Fermentation In certain embodiments, the present disclosure provides methods for producing a protein of interest, including fermentation of modified fungal cells, wherein the variant fungal cells secrete the protein of interest. Generally, fermentation methods well known in the art are used to ferment the variant fungal cells. In some embodiments, the fungal cells are grown under batch or continuous fermentation conditions. Classical batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and remains unchanged during the fermentation. At the start of the fermentation, the medium is inoculated with the desired organism. In this method, fermentation occurs without adding any components to the system. Generally, batch fermentation is considered "batch" with respect to the addition of a carbon source, and control of factors such as pH and oxygen concentration is frequently performed. The metabolite and biomass composition of a batch system changes constantly until the point at which the fermentation is stopped. Within a batch culture, cells progress through a static lag phase, a high-growth logarithmic phase, and eventually a stationary phase, where growth rate slows or stops. If left untreated, cells in the stationary phase eventually die. Generally, cells in log phase are responsible for the majority of product production.

[0260] A suitable variation on the standard batch system is the "fed-batch fermentation" system. In this variation of the typical batch system, substrate is added gradually as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit cellular metabolism and when a limited amount of substrate is desired in the medium. In fed-batch systems, the actual substrate concentration is difficult to measure and is therefore estimated based on changes in measurable factors such as pH, dissolved oxygen, and the partial pressure of waste gases such as CO2. Batch and fed-batch fermentation are common and well known in the art.

[0261] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant high density, with cells primarily in logarithmic growth phase. Continuous fermentation allows for the adjustment of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, a limiting nutrient, such as a carbon or nitrogen source, is maintained at a fixed ratio, while all other parameters are adjustable. In other systems, multiple factors affecting growth can be continuously varied while the cell concentration, measured by medium turbidity, remains constant. Continuous systems attempt to maintain steady-state growth conditions. Therefore, cell loss due to medium removal must be balanced against the cell growth rate during fermentation. Methods for adjusting nutrients and growth factors in continuous fermentation processes, as well as techniques for maximizing product formation rates, are well known in the art of industrial microbiology.

[0262] Certain embodiments of the present disclosure relate to fermentation procedures for culturing fungi. Fermentation procedures for the production of cellulase enzymes are known in the art. For example, cellulase enzymes can be produced by solid-state or submerged culture, including batch, fed-batch, and continuous-flow processes. Cultivation generally occurs in a growth medium containing an aqueous inorganic salts medium, organic growth factors, carbon and energy source materials, molecular oxygen, and, of course, a starting inoculum of the filamentous fungal host used.

[0263] To ensure proper microbial growth, maximize the uptake of carbon and energy sources by the cells in the microbial conversion process, and achieve maximum cell yield at maximum cell density in the fermentation medium, suitable amounts of inorganic nutrients must be supplied in appropriate proportions in addition to the carbon and energy sources, oxygen, assimilable nitrogen, and microbial inoculum.

[0264] The composition of the aqueous mineral medium can vary widely, depending in part on the microorganism and substrate used, as is known in the art. In addition to nitrogen, the mineral medium will contain suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium in suitable soluble, absorbable, ionic complex forms, and preferably also several trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, also in suitable soluble, absorbable forms, all of which are known in the art.

[0265] Fermentation reactions are aerobic processes in which the necessary molecular oxygen is supplied by a molecular oxygen-containing gas, such as air, oxygen-enriched air, or substantially pure molecular oxygen, provided to maintain the contents of the fermentor at a suitable oxygen partial pressure effective to support vigorous growth of the microbial species.

[0266] Fermentation temperatures can vary somewhat, but for filamentous fungi such as Trichoderma reesei, temperatures are generally within the range of about 20°C to 40°C, and generally preferably within the range of about 25°C to 34°C.

[0267] Microorganisms also require an assimilable nitrogen source. The assimilable nitrogen source can be any nitrogen-containing compound or any compound capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While various organic nitrogen source compounds, such as protein hydrolysates, can be used, typically inexpensive nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts, such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds, are utilized. Ammonia gas itself is convenient for large-scale operations and can be used by bubbling a suitable amount into the aqueous fermentation product (fermentation medium). At the same time, such ammonia can also be used to assist in pH control.

[0268] The pH range of the aqueous microbial fermentation (fermentation mixture) should be in the exemplary range of about 2.0 to 8.0. For filamentous fungi, the pH is typically in the range of about 2.5 to 8.0, and for Trichoderma reesei, the pH is typically in the range of about 3.0 to 7.0. The preferred pH range for a microorganism will depend to some extent on the medium and the particular microorganism used and will therefore vary somewhat with changes in the medium, as can be readily determined by one of ordinary skill in the art.

[0269] It is preferred to carry out the fermentation in such a way that the carbon-containing substrate can be controlled as the limiting factor, thereby providing good conversion of the carbon-containing substrate to the cells and avoiding contamination of the cells with significant amounts of unconverted substrate. The latter is not a problem with water-soluble substrates, as trace amounts of residue can be easily washed away. However, it can be a problem with water-insoluble substrates, necessitating additional product processing steps, such as suitable washing steps.

[0270] As noted above, the time to reach this level is not critical and may vary depending on the particular microorganism and fermentation process being carried out, however, methods for determining the carbon source concentration in a fermentation medium and whether the desired carbon source level has been achieved are well known in the art.

[0271] Fermentation can be carried out as a batch or continuous operation, with fed-batch operation being highly preferred for ease of control, production of uniform amounts of product, and the most economical use of all equipment.

[0272] If necessary, some or all of the carbon and energy source materials and / or some of the assimilable nitrogen source, such as ammonia, can be added to the aqueous mineral medium before it is fed to the fermenter.

[0273] Each of the streams introduced into the reactor is preferably controlled at a predetermined rate or according to needs that can be determined by monitoring the concentrations of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the fermentor off-gas, cell density as measured by dry cell weight, light transmittance, etc. The feed rates of the various materials can be varied to obtain the fastest possible cell growth rate and the highest possible yield of microbial cells relative to the substrate feed, consistent with efficient utilization of the carbon and energy sources.

[0274] In a batch or, preferably, fed-batch operation, all equipment, reactors, or fermentation means, tanks or vessels, piping, associated circulation or cooling devices, etc., are first sterilized, usually using steam, e.g., at about 121°C for at least about 15 minutes. The sterilized reactor is then inoculated with a culture of the selected microorganism in the presence of all necessary nutrients, including oxygen, and a carbon-containing substrate. The type of fermentor used is not critical.

[0275] Recovery and purification of enzymes (e.g., cellulases) from the fermentation broth can also be carried out by procedures known to those skilled in the art. The fermentation broth generally contains cellular debris, e.g., cells, various suspended solids and other biomass contaminants, as well as the desired cellulase enzyme product, which are preferably removed from the fermentation broth by means known in the art.

[0276] Suitable processes for such removal include conventional solid-liquid separation techniques such as, for example, centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes to produce a cell-free filtrate. Preferably, the fermentation broth, or cell-free filtrate, is further concentrated prior to crystallization using techniques such as ultrafiltration, evaporation, or precipitation.

[0277] Precipitation of the protein components of the supernatant or filtrate can be carried out with a salt, such as ammonium sulfate, followed by purification by various chromatographic methods, such as ion exchange chromatography, affinity chromatography, or similar art-recognized methods.

[0278] VII. Exemplary Embodiments Certain exemplary embodiments include the following:

[0279] 1. An isolated polynucleotide encoding a variant Ace3 transcription factor (TF) protein, wherein the variant Ace3 TF comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2 and comprises a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2.

[0280] 2. The polynucleotide of embodiment 1, wherein the variant Ace3 TF comprises a functional N-terminal zinc binuclear cluster (Zn2Cys6) DNA-binding domain comprising at least 95% sequence identity to SEQ ID NO:29.

[0281] 3. The polynucleotide of embodiment 1, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO: 2 is an amino acid deletion, an amino acid insertion, an amino acid substitution, or a combination thereof.

[0282] 4. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2.

[0283] 5. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last eight C-terminal amino acid positions 682-689 of SEQ ID NO:2.

[0284] 6. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last nine C-terminal amino acid positions 681-689 of SEQ ID NO:2.

[0285] 7. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last 10 C-terminal amino acid positions 680-689 of SEQ ID NO:2.

[0286] 8. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last 11 C-terminal amino acid positions 679-689 of SEQ ID NO:2.

[0287] 9. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last 12 C-terminal amino acid positions 678-689 of SEQ ID NO:2.

[0288] 10. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last 13 C-terminal amino acid positions 677-689 of SEQ ID NO:2.

[0289] 11. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last 14 C-terminal amino acid positions 676-689 of SEQ ID NO:2.

[0290] 12. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last 15 C-terminal amino acid positions 675-689 of SEQ ID NO:2.

[0291] 13. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2.

[0292] 14. The polynucleotide of embodiment 3, wherein the genetic modification of the one or more C-terminal amino acid residues comprises a deletion of at least the last 17 C-terminal amino acid positions 673 to 689 of SEQ ID NO:2.

[0293] 15. The polynucleotide of embodiment 3, wherein the variant Ace3 TF comprises an amino acid sequence selected from any one of SEQ ID NO: 9 to SEQ ID NO: 18.

[0294] 16. The polynucleotide of embodiment 3, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 is a deletion of one or more amino acid residues selected from positions 673 to 683 of SEQ ID NO:2.

[0295] 17. The polynucleotide of embodiment 3, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 is a deletion of two consecutive amino acid residues selected from positions 673 to 674 of SEQ ID NO:2, positions 674 to 675 of SEQ ID NO:2, positions 675 to 676 of SEQ ID NO:2, positions 676 to 677 of SEQ ID NO:2, positions 677 to 678 of SEQ ID NO:2, positions 678 to 679 of SEQ ID NO:2, positions 679 to 680 of SEQ ID NO:2, positions 680 to 681 of SEQ ID NO:2, positions 681 to 682 of SEQ ID NO:2, positions 682 to 683 of SEQ ID NO:2, and positions 683 to 684 of SEQ ID NO:2.

[0296] 18. The polynucleotide of embodiment 3, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 is a deletion of three consecutive amino acid residues selected from positions 673 to 675 of SEQ ID NO:2, positions 674 to 676 of SEQ ID NO:2, positions 675 to 677 of SEQ ID NO:2, positions 676 to 678 of SEQ ID NO:2, positions 677 to 679 of SEQ ID NO:2, positions 678 to 680 of SEQ ID NO:2, positions 679 to 681 of SEQ ID NO:2, positions 680 to 682 of SEQ ID NO:2, positions 681 to 683 of SEQ ID NO:2, positions 682 to 684 of SEQ ID NO:2, and positions 683 to 685 of SEQ ID NO:2.

[0297] 19. The polynucleotide of embodiment 3, wherein the variant Ace3 TF comprises a substitution of one or more C-terminal amino acid residues selected from amino acid positions 672 to 683 of SEQ ID NO:2.

[0298] 20. The polynucleotide of embodiment 19, wherein the variant Ace3 TF further comprises a deletion of at least the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2.

[0299] 21. The polynucleotide of embodiment 3, wherein the variant Ace3 TF comprises an insertion of one or more amino acid residues at a C-terminal amino acid position selected from amino acid positions 673 to 683 of SEQ ID NO:2.

[0300] 22. An isolated Trichoderma mutant cell comprising a mutant ace3 gene encoding a variant Ace3 transcription factor (TF) protein, wherein the variant Ace3 TF protein comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2 and comprises one or more mutated C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2.

[0301] 23. The mutant cell of embodiment 22, wherein the variant Ace3 TF protein upregulates expression of genes encoding lignocellulolytic enzymes in the absence of an inducing substrate when the mutant cell is fermented under conditions suitable for the production of lignocellulolytic enzymes.

[0302] 24. The mutant cell of embodiment 23, wherein the lignocellulolytic enzyme is selected from the group consisting of cellobiohydrolase, endoglucanase, and β-glucosidase.

[0303] 25. The mutant cell of embodiment 22, wherein the variant Ace3 TF protein comprises a functional N-terminal zinc binuclear cluster DNA-binding domain comprising at least 95% sequence identity to SEQ ID NO: 29.

[0304] 26. The mutant cell of embodiment 22, comprising an introduced expression cassette encoding a protein of interest (POI), the introduced cassette comprising an upstream (5') promoter sequence from a gene encoding a lignocellulolytic enzyme operably linked to a downstream (3') open reading frame (ORF) sequence encoding the POI.

[0305] 27. The mutant cell of embodiment 22, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO: 2 comprise an amino acid deletion, an amino acid insertion, an amino acid substitution, or a combination thereof.

[0306] 28. The mutant cell of embodiment 26, wherein the mutant cell is capable of producing the POI in the absence of an inducing substrate when fermented under conditions suitable for the production of the POI.

[0307] 29. A protein of interest (POI) produced by the mutant strain of any one of embodiments 22 to 28.

[0308] 30. A genetically modified Trichoderma fungal cell derived from a parent Trichoderma fungal cell, comprising an ace3 gene encoding an Ace3 transcription factor (TF) protein comprising at least 90% sequence identity to SEQ ID NO:2, wherein the genetically modified cell comprises a modified ace3 gene that encodes a variant Ace3TF protein comprising at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2, and that comprises a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2.

[0309] 31. The modified cell of embodiment 30, wherein the Ace3 TF protein comprises a functional N-terminal zinc binuclear cluster DNA-binding domain comprising at least 95% sequence identity to SEQ ID NO: 29.

[0310] 32. The modified cell of embodiment 30, wherein when fermented under conditions suitable for the production of lignocellulolytic enzymes, the variant Ace3 TF protein upregulates expression of genes encoding lignocellulolytic enzymes in the absence of an inducing substrate.

[0311] 33. The modified cell of embodiment 32, wherein the lignocellulolytic enzymes are selected from the group consisting of cellobiohydrolases, endoglucanases, and β-glucosidases.

[0312] 34. The modified cell of embodiment 30, comprising an introduced expression cassette encoding a protein of interest (POI), the introduced cassette comprising an upstream (5') promoter sequence from a gene encoding a lignocellulolytic enzyme operably linked to a downstream (3') open reading frame (ORF) sequence encoding the POI.

[0313] 35. The modified cell of embodiment 30, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO: 2 comprise an amino acid deletion, an amino acid insertion, an amino acid substitution, or a combination thereof.

[0314] 36. The modified cell of embodiment 34, wherein the modified cell produces the POI in the absence of an inducing substrate when fermented under conditions suitable for the production of the POI.

[0315] 37. The modified cell of embodiment 34, wherein the modified cell produces an increased amount of POI in the presence of an inducing substrate compared to the amount of the same POI produced by the parent cell when fermented under the same conditions for producing the POI in the presence of the inducing substrate.

[0316] 38. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last seven C-terminal amino acid positions 683 to 689 of SEQ ID NO:2.

[0317] 39. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2.

[0318] 40. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last nine C-terminal amino acid positions 681 to 689 of SEQ ID NO:2.

[0319] 41. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 10 C-terminal amino acid positions 680 to 689 of SEQ ID NO:2.

[0320] 42. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 11 C-terminal amino acid positions 679 to 689 of SEQ ID NO:2.

[0321] 43. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 12 C-terminal amino acid positions 678 to 689 of SEQ ID NO:2.

[0322] 44. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 13 C-terminal amino acid positions 677 to 689 of SEQ ID NO:2.

[0323] 45. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 14 C-terminal amino acid positions 676 to 689 of SEQ ID NO:2.

[0324] 46. ​​The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 15 C-terminal amino acid positions 675 to 689 of SEQ ID NO:2.

[0325] 47. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 16 C-terminal amino acid positions 674 to 689 of SEQ ID NO:2.

[0326] 48. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 17 C-terminal amino acid positions 673 to 689 of SEQ ID NO:2.

[0327] 49. The modified cell of embodiment 35, wherein the variant Ace3 TF comprises an amino acid sequence selected from any one of SEQ ID NO: 9 to SEQ ID NO: 18.

[0328] 50. The modified cell of embodiment 35, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO: 2 is a deletion of one or more amino acid residues selected from positions 673 to 683 of SEQ ID NO: 2.

[0329] 51. The modified cell of embodiment 35, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 is a deletion of two consecutive amino acid residues selected from positions 673 to 674 of SEQ ID NO:2, positions 674 to 675 of SEQ ID NO:2, positions 675 to 676 of SEQ ID NO:2, positions 676 to 677 of SEQ ID NO:2, positions 677 to 678 of SEQ ID NO:2, positions 678 to 679 of SEQ ID NO:2, positions 679 to 680 of SEQ ID NO:2, positions 680 to 681 of SEQ ID NO:2, positions 681 to 682 of SEQ ID NO:2, positions 682 to 683 of SEQ ID NO:2, and positions 683 to 684 of SEQ ID NO:2.

[0330] 52. The modified cell of embodiment 35, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 is a deletion of three consecutive amino acid residues selected from positions 673 to 675 of SEQ ID NO:2, positions 674 to 676 of SEQ ID NO:2, positions 675 to 677 of SEQ ID NO:2, positions 676 to 678 of SEQ ID NO:2, positions 677 to 679 of SEQ ID NO:2, positions 678 to 680 of SEQ ID NO:2, positions 679 to 681 of SEQ ID NO:2, positions 680 to 682 of SEQ ID NO:2, positions 681 to 683 of SEQ ID NO:2, positions 682 to 684 of SEQ ID NO:2, and positions 683 to 685 of SEQ ID NO:2.

[0331] 53. The modified cell of embodiment 35, wherein the variant Ace3 TF comprises a substitution of one or more C-terminal amino acid residues selected from amino acid positions 672 to 683 of SEQ ID NO: 2.

[0332] 54. The modified cell of embodiment 53, wherein the variant Ace3 TF further comprises a deletion of at least the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2.

[0333] 55. The modified cell of embodiment 35, wherein the variant Ace3 TF comprises an insertion of one or more amino acid residues at a C-terminal amino acid position selected from amino acid positions 673 to 683 of SEQ ID NO:2.

[0334] 56. The modified cell of any one of embodiments 32, 36, and 37, wherein the inducing substrate is selected from lactose, sophorose, gentiobiose, and cellulose.

[0335] 57. A lignocellulolytic enzyme produced by the modified cell of embodiment 32.

[0336] 58. A protein of interest (POI) produced by the modified cell of embodiment 34.

[0337] 59. A genetically modified Trichoderma fungal cell derived from a parent Trichoderma fungal cell, comprising a mutated ace3 gene encoding a variant Ace3 transcription factor (TF) protein comprising at least 90% sequence identity to SEQ ID NO:4, wherein the N-terminus of SEQ ID NO:4 does not comprise an intact zinc binuclear (Zn2Cys6) DNA binding sequence set forth in SEQ ID NO:29; the genetically modified cell comprises a modified ace3 gene encoding a variant Ace3 TF protein comprising at least 90% sequence identity to positions 1-672 of SEQ ID NO:2, wherein the N-terminus of SEQ ID NO:2 comprises an intact zinc binuclear (Zn2Cys6) DNA binding sequence set forth in SEQ ID NO:29; and the genetically modified cell comprises a genetic modification of one or more C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2.

[0338] 60. The modified cell of embodiment 59, wherein when fermented under conditions suitable for the production of lignocellulolytic enzymes, the variant Ace3 TF protein upregulates expression of genes encoding lignocellulolytic enzymes in the absence of an inducing substrate.

[0339] 61. The modified cell of embodiment 59, wherein the variant Ace3 TF protein upregulates expression of a gene encoding a lignocellulolytic enzyme in the presence of an inducing substrate, such that when the modified cell and the parent cell are fermented under the same conditions for production of the lignocellulolytic enzyme, the modified cell produces an increased amount of the lignocellulolytic enzyme compared to the amount of the same lignocellulolytic enzyme produced by the parent cell in the presence of the same inducing substrate.

[0340] 62. The modified cell of embodiment 59, comprising an introduced expression cassette encoding a protein of interest (POI), the introduced cassette comprising an upstream (5') promoter sequence from a gene encoding a lignocellulolytic enzyme operably linked to a downstream (3') open reading frame (ORF) sequence encoding the POI.

[0341] 63. The modified cell of embodiment 62, wherein the modified cell produces the POI in the absence of an inducing substrate when fermented under conditions suitable for the production of the POI.

[0342] 64. The modified cell of embodiment 62, wherein when the modified cell and parent cell are fermented under the same conditions for production of the POI in the presence of an inducing substrate, the modified cell produces an increased amount of the POI in the presence of an inducing substrate compared to a parent cell containing the same introduced expression cassette encoding the same POI.

[0343] 65. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO: 2 comprise an amino acid deletion, an amino acid insertion, an amino acid substitution, or a combination thereof.

[0344] 66. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last seven C-terminal amino acid positions 683 to 689 of SEQ ID NO:2.

[0345] 67. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2.

[0346] 68. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last nine C-terminal amino acid positions 681 to 689 of SEQ ID NO:2.

[0347] 69. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 10 C-terminal amino acid positions 680 to 689 of SEQ ID NO:2.

[0348] 70. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 11 C-terminal amino acid positions 679 to 689 of SEQ ID NO:2.

[0349] 71. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 12 C-terminal amino acid positions 678 to 689 of SEQ ID NO:2.

[0350] 72. The modified cell of embodiment 35, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 13 C-terminal amino acid positions 677 to 689 of SEQ ID NO:2.

[0351] 73. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 14 C-terminal amino acid positions 676 to 689 of SEQ ID NO:2.

[0352] 74. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 15 C-terminal amino acid positions 675 to 689 of SEQ ID NO:2.

[0353] 75. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 16 C-terminal amino acid positions 674 to 689 of SEQ ID NO:2.

[0354] 76. The modified cell of embodiment 59, wherein the one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 comprise a deletion of at least the last 17 C-terminal amino acid positions 673 to 689 of SEQ ID NO:2.

[0355] 77. The modified cell of embodiment 59, wherein the variant Ace3 TF comprises an amino acid sequence selected from any one of SEQ ID NO: 9 to SEQ ID NO: 18.

[0356] 78. The modified cell of embodiment 59, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO: 2 is a deletion of one or more amino acid residues selected from positions 673 to 683 of SEQ ID NO: 2.

[0357] 79. The modified cell of embodiment 59, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 is a deletion of two consecutive amino acid residues selected from positions 673 to 674 of SEQ ID NO:2, positions 674 to 675 of SEQ ID NO:2, positions 675 to 676 of SEQ ID NO:2, positions 676 to 677 of SEQ ID NO:2, positions 677 to 678 of SEQ ID NO:2, positions 678 to 679 of SEQ ID NO:2, positions 679 to 680 of SEQ ID NO:2, positions 680 to 681 of SEQ ID NO:2, positions 681 to 682 of SEQ ID NO:2, positions 682 to 683 of SEQ ID NO:2, and positions 683 to 684 of SEQ ID NO:2.

[0358] 80. The modified cell of embodiment 59, wherein the genetic modification of one or more C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2 is a deletion of three consecutive amino acid residues selected from positions 673 to 675 of SEQ ID NO:2, positions 674 to 676 of SEQ ID NO:2, positions 675 to 677 of SEQ ID NO:2, positions 676 to 678 of SEQ ID NO:2, positions 677 to 679 of SEQ ID NO:2, positions 678 to 680 of SEQ ID NO:2, positions 679 to 681 of SEQ ID NO:2, positions 680 to 682 of SEQ ID NO:2, positions 681 to 683 of SEQ ID NO:2, positions 682 to 684 of SEQ ID NO:2, and positions 683 to 685 of SEQ ID NO:2.

[0359] 81. The modified cell of embodiment 59, wherein the variant Ace3 TF comprises a substitution of one or more C-terminal amino acid residues selected from amino acid positions 672 to 683 of SEQ ID NO: 2.

[0360] 82. The modified cell of embodiment 81, wherein the variant Ace3 TF further comprises a deletion of at least the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2.

[0361] 83. The modified cell of embodiment 59, wherein the variant Ace3 TF comprises an insertion of one or more amino acid residues at a C-terminal amino acid position selected from amino acid positions 673 to 683 of SEQ ID NO: 2.

[0362] 84. The modified cell of any one of claims 61, 64, or 65, wherein the inducing substrate is selected from lactose, sophorose, gentiobiose, and cellulose.

[0363] 85. A lignocellulolytic enzyme produced by the modified cell of embodiment 61.

[0364] 86. A protein of interest (POI) produced by the modified cell of embodiment 63.

[0365] 87. A method for screening for a variant Ace3 TF protein that induces expression of a gene encoding a lignocellulolytic enzyme in the absence of an inducing substrate, the method comprising: (a) constructing a DNA library encoding a plurality of variant Ace3 TF proteins containing modified amino acid residues at one or more C-terminal amino acid positions selected from positions 673 to 683 of SEQ ID NO: 2; (b) transforming a plurality of Trichoderma fungal cells with DNA sequences from the DNA library of step (a); and (c) culturing and screening a plurality of transformed Trichoderma fungal cells under conditions suitable for production of a lignocellulolytic enzyme in the absence of an inducing substrate, wherein the screened Trichoderma fungal cells that express a gene encoding a lignocellulolytic enzyme are screened for a DNA library sequence encoding a variant Ace3 TF protein that induces expression of the lignocellulolytic enzyme gene in the absence of an inducing substrate.

[0366] 88. The method of embodiment 87, further comprising isolating transformed Trichoderma fungal cells comprising DNA library sequences encoding variant Ace3 TF proteins that induce expression of lignocellulolytic enzymes in the absence of an inducing substrate.

[0367] 89. The method of embodiment 88, further comprising the step of isolating a DNA sequence encoding the variant Ace3 TF protein.

[0368] 90. A method for screening for a variant Ace3 TF protein that upregulates the expression of a gene encoding a lignocellulolytic enzyme in the presence of an inducing substrate compared to the expression of the same gene encoding the same lignocellulolytic enzyme regulated by an Ace3 TF protein having a wild-type C-terminus of SEQ ID NO: 2 in the presence of the same inducing substrate, the method comprising the steps of: (a) constructing a DNA library encoding a plurality of variant Ace3 TF proteins comprising modified amino acid residues at one or more C-terminal amino acid positions selected from positions 673 to 683 of SEQ ID NO: 2; (b) transforming a plurality of Trichoderma fungal cells with DNA sequences from the DNA library of step (a); and (c) culturing and screening a plurality of transformed Trichoderma fungal cells in the presence of an inducing substrate under conditions suitable for the production of a lignocellulolytic enzyme, wherein the variant Ace3 TF protein upregulates the expression of the same gene encoding the same lignocellulolytic enzyme in the presence of the same inducing substrate compared to the expression of the same gene encoding the same lignocellulolytic enzyme regulated by an Ace3 TF protein having a wild-type C-terminus of SEQ ID NO: 2 in the presence of the same inducing substrate, the method comprising the steps of: (a) constructing a DNA library encoding a plurality of variant Ace3 TF proteins comprising modified amino acid residues at one or more C-terminal amino acid positions selected from positions 673 to 683 of SEQ ID NO: 2; (b) transforming a plurality of Trichoderma fungal cells with DNA sequences from the DNA library of step (a); and (c) culturing and screening a plurality of transformed Trichoderma fungal cells under conditions suitable for the production of a lignocellulolytic enzyme in the presence of an inducing substrate. The method further comprises identifying a DNA library sequence therein encoding a variant Ace3 TF protein that upregulates expression of the lignocellulolytic enzyme gene in the presence of an inducing substrate, wherein the screened Trichoderma fungal cells produce an increased amount of the lignocellulolytic enzyme compared to the amount of the same lignocellulolytic enzyme produced by a control Trichoderma fungal cell containing a gene encoding the TF protein.

[0369] 91. The method of embodiment 90, further comprising isolating transformed Trichoderma fungal cells comprising DNA library sequences encoding variant Ace3 TF proteins that upregulate expression of lignocellulolytic enzymes in the presence of an inducing substrate.

[0370] 92. The method of embodiment 91, further comprising isolating a DNA sequence encoding the variant Ace3 TF protein.

[0371] 93. A method for screening for variant Ace3 TF proteins that induce expression of a gene encoding a reporter protein in the absence of an inducing substrate, comprising: (a) screening multiple variant Ace3 TF proteins containing modified amino acid residues at one or more C-terminal amino acid positions selected from positions 673 to 683 of SEQ ID NO: 2; 1. A method for producing a TF protein comprising: (a) constructing a DNA library encoding the TF protein; (b) transforming a plurality of Trichoderma cells with DNA sequences from the DNA library of step (a), wherein the transformed Trichoderma cells contain an introduced expression cassette comprising an upstream (5') cellulase promoter operably linked to an open reading frame (ORF) encoding a reporter protein; and (c) culturing and screening the plurality of transformed Trichoderma cells for expression of the reporter protein-encoding ORF in the absence of an inducing substrate, wherein the screened Trichoderma cells expressing the reporter protein-encoding ORF identify DNA library sequences therein encoding variant Ace3 TF proteins that induce expression of the reporter protein-encoding ORF in the absence of an inducing substrate.

[0372] 94. The method of embodiment 93, further comprising isolating transformed Trichoderma fungal cells comprising DNA library sequences encoding variant Ace3 TF proteins that induce expression of an ORF encoding a reporter protein in the absence of an inducing substrate.

[0373] 95. The method of embodiment 93, further comprising isolating a DNA sequence encoding the variant Ace3 TF protein.

[0374] 96. A method for screening for variant Ace3 TF proteins that upregulate the expression of a gene encoding a reporter protein in the presence of an inducing substrate compared to the expression of the same gene encoding the same reporter protein regulated by an Ace3 TF protein having a wild-type C-terminus of SEQ ID NO: 2 in the presence of the same inducing substrate, comprising: (a) screening multiple variant Ace3 TF proteins that contain modified amino acid residues at one or more C-terminal amino acid positions selected from positions 673 to 683 of SEQ ID NO: 2; (b) constructing a DNA library encoding a TF protein; (b) transforming a plurality of Trichoderma fungal cells with DNA sequences from the DNA library of step (a), wherein the transformed Trichoderma fungal cells contain an introduced expression cassette comprising an upstream (5') cellulase promoter operably linked to an open reading frame (ORF) encoding a reporter protein, and optionally a downstream (3') terminator sequence operable in Trichoderma fungi; and (c) culturing and screening the plurality of transformed Trichoderma fungal cells for expression of the ORF encoding the reporter protein in the presence of an inducing substrate, wherein the transformed Trichoderma fungal cells contain Ace3 having a wild-type C-terminus of SEQ ID NO:2. The method further comprises identifying a DNA library sequence therein encoding a variant Ace3 TF protein that produces an increased amount of reporter protein compared to the amount of the same reporter protein produced by a control Trichoderma cell containing a gene encoding the TF protein, and wherein the screened Trichoderma cell containing the expression cassette of step (b) upregulates expression of the ORF encoding the reporter protein in the presence of an inducing substrate.

[0375] 97. The method of embodiment 96, further comprising isolating transformed Trichoderma fungal cells comprising DNA library sequences encoding variant Ace3 TF proteins that upregulate expression of an ORF encoding a reporter protein in the presence of an inducing substrate.

[0376] 98. The method of embodiment 97, further comprising isolating a DNA sequence encoding the variant Ace3 TF protein.

[0377] 99. The method of embodiment 87 or embodiment 90, wherein the lignocellulolytic enzymes are selected from the group consisting of cellobiohydrolases, endoglucanases, and β-glucosidases.

[0378] 100. The method of embodiment 93 or embodiment 96, wherein the cellulase promoter is selected from the group consisting of a cellobiohydrolase promoter sequence, an endoglucanase promoter sequence, a β-glucosidase promoter sequence, and a xylanase promoter sequence.

[0379] 101. The method of any one of embodiments 87, 90, 93, and 96, wherein the inducing substrate is selected from lactose, sophorose, gentiobiose, and cellulose.

[0380] 102. A method for producing lignocellulolytic enzymes in Trichoderma fungal cells in the absence of an inducing substrate, comprising: (a) isolating T. reesei mutant cells containing a mutant ace3 gene encoding a variant Ace3 transcription factor (TF) protein, wherein the variant Ace3 TF protein contains at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2 and contains one or more mutated C-terminal amino acid residues selected from positions 673-689 of SEQ ID NO:2; and (b) fermenting the isolated mutant cells under conditions suitable for production of lignocellulolytic enzymes, wherein the suitable fermentation conditions do not contain an inducing substrate.

[0381] 104. The method of embodiment 103, wherein the lignocellulolytic enzyme is selected from the group consisting of cellobiohydrolases, endoglucanases, and β-glucosidases.

[0382] 105. A method for producing lignocellulolytic enzymes in a genetically modified Trichoderma fungal cell in the absence of an inducing substrate, comprising: (a) obtaining a Trichoderma fungal cell containing an ace3 gene encoding a wild-type Ace3 transcription factor (TF) protein containing at least 90% sequence identity to amino acid positions 1-689 of SEQ ID NO:2;

[0383] (b) genetically modifying the ace3 gene of the Trichoderma fungal cell of step (a), wherein the modified ace3 gene encodes a variant Ace3 TF protein that comprises at least 90% sequence identity to amino acid positions 1 to 672 of SEQ ID NO:2 and that comprises one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2; and (c) fermenting the modified Trichoderma fungal cell of step (b) under conditions suitable for production of lignocellulolytic enzymes, wherein the suitable fermentation conditions do not include an inducing substrate.

[0384] 106. The method of embodiment 105, wherein the C-terminus of the wild-type Ace3 TF protein comprises at least 95% sequence identity to amino acid positions 673-689 of SEQ ID NO:2.

[0385] 107. A method for producing a lignocellulolytic enzyme in a genetically modified Trichoderma fungal cell in the absence of an inducing substrate, comprising: (a) obtaining a Trichoderma fungal cell containing an ace3 gene encoding a variant Ace3 TF protein comprising at least 90% sequence identity to the Ace3-S TF protein of SEQ ID NO: 4; (b) genetically modifying the ace3 gene of the Trichoderma fungal cell of step (a), wherein the modified ace3 gene encodes a variant Ace3 TF protein comprising at least 90% sequence identity to amino acid positions 1 to 672 of SEQ ID NO: 2 and comprising one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO: 2; and (c) fermenting the modified Trichoderma fungal cell of step (b) under conditions suitable for production of the lignocellulolytic enzyme, wherein the suitable fermentation conditions do not include an inducing substrate.

[0386] 108. The method of embodiment 107, wherein the variant Ace3TF protein comprising at least 90% sequence identity to the Ace3-S TF protein of SEQ ID NO: 4 does not comprise an intact zinc binuclear (Zn2Cys6) DNA binding as set forth in SEQ ID NO: 29.

[0387] 109. The method of embodiment 107, wherein the genetically modified ace3 gene encoding a variant Ace3 TF protein comprising at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2 comprises an intact zinc binuclear (Zn2Cys6) DNA binding as set forth in SEQ ID NO:29.

[0388] 110. A method for producing a protein of interest (POI) in an engineered Trichoderma fungal cell in the absence of an inducing substrate, comprising: (a) obtaining a Trichoderma fungal cell containing an ace3 gene encoding a wild-type Ace3 transcription factor (TF) protein having at least 90% sequence identity to amino acid positions 1 to 689 of SEQ ID NO:2; (b) genetically modifying the ace3 gene of the Trichoderma fungal cell of step (a), wherein the engineered ace3 gene encodes a variant Ace3 TF protein having at least 90% sequence identity to amino acid positions 1 to 672 of SEQ ID NO:2 and one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO:2. (c) introducing an expression cassette encoding a POI into the modified Trichoderma fungal cell of step (b), wherein the expression cassette comprises an upstream (5') promoter sequence from a gene encoding a lignocellulolytic enzyme operably linked to a downstream (3') open reading frame (ORF) sequence encoding the POI; and (d) fermenting the modified Trichoderma fungal cell of step (c) under conditions suitable for production of the POI, wherein the suitable fermentation conditions do not include an inducing substrate.

[0389] 111. The method of embodiment 110, wherein steps (b) and (c) are performed simultaneously or in either order.

[0390] 112. A method for producing a protein of interest (POI) in an engineered Trichoderma fungal cell in the absence of an inducing substrate, comprising: (a) obtaining a Trichoderma fungal cell containing an ace3 gene encoding a variant Ace3 TF protein having at least 90% sequence identity to the Ace3-S TF protein of SEQ ID NO: 4; (b) genetically modifying the ace3 gene of the Trichoderma fungal cell of step (a), wherein the engineered ace3 gene encodes a variant Ace3 TF protein having at least 90% sequence identity to amino acid positions 1 to 672 of SEQ ID NO: 2 and containing one or more mutated C-terminal amino acid residues selected from positions 673 to 689 of SEQ ID NO: 2. (c) introducing an expression cassette encoding a POI into the modified Trichoderma fungal cell of step (b), wherein the expression cassette comprises an upstream (5') promoter sequence from a gene encoding a lignocellulolytic enzyme operably linked to a downstream (3') open reading frame (ORF) sequence encoding the POI; and (d) fermenting the modified Trichoderma fungal cell of step (c) under conditions suitable for production of the POI, wherein the suitable fermentation conditions do not include an inducing substrate.

[0391] 113. The method of embodiment 110, wherein steps (b) and (c) are performed simultaneously or in either order. [Example]

[0392] The following examples illustrate embodiments of the present disclosure, but it should be understood that they are given by way of illustration only. From the above discussion and these examples, those skilled in the art will be able to make various changes and modifications to the present disclosure to adapt it to various uses and conditions. Such modifications are also intended to fall within the scope of the invention as defined in the claims.

[0393] Example 1 ACE3-L protein containing C-terminal truncations A. Overview Ace3 is a T. reesei transcription factor that regulates cellulase and hemicellulase production under inducing conditions (e.g., in the presence of lactose). Similarly, a highly functional form of the Ace3 transcription factor, designated "Ace3L," has been identified and described in International Publication No. WO 2018 / 067599. For example, as described in International Publication No. WO 2018 / 067599, T. reesei strains expressing the Ace3L transcription factor (e.g., SEQ ID NO: 6) not only exhibited enhanced (increased) protein production in the presence of an inducer (e.g., lactose, sophorose), but also showed a significant increase in protein production in the absence of the inducer.

[0394] The Ace3 transcription factor has two known variants at its C-terminus. For example, the ace3 gene in T. reesei strain QM6a encodes the Ace3-S protein (Figure 1) containing the full-length (wild-type) C-terminus encoded by the ace3 gene, whereas the same ace3 gene in T. reesei strains RL-P37 and RUT-C30 contains a premature stop codon resulting in the truncation of 11 amino acids in the encoded protein (Figure 1; SEQ ID NO: 6). Furthermore, as shown in Figure 1, the Ace3L protein contains an intact zinc binuclear cluster (Zn2Cys6) DNA-binding domain at the N-terminus (Figure 3; SEQ ID NO: 6) compared to the Ace3-S protein (SEQ ID NO: 4). 4). The Ace3-LC mutant was generated by reverting the premature stop codon to a glutamine codon and thus contains an intact zinc binuclear cluster (Zn2Cys6) DNA-binding domain at the N-terminus and the full-length (wild-type) sequence at the C-terminus.

[0395] As described and exemplified herein, both the truncated C-terminus of the Ace3 protein and the intact N-terminal zinc binuclear cluster (Zn2Cys6) DNA-binding domain of the Ace3 protein are essential features for its function of increasing protein production both in the absence and presence of an inducer. In this example, Applicant further evaluated the C-terminal amino acid region of the Ace3 protein by selectively truncating the C-terminal amino acid residues, as described below. The T. reesei parental cells shown in the following examples were derived from T. reesei strain RL-P37 (NRRL Accession No. 15709) in which the T. reesei pyr4 gene was deleted, as generally described by Sheir-Neiss and Montenecourt, 1984.

[0396] B. Construction of Expression Vectors and Strains Two sets of expression vectors with Ace3 C-terminal truncations were constructed. The first set of six vectors contains C-terminal truncations ranging from 0 to 25 amino acids in 5 amino acid increments (see Table 1; Set 1). The second set of 12 vectors contains truncations ranging from 6 to 19 amino acids in 1 amino acid increments (see Table 1; Set 2).

[0397] These 18 Ace3-LC_C-term variant expression vectors were constructed using standard molecular biology techniques. These vectors were designed to enable targeted integration of the ace3 expression cassette into the glucoamylase (gla1) locus of T. reesei. They contain the 5' and 3' flanks of the T. reesei gla1 locus required for targeted integration. The 5' flank sequence contains a 1.5-kb homology box homologous to the DNA sequence corresponding to scaffold_1:1489226-1490662. The 3' flank sequence contains a 1.5-kb homology box homologous to the DNA sequence corresponding to scaffold_1:1492835-1494335. These sequences were designed to target the gla1 gene (JGI protein ID Trire2_1885) and replace the genomic nucleotides between the 5' and 3' flanks (scaffold_1:1490663-1492834) with the intervening cassette sequence. The expression vectors also contain a heterologous T. reesei dic1 promoter sequence (scaffold_8:1376871-1378833, see, e.g., WO 2018 / 067599) operably linked to the ace3 ORF coding sequence with the native ace3 gene terminator. Additionally, these vectors contain the pyr4 marker with its native promoter and terminator for selection of T. reesei transformants. Repeats of the pyr4 promoter were included to allow for excision of the pyr4 gene after integration into the gla1 locus. The vector also contains the bacterial ColE1 ori and AmpR gene for replication and selection in E. coli, as well as the yeast 2μ ori and ura3 gene for replication and selection in S. cerevisiae. A representative vector map is shown in Figure 2, which depicts vector pYL72, which contains the ORF encoding the Ace3-LC protein (SEQ ID NO: 2; i.e., including the full-length wild-type C-terminus).Other vectors (eg, pYL73, pYL74, etc.) contain ORF sequences encoding various C-terminally truncated Ace3 proteins (Table 1).

[0398] The expression vectors shown in Table 1 were digested with PmeI to release fragments for targeted integration and separated by agarose gel electrophoresis. The correct fragments were isolated from the gel using a gel extraction kit (Qiagen) according to the manufacturer's protocol. Cas9 nuclease and a synthetic single-guide RNA (sgRNA) (SEQ ID NO: 70) targeting a 23-bp sequence within the gla1 gene were constructed in vitro according to the manufacturer's protocol (Synthego), as generally described in WO 2016 / 100568. Approximately 10 μg of the purified fragment and the constructed Cas9-sgRNA complex were used to transfect the pyr4 T. reesei (RL-P37) strain. - Protoplasts of the mutants were transformed using a polyethylene glycol (PEG)-mediated protoplast transformation protocol (Ouedraogo et al., 2015; Penttila et al., 1987).

[0399] Transformants were grown on Vogel's minimal medium agar plates and selected for uridine prototrophy, conferred by the pyr4 marker. Grown clones were screened for correct integration by PCR using the primers listed in Table 2. Clones giving the expected signal were purified to single-cell clones and rescreened for correct integration and clonal purity by PCR using the primers listed in Table 2. PCR products were sequenced by Sanger sequencing to confirm the presence of the designated ace3 C-terminal truncation variants. The purified and sequence-verified Ace3 C-terminal truncation variants are listed in Table 3. [Table 1] [Table 2] [Table 3]

[0400] C. Culture of various ace3 transformants The parental (LT338) and transformed (daughter) T. reesei cells (Table 3) described above were tested under both "uninducing" (glucose) and "inducing" (glucose / sophorose or lactose) conditions. For example, in the "uninducing" condition, cells were grown in 1.25 ml liquid broth of defined medium supplemented with 2% glucose (wt / vol) in a standard 24-well microtiter plate (MTP). In the "inducing" condition, cells were grown in 1.25 ml liquid broth of defined medium supplemented with 2% lactose or 2% glucose / sophorose (wt / vol) in a 24-well MTP, where lactose or sophorose act as strong inducers of cellulase enzyme expression.

[0401] MTP cultures were incubated at 28°C, 250 rpm, and 85% humidity for 5 days. Following incubation, supernatants were collected from all cultures, and total secreted protein was measured by Bradford dye-binding assay at 595 nm using Bio-Rad reagent (Thermo Scientific®; catalog no. 23236) and a 5-fold dilution of bovine serum albumin (BSA) as a standard, according to the manufacturer's protocol.

[0402] D. Ace3-LC variants with 10-15 amino acid C-terminal truncations are functional As shown in FIG. 7, Applicant evaluated total protein production by a first set of strains (Table 1; Set 1 and Table 3) expressing Ace3-LC variants with C-terminal truncations of 5, 10, 15, 20, and 25 amino acids (see, e.g., Table 1; Set 1; Table 3 and FIG. 7). As shown in FIG. 7, the parental control strain RL-P37 was only able to produce large amounts of protein on lactose (i.e., under inducing conditions) and produced minimal basal levels of protein on glucose (i.e., under non-inducing conditions). Daughter cells expressing Ace3-L with the 10-amino acid truncation (SEQ ID NO: 12) or the 15-amino acid truncation (SEQ ID NO: 16) showed increased protein production under both lactose (inducing) and glucose (non-inducing) conditions. Thus, as shown in FIG. 7, the fold change of increase is approximately 1.8-fold on glucose and approximately 3-fold on lactose compared to the protein levels produced by the parent strain (RL-P37) on lactose.

[0403] For example, this improvement in total protein production is similar to that observed with the Ace3-L protein (SEQ ID NO: 6), which contains an 11-amino acid truncation, as described in WO 2018 / 067599. A 5-amino acid truncation (SEQ ID NO: 7) demonstrated protein expression levels similar to those of the parent strain on both lactose and glucose. A 0-amino acid truncation (i.e., Ace3-LC; WT C-terminus; SEQ ID NO: 2), a 20-amino acid truncation (SEQ ID NO: 21), or a 25-amino acid truncation (SEQ ID NO: 22) significantly reduced total protein production on both lactose and glucose. These results indicate that truncation of the C-terminus of the Ace3 protein is essential for its ability to upregulate protein production. Similarly, as shown in Figure 7, there are upper and lower limits to the number of amino acid truncations tolerated at the Ace3 C-terminus.

[0404] E. Ace3-LC variants with 7-17 amino acid C-terminal truncations are functional To further explore the upper and lower limits of acceptable Ace3 C-terminal amino acid truncations, we constructed and tested T. reesei strains containing Ace3-LC with C-terminal truncations of 5 to 20 amino acids (i.e., using truncations in one-amino acid increments; see, e.g., Tables 1, 3, and Figure 8). For example, truncations of 5 or 6 amino acids (Figure 8) showed results similar to those of the parent strain (i.e., basal levels of protein production on glucose and high levels on lactose). In contrast, truncations of at least 7 to 17 amino acids showed improved production on both glucose and lactose, similar to previous observations with the 11-amino acid truncation in the Ace3-L protein (Figure 8). Furthermore, truncations of 18 or more C-terminal amino acids significantly reduced protein production to basal levels on both lactose and glucose (Figure 8).

[0405] Thus, as described above, the (Ace3) C-terminus plays an essential role in the function of the Ace3 protein (i.e., regulating protein production) both in the presence and absence of an inducer. For example, strains expressing Ace3-LC proteins with truncations of 7 to 17 amino acids showed an approximately two-fold improvement in total protein production under glucose (non-inducing) conditions compared to the parent strain RL-P37 under lactose (inducing) conditions, and also showed an approximately three-fold improvement in total protein production under lactose (inducing) conditions compared to the parent strain RL-P37 under lactose (inducing) conditions. In contrast, strains expressing Ace3-LC with a wild-type C-terminus or a C-terminus with truncations of 5, 6, 18, or more amino acids produced minimal amounts of protein under glucose (non-inducing) conditions, and under lactose (inducing) conditions, the amount of protein produced was similar or reduced compared to the parent strain under lactose (inducing) conditions. Thus, as shown herein, T. reesei strains containing an Ace3-LC protein having at least a 7 amino acid truncation (SEQ ID NO: 9) and up to a 17 amino acid truncation (SEQ ID NO: 18) exhibit an enhanced protein production phenotype (compared to other strains tested), and the enhanced protein production phenotype was observed both in the presence and absence of an inducer.

[0406] Example 2 Reversion of ACE3 truncation A. Overview The industrially important Trichoderma strains Rut-C30 and RL-P37 are mutagenized derivatives of the Trichoderma natural isolate QM6a (Le Crom et al., 2009; Sheir-Neiss and Montenecourt, 1984), with strain NG14 being their last common ancestor. Genomic comparison of NG14 and QM6A identified 126 single nucleotide polymorphisms (SNPs) and 22 insertions and deletions (indels) between them and the original strain QM6A (PMID: 19805272).

[0407] Among these, a point mutation in a gene encoding a transcription factor was predicted to result in an 11-amino acid C-terminal truncation based on the computationally predicted gene structure available at the time. This transcription factor was later named Ace3 after gene knockout resulted in a significant loss of cellulase and hemicellulase production under inducing conditions (e.g., in the presence of lactose; Hakkinen et al., 2014). Applicants previously showed that the 11-amino acid truncation, when overexpressed, was essential for the function of Ace3-L (a specific variant of Ace3) in improving protein production both in the presence and absence of an inducer (see, e.g., WO 2018 / 067599).

[0408] As described in Example 1 above, Applicants determined the exact number of C-terminal (amino acid) truncations (i.e., 7 to 17 amino acids) required for improved protein production. In this example, Applicants molecularly reverted the C-terminal truncation point mutation in the ace3 locus to the "wild-type" (C-terminal) sequence of QM6a, which encodes a full-length Ace3 protein with a wild-type N-terminus (FIG. 1A; SEQ ID NO: 2) and a wild-type C-terminus terminating in glycine (G) (FIG. 1B; SEQ ID NO: 2). As described below, strains expressing the full-length Ace3 transcription factor (SEQ ID NO: 2) exhibited reduced protein production.

[0409] T. reesei host strain The T. reesei parent strain described in this example was derived from T. reesei strain RL-P37 (NRRL Accession No. 15709), as described by Sheir-Neiss and Montenecourt (1984). The strain designated herein as "T4abc pyr2" is a mutagenized derivative of RL-P37, with significant mutations in pyr2 that cause the strain to require uridine for growth and the nik1 (M743T) mutation, which increases total protein production (see, e.g., U.S. Patent Publication No. 2018 / 0037919).

[0410] C. Construction of Expression Vectors and Strains A Trichoderma ace3 reversion cassette plasmid, pRATT346, was prepared using standard molecular biology procedures, so that one of skill in the art could easily recreate this plasmid from the relevant DNA components disclosed herein. The pRATT346 plasmid contained a DNA sequence with a 2.0 kb homology box homologous to DNA sequence corresponding to scaffold 8, 422475-424516 (left flank). The nucleotide corresponding to scaffold 8, 424132, within this left flank was a cytosine (C) as in the QM6a genome, as opposed to a thymine (T) as in the genomes of NG14 and its derivatives, including T4abc. The plasmid also contained a 1.8 kb homology box homologous to DNA sequence corresponding to scaffold 8, 424588-426381 (right flank). These sequences were designed to target the ace3 gene and replace the region of the genome between the left and right flanks (Scaffold 8, 424517 to 424587) with an intervening cassette sequence.

[0411] These intervening cassette sequences contained the pyr2 selection marker from Trichoderma atroviride, which was intended to minimize homology with the endogenous T. reesei pyr2 in the genome of the strain to be transformed. Immediately upstream of the pyr2 selection marker was a direct repeat duplication of the 3' end of this marker, facilitating subsequent marker loss and isolation of useful pyr2 mutant derivatives of the transformants / disruptants. In a subset of transformants with the correctly targeted intervening cassette sequence, a cytosine (C) nucleotide on the left flank would also be integrated into the genome in place of the mutant thymine (T) nucleotide (Scaffold 8, 424132). The ace3 revertant allele described herein (encoding full-length Ace3; SEQ ID NO:2) contains both a reversion of the nucleotide at Scaffold 8, 424132, and an insertion of the repeat-flanked pyr2 marker between Scaffold 8, 424517-424587.

[0412] Therefore, strain T4abc pyr2 was transformed with the ace3 reversion cassette from pRATT346 using PEG-mediated transformation and plated on Vogel's minimal medium (Vogel, 1956) containing 1.2 M sorbitol to select for candidates based on uridine prototrophy conferred by the pyr2 marker. Trichoderma transformation methods are well known and described in the art (see, e.g., U.S. Pat. No. 5,246,853). Individual transformants were isolated, transferred to Vogel's minimal medium, and grown. PCR analysis was used to identify transformants in which the ace3 reversion cassette had integrated by homologous recombination at the ace3 locus, using methods known to those skilled in the art with the following guidance:

[0413] To precisely target the disruption of a gene of interest, only a subset of recombinant cells will be able to successfully utilize the homologous flanks, so it may be necessary to screen a large number of transformants to identify transformants with the desired event. PCR can be used to test which recombinant cells have the desired targeted disruption. Primers must be designed to amplify across each of the homology box regions, with one primer priming at a location within the selectable marker more than 100 bp from the nearest end, and the other primer priming at a location 100 bp beyond the end of the homology box region within the adjacent genomic sequence. Cells likely to contain the correct targeted disruption will succeed in generating PCR products spanning the left and right flanks of the disruption cassette, while unsuccessful transformation events will not produce products of the expected size. At this stage, the culture is a mixture of transformed and untransformed cells, so a purification step may be necessary. Purified cultures can be tested by PCR for the disappearance of short PCR products spanning the disruption site.

[0414] After spore purification, further PCR analysis was performed to confirm that integration had occurred correctly and that the transformants were homonuclear. The portion of the ace3 gene spanning the reversion mutation was then PCR amplified and sequenced to determine whether this mutation had been incorporated into the left flank during the integration event. The resulting strain with confirmed homologous integration of the ace3 reversion cassette was designated "T4abc ace3_rev."

[0415] Fermentation of D.ace3 revertants Three strains were evaluated for their protein production in shake flask fermentation. These included the "T4abc" strain (containing the ace3 truncation allele), the "T4abc ace3_rev" strain (containing the ace3 full-length allele), and the "T4abc del-cbh1" strain (containing a deletion of the cbh1 cellobiohydrolase gene). Each strain was evaluated in two independent shake flasks (e.g., Flask A and Flask B).

[0416] The liquid defined (LD) culture medium (see, e.g., U.S. Pat. No. 8,455,631) contained the following components: casamino acids, 9 g / L; (NH4)2SO4, 5 g / L; MgSO4·7H2O, 1 g / L; KH2PO4, 4.5 g / L; CaCl2·2H2O, 1 g / L; PIPPS, 33 g / L; 400× T. reesei trace elements, 2.5 mL / L; pH adjusted to 5.5 with NaOH. After sterilization, lactose or a glucose / sophorose mixture was added to a final concentration of 1.6 w / v%.

[0417] To create seed cultures, spores of each strain were individually added to 50 mL of YEG (5 g / L yeast extract, 22 g / L glucose, HO) in a 250 mL flask. The cultures were grown for 36–48 hours at 28°C and 200 rpm in a shaking incubator. After incubation, 0.3 mL of the seed culture was added to 50 mL of LD medium in a baffled shake flask. This production culture was grown for 5 days at 28°C and 180 rpm. Secreted proteins were harvested by centrifugation to pellet the cells and then collect the supernatant. Proteins were precipitated from the supernatant using an equal volume of trichloroacetic acid (TCA) and then dissolved in 0.1 N sodium hydroxide (NaOH). Total protein was then measured using the BCA protein assay according to the manufacturer's protocol (ThermoFisher Scientific, Grand Island, NY, USA). BCA assay numbers were normalized to the average value of T4abc cultures run in parallel to minimize the effect of any week-to-week variations in total protein production efficiency.

[0418] As shown in Figure 9, when the ace3 mutation was reverted in a T4abc background to encode full-length Ace3 (e.g., SEQ ID NO: 2), the total secreted protein titer was reduced by 73% compared to the T4abc parent strain encoding a truncated form of the Ace3 C-terminus (e.g., SEQ ID NO: 6). In contrast, deletion of the gene encoding the most prominent secreted protein (Cbh1) reduced the total secreted protein titer by only 47%.

[0419] Example 3 ACE3 C-terminal amino acid variants As described above in Examples 1 and 2, applicants have demonstrated that truncations of a defined number of amino acids (e.g., 7-17) at the Ace3LC C-terminus are necessary to improve protein productivity. Furthermore, as briefly described above in the detailed description, applicants contemplate that other genetic modifications of the Ace3 C-terminus (e.g., substitutions, insertions, internal (C-terminal) deletions, combinations thereof, etc.) are equally suitable genetic modifications for improving protein productivity.

[0420] This example describes compositions and methods for constructing Ace3 C-terminal variant libraries and screening such libraries to improve protein productivity. For example, a C-terminal variant library for Ace3 can be generated by one of skill in the art by substituting the coding sequence of Ace3-LC, encoding amino acids 641-689 of SEQ ID NO:2, from plasmid pYL72 (Figure 6) and by referencing the sequence identification numbers (SEQ ID NOs) provided for each library and the exemplary plasmid pA3L02 for SEQ ID NO:32 (Figure 11). Accordingly, Applicants describe herein methods for constructing and screening three different Ace3 C-terminal libraries, including a site-substitution library (Library 1), a scanning insertion library (Library 2), and a scanning deletion library (Library 3). Therefore, the three example libraries described herein are not intended to be limiting but rather to illustrate the overall approach, which can be easily adapted by one of skill in the art to design / construct other Ace3 C-terminal variant libraries and screen them to improve protein productivity.

[0421] Library 1 is a site-substitution library in which the codon for each amino acid corresponding to amino acid positions 673-683 of SEQ ID NO:2 is replaced with an "NNK" codon (mut) (where "N" is any nucleotide and "K" is a "G" or "T" nucleotide). The degenerate DNA sequences of Library 1 for each encoded amino acid position corresponding to amino acids 641-689 of SEQ ID NO:2 are set forth as SEQ ID NOs:32-42, respectively, and are shown in Table 4 below. For comparison, SEQ ID NO:31 corresponds to the native DNA sequence for each encoded amino acid position corresponding to amino acids 641-689 of SEQ ID NO:2 (e.g., SEQ ID NO:30, inclusive of amino acid positions 641-689 of SEQ ID NO:2). [Table 4]

[0422] Library 2 is a scanning insertion library in which the three codon sequence "NDT" (codon 1)-"NDT" (codon 2)-"NNK" (codon 3) is inserted after each codon (3') corresponding to amino acids 672-683 of SEQ ID NO:2 (where "N" is any nucleotide, "D" is an "A," "G," or "T" nucleotide, and "K" is a "G" or "T" nucleotide). The degenerate DNA sequences of Library 2 for each encoded amino acid position corresponding to amino acids 641-689 of SEQ ID NO:2 are set forth as SEQ ID NOs:43-54, respectively, and are shown in Table 5 below. [Table 5] Library 3 is a scanning deletion library in which each codon corresponding to amino acid positions 673-683 of SEQ ID NO:2 is modified as follows: starting with the codon corresponding to amino acid position 673 of SEQ ID NO:2, the codon at position 673 and the next two adjacent downstream (3') codons (i.e., a total of 9 nucleotides) are deleted from the Ace3 coding sequence. The DNA sequences of Library 3 for each encoded amino acid position corresponding to amino acids 641-689 of SEQ ID NO:2 are set forth as SEQ ID NOs:55-65 and are shown in Table 6 below. [Table 6]

[0423] By pooling degenerate library plasmid clones containing mutated amino acid positions (e.g., from Library 1), one skilled in the art can reduce the number of Trichoderma reesei transformations required. For example, the entire Library 3 can be reasonably pooled for Trichoderma reesei transformation. Thus, pooling can be performed after isolation and sequencing of individual plasmid clones or by pooling random E. coli transformants prior to plasmid extraction, providing high confidence in the correct modification of the pYL72 plasmid in over 90% of the E. coli transformants and over 100 E. coli transformants per pool.

[0424] Therefore, Trichoderma reesei cells (e.g., RL-P37) are transformed as described in Example 1 above, but with the pooled construct library described above, targeting Ace3 variants to the gla1 locus. For each transformation library, over 60 transformants are cultured and assayed for protein production as described in Example 1. Cell cultures can be stored to allow for molecular characterization of the desired transformants. Transformants resulting from integration of plasmids pYL72 (Ace3-LC) and pYL88 (Ace3-LC C-term-11) are included in at least triplicate per 24-well plate as negative and positive controls. For example, due to the "NNK" degeneracy in Library 1, 1 / 32 clones are expected to have a stop codon and thereby encode one of the truncated Ace3 variants described in Example 1. These clones (truncated Ace3 variants) serve as internal positive controls and validation of the methodology performed.

[0425] Similarly, some Trichoderma transformants harboring Ace3 variants (e.g., pYL88(Ace3-LC C-term-11) transformants) can produce greater amounts of secreted cellulases and hemicellulases during fermentation in the absence of an inducer, compared to pYL72(Ace3-LC) transformants. To determine the sequence of the Ace3 variant integrated into the gla1 locus in these transformants, the variants can be molecularly characterized by methods known in the art, as follows.

[0426] Genomic DNA is extracted from each transformant, and the C-terminal portion of the Ace3 variant at the gla1 locus is PCR-amplified from each transformant individually using a forward primer (SEQ ID NO: 23, Table 2) that primes approximately 500 bp upstream (5') of the codon encoding amino acid position 670 of SEQ ID NO: 2 and a reverse primer (SEQ ID NO: 24, Table 2) that specifically primes in the pyr4 gene and approximately 900 bp downstream (3') of the stop codon for the Ace3 variant. The purified PCR products are then sequenced using additional nested primers or by other sequencing methods known to those skilled in the art. Subsequent analysis of the sequencing results readily identifies Ace3 variants that exhibit enhanced protein production in the absence of an inducer.

[0427] As will be appreciated by those skilled in the art, some false positives may be obtained in screening. Therefore, one skilled in the art can generate derivatives of pYL72 containing the specific Ace3 variant identified and evaluate additional transformants with the specific Ace3 variant as described above for pools.

[0428] If transformants carrying various Ace3 variants show inducer-independent phenotypic intermediates between the pYL72(Ace3-LC) and pYL88(Ace3-LC C-term-11) control transformants (if practically possible), the individual mutations of these variants can be combined and similarly evaluated as described above.

[0429] Similarly, the optimal amino acid sequences of any Ace3 variants identified from the screened library pools described above (e.g., from Library 1, Library 2, and / or Library 3) can be combined and further subjected to subsequent rounds of focal library screening and evaluated as described above. For example, depending on the sample size of the screened transformants, some amino acid substitutions at a given position may not be represented in the site-substitution library (Library 1). There may be an incentive to isolate or generate individual plasmid clones with variants for the most useful positions, so that all possible substitutions can be evaluated individually (as opposed to, e.g., the pool initially used for Library 1). Furthermore, for useful Ace3 variants identified from the scanning insertion library (Library 2), there may be an incentive to test additional variants at the most useful positions, for example, by increasing the insert length to six, nine, or more amino acid residues and / or reducing the insertion to two or one amino acid residue. Furthermore, for useful variants identified from the scanning deletion library (Library 3), there may be an incentive to generate and test additional variants at the most useful positions, e.g., by actually increasing the number of adjacent deleted residues to 4, 6, 8, etc. amino acid residues and / or decreasing the deletion size to 2 or 1 amino acid residue.

[0430] Example 4 ACE3 with C-terminal substitutions A. Overview In this example, Applicant molecularly replaced the last 11 amino acid residues at the C-terminus of Ace3-LC (SEQ ID NO: 2) with a V5 epitope tag or a V5-(6xHis) tandem tag (Figure 12). As described below, Trichoderma strains expressing Ace3-LC with a C-terminal V5 tag substitution (i.e., Ace3-LC-V5) and Ace3-LC with a C-terminal V5-(6xHis) tandem tag substitution (i.e., Ace3-LC-V5-(6xHis)) showed increased protein production both in the presence and absence of an inducing substrate.

[0431] B. Host strains, expression vectors and strain construction. T. reesei strain T4abc (e.g., as described in Example 2 above) was used as the parent strain in this example. Trichoderma ace3 C-terminal substitution cassette plasmids pYL18 (Ace3-LC-V5) and pYL19 (Ace3-LC-V5-(6xHis)) were prepared using standard molecular biology procedures so that one of skill in the art could easily recreate this plasmid from the relevant DNA segments disclosed. More specifically, the DNA sequences of the coding sequences for the Ace3 C-terminal substitution variants, beginning with the codon corresponding to amino acid residue 641 of SEQ ID NO:2, are set forth as SEQ ID NO:67 for Ace3-LC-V5 and SEQ ID NO:69 for Ace3-LC-V5-(6xHis).

[0432] The expression vector contains a vector backbone with the bacterial ColE1 ori and AmpR genes for replication and selection in E. coli, and the 2μ ori and URA3 genes for replication and selection in Saccharomyces cerevisiae. Additionally, there is a T. reesei telomere sequence ("TrTEL"), a T. reesei pyr2 selectable marker, the T. reesei promoter sequence for the gene dic1, and an Ace3-LC variant with its native terminator sequence. A representative vector map is shown in Figure 13, depicting vector pYL18, which contains the Ace3-LC variant with the last 11 amino acids replaced with a 14-amino acid V5 tag sequence.

[0433] This expression vector was inserted (transformed) into the T. reesei parent host strain (containing a nonfunctional pyr2 gene) by polyethylene glycol (PEG)-mediated protoplast transformation (Ouedraogo et al., 2015; Penttila et al., 1987). Transformants were grown on Vogel's minimal medium agar plates and selected for uridine prototrophy, acquired by the pyr2 marker. Stable transformants were obtained by two successive transfers on Vogel's agar plates, followed by two successive growths on nonselective PDA plates and one growth on Vogel's agar plates. Single colonies were then obtained by plating dilutions of the spore suspension.

[0434] Fermentation of C.Ace3 C-terminally substituted transformants The parent and transformed (daughter) T. reesei host cells were tested under both "uninduced" and "induced" conditions, as described in Example 1 above. For example, as shown in Figure 14, the parent T. reesei cells produced high levels of secreted protein only in the presence of the sophorose inducer. In contrast, the variant (daughter) T. reesei cells containing and expressing Ace3-L (i.e., containing the 11 amino acid truncation) produced large amounts of secreted protein under both induced (Glu / Sop) and uninduced (Glu) conditions. Furthermore, variant (daughter) Treesei cells containing and expressing the Ace3-LC-V5 tag (i.e., containing 14 amino acid substitutions in the last 11 amino acids) and T. reesei cells containing and expressing the Ace3-LC-V5-(6xHis) dual tag (i.e., containing 23 amino acid substitutions in the last 11 amino acids) also showed high protein productivity, although approximately 10% lower than that of the Ace3-L variant, under both induced (Glu / Sop) and uninduced (Glu) conditions (Figure 14).

[0435] Based on the above, these results indicate that additional genetic modifications of the Ace3 C-terminus, including but not limited to substitutions, insertions, internal (C-terminal) deletions, and combinations thereof, are equally suitable for improving protein productivity in Trichoderma cells under both inducing (Glu / Sop) and non-inducing (Glu) substrate conditions.

[0436] References International Publication No. 1992 / 06183 Pamphlet International Publication No. 1992 / 06209 Pamphlet International Publication No. 1992 / 06221 Pamphlet International Publication No. 1992 / 10581 Pamphlet International Publication No. 1998 / 15619 Pamphlet International Publication No. 2002 / 12465 Brochure International Publication No. 2005 / 028636 Pamphlet International Publication No. 2006 / 074005 Brochure International Publication No. 2006 / 74005 Brochure International Publication No. 2016 / 100568 Brochure U.S. Patent No. 6,022,725 U.S. Patent No. 6,268,328 Deuschle et al., EMBO J.3:1581-1585, 1984. Cao et al.,Science,9:991-1001,2000. Campbell et al., Curr. Genet., 16:53-56, 1989. Chen et al., “Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3”, Biotechnol Biofuels 13:62, 2020. Colot et al., PNAS 103(27):10352-10357, 2006. Devereux et al., Nucleic Acids Res. 12:387-395, 1984. Hakkinen, M., Valkonen, MJ, Westerholm-Parvinen, A., Aro, N., Arvas, M., Vitikainen, M., Penttila, M., Saloheimo, M., and Pakula, TM, “Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production”, Biotechnol Biofuels 7,14,2014. Harkki et al.,BioTechnol.,7:596-603,1989. Harkki et al.,Enzyme Microb.Technol.,13:227-233,1991. Ilmen et al.,“Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei”,Applied and Environmental Microbiology,63(4)-1298-1306,1997. Kriegler,Gene Transfer and Expression:A Laboratory Manual,1990. Le Crom et al.,“Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing”,PNAS 106(38):16151-16156,2009. Mullaney et al.,MGG 199:3745,1985. Needleman and Wunsch,J.Mol.Biol.,48:443,1970. Nunberg et al.,Mol.Cell Biol.4:2306,1984. Ouedraogo,J.P.,Arentshorst,M.,Nikolaev,I.,Barends,S.,and Ram,A.F.,“I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei” Applied microbiology and biotechnology 99,10083-10095,2015. Pearson and Lipman,Proc.Natl.Acad.Sci.USA 85:2444,1988. Penttila,M.,Nevalainen,H.,Ratto,M.,Salminen,E.,and Knowles,J.,“A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei”,Gene 61,155-164,1987. Sambrook et al.,Molecular Cloning,A Laboratory Manual,2 nd Edition,Cold Spring Harbor Laboratory Press,Cold Spring,New York,1989. Sambrook et al.,Molecular Cloning,A Laboratory Manual,4 th Edition,Cold Spring Harbor Laboratory Press,Cold Spring,New York,2012. 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. Smith and Waterman,Adv.Appl.Math.2:482,1981. Yelton et al.,PNAS USA 81:1470-1474,1984. Zhang et al.,“The transcription factor ACE3 controls cellulase activities and lactose metabolism via two additional regulators in the fungus Trichoderma reesei”,J.Biol.Chem.,294(48):18435-18450,2019.

Claims

1. 1. An isolated polynucleotide encoding a variant Ace3 transcription factor (TF) protein, said variant Ace3 TF comprising at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2, and including a deletion of the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2, a deletion of the last eight C-terminal amino acid positions 682-689 of SEQ ID NO:2, a deletion of the last nine C-terminal amino acid positions 681-689 of SEQ ID NO:2, a deletion of the last ten C-terminal amino acid positions 680-689 of SEQ ID NO:2, a deletion of the last twelve C-terminal amino acid positions 678-689 of SEQ ID NO:2, a deletion of the last thirteen C-terminal amino acid positions 677-689 of SEQ ID NO:2, a deletion of the last fourteen C-terminal amino acid positions 676-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 678-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 679-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 681-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 682-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 683-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 684-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 685-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 686-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions 687-689 of SEQ ID NO:2, a deletion of the last C-terminal amino acid positions a deletion of the last 15 C-terminal amino acid positions 675-689 of SEQ ID NO:2, a deletion of the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2, a deletion of the last 17 C-terminal amino acid positions 673-689 of SEQ ID NO:2, or an amino acid substitution of the last 11 amino acid residues at the C-terminus of SEQ ID NO:2 with a V5 epitope tag or a V5-(6xHis) tandem tag, wherein the amino acid residues from amino acid position 673 corresponding to SEQ ID NO:2 to the site of the deletion or substitution, if present, in the variant Ace3 transcription factor (TF) protein are identical to the corresponding amino acid residues in SEQ ID NO:

2.

2. The polynucleotide of claim 1, wherein the variant Ace3 TF comprises an amino acid sequence selected from any one of SEQ ID NOs: 9 to 18.

3. 1. A genetically modified Trichoderma fungal cell derived from a parent Trichoderma fungal cell comprising an ace3 gene encoding an Ace3 transcription factor (TF) protein comprising at least 90% sequence identity to SEQ ID NO:2, wherein the genetically modified cell comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2, and further comprises: a deletion of the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2; a deletion of the last eight C-terminal amino acid positions 682-689 of SEQ ID NO:2; a deletion of the last nine C-terminal amino acid positions 681-689 of SEQ ID NO:2; a deletion of the last ten C-terminal amino acid positions 680-689 of SEQ ID NO:2; a deletion of the last twelve C-terminal amino acid positions 678-689 of SEQ ID NO:2; a deletion of the last C-terminal amino acid positions 679-689 of SEQ ID NO:2; a deletion of the last 14 C-terminal amino acid positions 676 to 689 of SEQ ID NO:2, a deletion of the last 15 C-terminal amino acid positions 675 to 689 of SEQ ID NO:2, a deletion of the last 16 C-terminal amino acid positions 674 to 689 of SEQ ID NO:2, a deletion of the last 17 C-terminal amino acid positions 673 to 689 of SEQ ID NO:2, or an amino acid substitution of the last 11 amino acid residues at the C-terminus of SEQ ID NO:2 with a V5 epitope tag or a V5-(6xHis) tandem tag, wherein the amino acid residues from amino acid position 673 corresponding to SEQ ID NO:2 to the site of said deletion or substitution in said variant Ace3 TF protein, if present, are identical to the corresponding amino acid residues in SEQ ID NO:

2.

4. 4. The modified cell of claim 3, wherein the Ace3 TF protein comprises a functional N-terminal zinc binuclear cluster DNA-binding domain that comprises at least 95% sequence identity to SEQ ID NO:

29.

5. 4. The modified cell of claim 3, wherein the variant Ace3 TF protein upregulates expression of a gene encoding a lignocellulolytic enzyme in the absence of an inducing substrate when fermented under conditions suitable for the production of the lignocellulolytic enzyme.

6. 6. The modified cell of claim 5, wherein the lignocellulolytic enzyme is selected from the group consisting of cellobiohydrolase, endoglucanase, and β-glucosidase.

7. The modified cell of claim 3, comprising an introduced expression cassette encoding a protein of interest (POI), the introduced cassette comprising an upstream (5') promoter sequence from a gene encoding a lignocellulolytic enzyme operably linked to a downstream (3') open reading frame (ORF) sequence encoding the POI.

8. 8. The modified cell of claim 7, wherein the modified cell produces the POI in the absence of an inducing substrate when fermented under conditions suitable for the production of the POI.

9. The modified cell of claim 7, wherein the modified cell produces an increased amount of the POI in the presence of an inducing substrate compared to the amount of the same POI produced by the parent cell when fermented under the same conditions for producing the POI in the presence of an inducing substrate.

10. The modified cell of claim 3, wherein the variant Ace3 TF comprises an amino acid sequence selected from any one of SEQ ID NOs: 9 to 18.

11. 1. A genetically modified Trichoderma fungal cell derived from a parent Trichoderma fungal cell comprising a mutated ace3 gene encoding a variant Ace3 transcription factor (TF) protein comprising at least 90% sequence identity to SEQ ID NO:4, wherein the N-terminus of SEQ ID NO:4 is an intact zinc dinuclear (Zn) ATPase as set forth in SEQ ID NO:

29. ２ Cys ６ The genetically modified cell does not contain a DNA linkage, and the genetically modified cell contains a modified ace3 gene encoding a variant Ace3 TF protein comprising at least 90% sequence identity to positions 1-672 of SEQ ID NO:2, wherein the N-terminus of SEQ ID NO:2 is an intact zinc dinuclear (Zn) TF protein as set forth in SEQ ID NO:

29. ２ Cys ６ ) comprising DNA binding, and a deletion of the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2, a deletion of the last eight C-terminal amino acid positions 682-689 of SEQ ID NO:2, a deletion of the last nine C-terminal amino acid positions 681-689 of SEQ ID NO:2, a deletion of the last ten C-terminal amino acid positions 680-689 of SEQ ID NO:2, a deletion of the last twelve C-terminal amino acid positions 678-689 of SEQ ID NO:2, a deletion of the last thirteen C-terminal amino acid positions 677-689 of SEQ ID NO:2, a deletion of the last fourteen C-terminal amino acid positions 676-689 of SEQ ID NO:2, a deletion of the last fifteen C-terminal amino acid positions 678-689 of SEQ ID NO:2, 2, a deletion of the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2, a deletion of the last 17 C-terminal amino acid positions 673-689 of SEQ ID NO:2, or an amino acid substitution of the last 11 amino acid residues at the C-terminus of SEQ ID NO:2 with a V5 epitope tag or a V5-(6xHis) tandem tag, wherein in said variant Ace3 transcription factor (TF) protein, the amino acid residues from amino acid position 673 corresponding to SEQ ID NO:2 to the site of said deletion or substitution, if present, are identical to the corresponding amino acid residues in SEQ ID NO:

2.

12. 12. The modified cell of claim 11, wherein the variant Ace3 TF protein upregulates expression of a gene encoding a lignocellulolytic enzyme in the absence of an inducing substrate when fermented under conditions suitable for the production of the lignocellulolytic enzyme.

13. 1. A method for producing lignocellulolytic enzymes in Trichoderma fungal cells in the absence of an inducing substrate, comprising: (a) isolating a T. reesei mutant cell comprising a mutant ace3 gene encoding a variant Ace3 transcription factor (TF) protein, wherein the variant Ace3 TF protein comprises at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO:2, and wherein the variant Ace3 TF protein has at least one of the following: a deletion of the last seven C-terminal amino acid positions 683-689 of SEQ ID NO:2; a deletion of the last eight C-terminal amino acid positions 682-689 of SEQ ID NO:2; a deletion of the last nine C-terminal amino acid positions 681-689 of SEQ ID NO:2; a deletion of the last ten C-terminal amino acid positions 680-689 of SEQ ID NO:2; a deletion of the last twelve C-terminal amino acid positions 678-689 of SEQ ID NO:2; a deletion of the last thirteen C-terminal amino acid positions 677-689 of SEQ ID NO:2; a deletion of the last fourteen C-terminal amino acid positions 676-689 of SEQ ID NO:2; a deletion of the last 15 C-terminal amino acid positions 675-689 of SEQ ID NO:2, a deletion of the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2, a deletion of the last 17 C-terminal amino acid positions 673-689 of SEQ ID NO:2, or an amino acid substitution of the last 11 amino acid residues at the C-terminus of SEQ ID NO:2 with a V5 epitope tag or a V5-(6xHis) tandem tag, wherein in said variant Ace3 transcription factor (TF) protein, the amino acid residues from amino acid position 673 corresponding to SEQ ID NO:2 to the site of said deletion or substitution, if present, are identical to the corresponding amino acid residues in SEQ ID NO:2; and (b) fermenting the isolated mutant cells under conditions suitable for the production of lignocellulolytic enzymes, wherein the suitable fermentation conditions do not include an inducing substrate; A method comprising:

14. The method of claim 13, wherein the lignocellulolytic enzyme is selected from the group consisting of cellobiohydrolases, endoglucanases, and β-glucosidases.

15. 1. A method for producing lignocellulolytic enzymes in a genetically modified Trichoderma fungal cell in the absence of an inducing substrate, comprising: (a) obtaining a Trichoderma fungal cell comprising an ace3 gene encoding a wild-type Ace3 transcription factor (TF) protein comprising at least 90% sequence identity to amino acid positions 1-689 of SEQ ID NO:2; (b) genetically modifying the ace3 gene of the Trichoderma fungal cell of step (a), wherein the modified ace3 gene comprises at least 90% sequence identity to amino acid positions 1 to 672 of SEQ ID NO:2, and has one of the following deletions: the last seven C-terminal amino acid positions 683 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2, the last nine C-terminal amino acid positions 681 to 689 of SEQ ID NO:2, the last ten C-terminal amino acid positions 680 to 689 of SEQ ID NO:2, the last twelve C-terminal amino acid positions 678 to 689 of SEQ ID NO:2, the last thirteen C-terminal amino acid positions 677 to 689 of SEQ ID NO:2, the last eight ... eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 681 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 681 to 689 of a deletion of the last 14 C-terminal amino acid positions 676-689 of SEQ ID NO:2, a deletion of the last 15 C-terminal amino acid positions 675-689 of SEQ ID NO:2, a deletion of the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2, a deletion of the last 17 C-terminal amino acid positions 673-689 of SEQ ID NO:2, or an amino acid substitution of the last 11 amino acid residues at the C-terminus of SEQ ID NO:2 with a V5 epitope tag or a V5-(6xHis) tandem tag, wherein the amino acid residues from amino acid position 673 corresponding to SEQ ID NO:2 to the site of said deletion or substitution, if present, are identical to the corresponding amino acid residues in SEQ ID NO:2; (c) fermenting the modified Trichoderma fungal cells of step (b) under conditions suitable for the production of lignocellulolytic enzymes. Including, The method, wherein the suitable fermentation conditions do not include an inducing substrate.

16. 16. The method of claim 15, wherein the C-terminus of the wild-type Ace3 TF protein comprises at least 95% sequence identity to amino acid positions 673-689 of SEQ ID NO:

2.

17. 1. A method for producing lignocellulolytic enzymes in a genetically modified Trichoderma fungal cell in the absence of an inducing substrate, comprising: (a) obtaining a Trichoderma fungal cell comprising an ace3 gene encoding a variant Ace3 TF protein comprising at least 90% sequence identity to the Ace3-S TF protein of SEQ ID NO: 4; (b) genetically modifying the ace3 gene of the Trichoderma fungal cell of step (a), wherein the modified ace3 gene comprises at least 90% sequence identity to amino acid positions 1 to 672 of SEQ ID NO:2, and has one of the following deletions: the last seven C-terminal amino acid positions 683 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2, the last nine C-terminal amino acid positions 681 to 689 of SEQ ID NO:2, the last ten C-terminal amino acid positions 680 to 689 of SEQ ID NO:2, the last twelve C-terminal amino acid positions 678 to 689 of SEQ ID NO:2, the last thirteen C-terminal amino acid positions 677 to 689 of SEQ ID NO:2, the last eight ... eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 681 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 681 to 689 of a deletion of the last 14 C-terminal amino acid positions 676-689 of SEQ ID NO:2, a deletion of the last 15 C-terminal amino acid positions 675-689 of SEQ ID NO:2, a deletion of the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2, a deletion of the last 17 C-terminal amino acid positions 673-689 of SEQ ID NO:2, or an amino acid substitution of the last 11 amino acid residues at the C-terminus of SEQ ID NO:2 with a V5 epitope tag or a V5-(6xHis) tandem tag, wherein the amino acid residues from amino acid position 673 corresponding to SEQ ID NO:2 to the site of said deletion or substitution, if present, are identical to the corresponding amino acid residues in SEQ ID NO:2; (c) fermenting the modified Trichoderma fungal cells of step (b) under conditions suitable for the production of lignocellulolytic enzymes. Including, The method, wherein the suitable fermentation conditions do not include an inducing substrate.

18. The variant Ace3 TF protein comprising at least 90% sequence identity to the Ace3-S TF protein of SEQ ID NO: 4 is an intact zinc dinuclear (Zn) TF protein as set forth in SEQ ID NO:

29. ２ Cys ６ 18. The method of claim 17, wherein the method does not involve DNA binding.

19. The genetically modified ace3 gene encoding a variant Ace3 TF protein comprising at least 90% sequence identity to amino acid positions 1-672 of SEQ ID NO: 2 is an intact zinc binuclear (Zn) protein as set forth in SEQ ID NO:

29. ２ Cys ６ 18. The method of claim 17, comprising DNA binding.

20. 1. A method for producing a protein of interest (POI) in an engineered Trichoderma fungal cell in the absence of an inducing substrate, comprising: (a) obtaining a Trichoderma fungal cell comprising an ace3 gene encoding a wild-type Ace3 transcription factor (TF) protein comprising at least 90% sequence identity to amino acid positions 1-689 of SEQ ID NO:2; (b) genetically modifying the ace3 gene of the Trichoderma fungal cell of step (a), wherein the modified ace3 gene comprises at least 90% sequence identity to amino acid positions 1 to 672 of SEQ ID NO:2, and has one of the following deletions: the last seven C-terminal amino acid positions 683 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2, the last nine C-terminal amino acid positions 681 to 689 of SEQ ID NO:2, the last ten C-terminal amino acid positions 680 to 689 of SEQ ID NO:2, the last twelve C-terminal amino acid positions 678 to 689 of SEQ ID NO:2, the last thirteen C-terminal amino acid positions 677 to 689 of SEQ ID NO:2, the last eight ... eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 681 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 681 to 689 of a deletion of the last 14 C-terminal amino acid positions 676-689 of SEQ ID NO:2, a deletion of the last 15 C-terminal amino acid positions 675-689 of SEQ ID NO:2, a deletion of the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2, a deletion of the last 17 C-terminal amino acid positions 673-689 of SEQ ID NO:2, or an amino acid substitution of the last 11 amino acid residues at the C-terminus of SEQ ID NO:2 with a V5 epitope tag or a V5-(6xHis) tandem tag, wherein the amino acid residues from amino acid position 673 corresponding to SEQ ID NO:2 to the site of said deletion or substitution, if present, are identical to the corresponding amino acid residues in SEQ ID NO:2; (c) introducing an expression cassette encoding a POI into the modified Trichoderma fungal cell of step (b), wherein the expression cassette comprises an upstream (5') promoter sequence from a gene encoding a lignocellulolytic enzyme operably linked to a downstream (3') open reading frame (ORF) sequence encoding a POI; and (d) fermenting the modified Trichoderma fungal cells of step (c) under conditions suitable for the production of the POI. Including, The method, wherein the suitable fermentation conditions do not include an inducing substrate.

21. 21. The method of claim 20, wherein steps (b) and (c) are performed simultaneously or in either order.

22. 1. A method for producing a protein of interest (POI) in an engineered Trichoderma fungal cell in the absence of an inducing substrate, comprising: (a) obtaining a Trichoderma fungal cell comprising an ace3 gene encoding a variant Ace3 TF protein comprising at least 90% sequence identity to the Ace3-S TF protein of SEQ ID NO: 4; (b) genetically modifying the ace3 gene of the Trichoderma fungal cell of step (a), wherein the modified ace3 gene comprises at least 90% sequence identity to amino acid positions 1 to 672 of SEQ ID NO:2, and has one of the following deletions: the last seven C-terminal amino acid positions 683 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2, the last nine C-terminal amino acid positions 681 to 689 of SEQ ID NO:2, the last ten C-terminal amino acid positions 680 to 689 of SEQ ID NO:2, the last twelve C-terminal amino acid positions 678 to 689 of SEQ ID NO:2, the last thirteen C-terminal amino acid positions 677 to 689 of SEQ ID NO:2, the last eight ... eight C-terminal amino acid positions 682 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 681 to 689 of SEQ ID NO:2, the last eight C-terminal amino acid positions 681 to 689 of a deletion of the last 14 C-terminal amino acid positions 676-689 of SEQ ID NO:2, a deletion of the last 15 C-terminal amino acid positions 675-689 of SEQ ID NO:2, a deletion of the last 16 C-terminal amino acid positions 674-689 of SEQ ID NO:2, a deletion of the last 17 C-terminal amino acid positions 673-689 of SEQ ID NO:2, or an amino acid substitution of the last 11 amino acid residues at the C-terminus of SEQ ID NO:2 with a V5 epitope tag or a V5-(6xHis) tandem tag, wherein the amino acid residues from amino acid position 673 corresponding to SEQ ID NO:2 to the site of said deletion or substitution, if present, are identical to the corresponding amino acid residues in SEQ ID NO:2; (c) introducing an expression cassette encoding a POI into the modified Trichoderma fungal cell of step (b), wherein the expression cassette comprises an upstream (5') promoter sequence from a gene encoding a lignocellulolytic enzyme operably linked to a downstream (3') open reading frame (ORF) sequence encoding a POI; and (d) fermenting the modified Trichoderma fungal cells of step (c) under conditions suitable for the production of the POI. Including, The method, wherein the suitable fermentation conditions do not include an inducing substrate.

23. 23. The method of claim 22, wherein steps (b) and (c) are performed simultaneously or in either order.

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