Compositions and methods for enhanced selection of recombinant fungal cells

Abstract

The present disclosure is generally related to methods and compositions for constructing, engineering, transforming, selecting, growing, fermenting, and the like filamentous fungal cells (strains). Certain embodiments therefore provide, inter alia, nucleic acids, polynucleotides, plasmids, vectors, expression constructs (cassettes), gene coding sequences (CDS), recombinant (modified) host cells, methods thereof and the like.

Description

COMPOSITIONS AND METHODS ENHANCED SELECTION OF RECOMBINANT FUNGAL CELLS FIELD

[0001] The present disclosure is generally related to the fields of molecular biology, biochemistry, regulatory proteins, industrial fermentation, protein production, filamentous fungi and the like. Certain embodiments of the disclosure are related to modified (mutant) filamentous fungal cells and methods thereof for use in the enhanced production of proteins of interest.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit to U.S. Provisional Patent Application No. 63 / 754,347, filed February 5, 2025, which is incorporated herein by referenced in its entirety SEQUENCE LISTING

[0003] The sequence listing submitted herewith contains the file “NB42125-US-PSP_SequenceListing” created on February 03, 2025, which is 119,808 bytes in size. This sequence listing complies with 37 C.ER. § 1.52(e) and is incorporated herein by reference in its entirety.BACKGROUND

[0004] Filamentous fungi (e.g., Aspergillus sp., Penicillium sp., Talaromyces sp., Fupositionsarium sp., Myceliophthora sp., Neurospora sp., Candida sp., Trichoderma sp.. and die like) are capable of expressing native and heterologous proteins to high levels, making them well-suited for the large-scale production of proteins ( e.g., enzymes, antibodies, peptides, etc.) and / or metabolites for industrial and / or commercial applications such as pharmaceutical applications, animal health applications, food applications, beverage applications, laundry and textile applications, and the like. For example, recombinant gene expression in fungal host strains is a common method for the production of proteins of interest and as such, any improvements in host strain construction, transformation, selection, fermentation, protein productivity, and the like are highly desirable. As set forth and described hereinafter, the present disclosure addresses ongoing and unmet needs in the art.SUMMARY

[0005] As generally set forth herein, certain embodiments of die disclosure are related to methods and compositions for constructing, engineering, transforming, selecting, growing, fermenting, and the like filamentous fungal cells / strains. Thus, certain embodiments of the disclosure provide, inter alia, nucleic acids, polynucleotides, plasmids, vectors, expression constructs, recombinant (modified) host cells and related methods for selecting modified cells thereof.

[0006] In particular embodiments, the disclosure relates to modified Trichoderma sp. cells comprising an introduced mutation at a beta-tubulin 1 (tbbl) gene locus, wherein the mutated tbbl gene locus encodes a variant tbbl protein comprising an amino acid substitution at position 198 of SEQ ID NO:3. In related embodiments, the modified cells are selected by carbendazim resistance or diethofencarb sensitivity. In certain embodiments, the substitution at position 198 of the tbbl protein is a glutamic acid (E) to glycine (G) substitution (E198G). In other embodiments, the substitution at position 198 of tbbl protein is a glutamic acid (E) to lysine (G) substitution (E198K). In yet other embodiments, the substitution at position 198 of tbbl protein is a glutamic acid (E) to lysine (G) substitution (E198D).

[0007] In certain other embodiments, the modified Trichoderma sp. cells are constructed from parental / control (isogenic) Trichoderma sp. cells comprising a beta-tubulin 1 (tbbl) gene locus encoding a native tbbl protein comprising a glutamic acid (E) at amino acid position 198 (E 198) of SEQ ID NO: 3. In related embodiments, the parental cells expresses one or more endogenous proteins of interest and / or one or more heterologous proteins of interest. In other embodiments, the parental cells are selected by carbendazim sensitivity or diethofencarb resistance. In certain other embodiments, the modified cells produce at least an equivalent amount of the endogenous and / or heterologous proteins of interest as compared to the parental cells fermented under the same conditions. In other embodiments, Trichoderma sp. cells of the disclosure comprise at least one introduced expression cassette encoding a heterologous protein on interest (POI).

[0008] Certain other embodiments are therefore directed to a mutant tbbl gene comprising at least 95% identity to SEQ ID NO: 1 and encoding a variant tbbl protein having a glutamic acid (E) to glycine (G) substitution at amino acid position 198 (E198G). In other embodiments, the disclosure provides a tbbl gene coding sequence (CDS) comprises at least about 95% identity to SEQ ID NO: 2. In certain other embodiments, a mutant tbbl gene encodes a variant tbbl protein comprising at least 95% identity to SEQ ID NO: 3 and having an E198G substitution.

[0009] Certain other embodiments of the disclosure are related to a methods for co-editing gene loci in Trichoderma sp. cells, methods for iterative editing of multiple gene loci in Trichoderma sp. cells and the like. Thus, certain one or more embodiments are directed to a method for co-editing gene loci in a Trichoderma sp. cell comprising co-transforming a plurality of Trichoderma sp. cells with at least two homology directed repair donor (HDRD) fragments, wherein the first HDRD fragment edits a betatubulin 1 (tbbl) gene locus resulting in a mutant tbbl gene locus encoding a variant tbbl protein comprising a substitution of the glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, and the second HDRD fragment edits a first gene locus of interest (GLOI), culturing the plurality of cells in a media comprising carbendazim and selecting transformants thereof resistant to carbendazim, isolating and analyzing the selected transformants for the targeted edit at the first GLOI, and selecting and isolating at least one transformant having the targeted edit at the first GLOI, wherein the isolated cell comprises the mutant tbbl gene locus and targeted edit at the first GLOI. In certain embodiments of the method, editing is stimulated by at least two guide RNA / Cas (RNA / Cas) complexes, wherein one targets the tbbl gene locus and the other targets the first gene locus of interest (GLOI). In other embodiments of the method, the plurality of Trichoderma sp. cells are co-transformed with at least three HDRD fragments and at least three sgRNA / Cas complexes, wherein the first HDRD fragmentand sgRNA / Cas complex target and edit the WT tbbl gene locus resulting in a mutant tbbl gene locus encoding a variant tbbl protein comprising a substitution of the glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, the second HDRD fragment and sgRNA / Cas complex target and edit a first GLOI, and the third HDRD fragment and sgRNA / Cas complex target and edit a second GLOI, culturing tire plurality of cells in a media comprising carbendazim and selecting transformants thereof resistant to carbendazim, isolating and analyzing the selected transformants for the targeted edits at the first and second GLOI. and selecting and isolating at least one transformant having the targeted edits at the first and second GLOI, wherein the isolated cell comprises the mutant tbbl gene locus and targeted edit at the first and second GLOI. Certain other embodiments provide a method for iterative editing of multiple gene loci in a Trichoderma sp. cells. In related embodiments, the methods comprise cotransforming the plurality of the Trichoderma sp. cells with at least one expression construct encoding a heterologous protein of interest. In certain other embodiments, the Trichoderma sp. cells express one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.

[0010] In yet other embodiments, the disclosure relates modified Trichoderma sp. cells comprising an introduced polynucleotide expressing a mutant ergl 1 protein, wherein the modified cell can be selected by fluconazole resistance. In certain embodiments, the mutant erg 11 protein comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% to about 100% identity to the native ergll protein of SEQ ID NO: 85 and comprises at least one amino acid substitution at a position corresponding to position 137, 148, 464 or 467 of SEQ ID NO: 85. In certain other embodiments, the substitution at position 137 is a tyrosine (Y) to phenylalanine (F) substitution (Y137F), tire substitution at position 148 is lysine (K) to arginine (R) substitution (K148R), or a lysine (K) to glutamine (Q) substitution (K148Q), the substitution at position 464 is a glycine (G) to serine(S) substitution, and / or the substitution at position 467 is a arginine (R) to lysine (K) substitution. In other embodiments, tire mutant ergl 1 protein comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% identity to the native ergll protein of SEQ ID NO: 86 or SEQ ID NO: 87, and comprises at least one amino acid substitution at a position corresponding to position 132, 464 and / or 467 of SEQ ID NO: 86 or SEQ ID NO: 87. hi related embodiments, (lie substitution al position 132 is a tyrosine (Y) to phenylalanine (F) substitution (Y132F), or a tyrosine (Y) to histidine (H) substitution (Y132H), the substitution at position 464 is glycine (G) to serine (S) substitution (G464S), and / or the substitution at position 467 is arginine (R) to lysine (K) substitution (R467K). In certain other embodiments, the polynucleotide expressing the mutant ergll protein comprises at least about 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%. 89%, 90%, 91%, 92%, 93%.94%, 95%, 96%, 97% , 98%. 99% to about 100% identity to a polynucleotide of any one of SEQ ID NO: 79-84. In other one or more embodiments, the modified cell is constructed from a parental (isogenic) Trichoderma sp. cell which does not comprise an introduced polynucleotide expressing a mutant er l 1 protein, wherein parental cell can be selected by fluconazole sensitivity. In other embodiments, modified cells comprising an introduced polynucleotide expressing a mutant ergll protein further express / produce one or more endogenousproteins of interest and / or one or more heterologous proteins of interest. In certain other embodiments, modified cells comprise a polynucleotide expressing the mutant erg 11 protein and a polynucleotide encoding a protein of interest (POI) operably linked polynucleotide expressing tire mutant erg 11 protein.

[0011] In yet other embodiments, the disclosure provides isolated polynucleotides encoding a mutant ergl l protein comprising at least about 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97% , 98%, 99% to about 100% identity to SEQ ID NO: 85 and having at least one substitution at an amino acid position selected from 137, 148, 464 and / or 467 of SEQ ID NO: 85. In certain embodiments, the substitution at position 137 is a tyrosine (Y) to phenylalanine (F) substitution (Y137F), the substitution at position 148 is lysine (K) to arginine (R) substitution (K148R), or a lysine (K) to glutamine (Q) substitution (K148Q), the substitution at position 464 is a glycine (G) to serine (S) substitution, and / or the substitution at position 467 is a arginine (R) to lysine (K) substitution. In other one or more embodiments, polynucleotides encoding a mutant ergll protein comprise at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% to about 100% identity to any one of SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84.

[0012] Thus, certain other embodiments of the disclosure are directed to methods for selecting a transformed Trichoderma sp. cell comprising transforming a plurality of Trichoderma sp. cells with a polynucleotide comprising a gene coding sequence (CDS) encoding a mutant ergll protein, wherein the gene CDS comprises upstream and downstream homologous recombination (HR) regions flanking the gene CDS to enable integration at a selected gene locus, culturing the plurality of cells in a media comprising fluconazole, selecting and isolating at least one transformed cell thereof resistant to fluconazole, wherein the isolated cell comprises the gene CDS cassette integrated tire selected gene locus. In certain one or more embodiments of the methods, the transformed polynucleotide further comprises an operably linked polynucleotide encoding a protein of interest. Certain other one or more embodiments provide methods for selecting a transformed Trichoderma sp. cell comprising transforming a plurality of Trichoderma sp. cells with a polynucleotide expression construct encoding a mutant ergll protein, wherein the construct comprises an upstream promoter operably linked to a downstream mutant ergl 1 gene coding sequence (CDS) and upstream and downstream homologous recombination (HR) regions flanking the construct to enable integration at a selected gene locus, culturing the plurality of cells in a media comprising fluconazole, selecting and isolating at least one transformed cell thereof resistant to fluconazole, wherein the isolated cell comprises the ergll construct integrated the selected gene locus. In one or more related embodiments of the methods, the transformed polynucleotide further comprises an operably linked polynucleotide encoding a protein of interest. In yet other embodiments of the methods the plurality of cells are co-transformed with a sgRNA / Cas complex that generates a double strand break at the selected gene locus. In other embodiments of the methods, the plurality of cells express one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows Trichoderma growth on Vogel’s minimal media agar plates with differing concentrations of beta-tubulin inhibitors carbendazim (cdz) and diethofencarb (dfc) ranging from 0 to 1 microgram per milliliter (ug / ml). Confluent growth was observed on 0 ug / ml beta-tubulin inhibitor and 1 ug / ml diethofencarb. Carbendazim was strongly inhibitory at 1 ug / ml with four spontaneous resistant colonies developing.

[0014] Figure 2 presents schematic diagrams showing the tbbl locus and alleles. More particularly, FIG.2 shows a graphical representation of the tbbl locus, wherein the locus coordinates correspond to SEQ ID NO: 1. Below die coordinates is a graphical representation of the regions corresponding to die tbbl coding sequence (TBB1 CDS), illustrated as a series of arrows representing exons and lines representing introns. Below this, die two amino acid positions identified (i.e., Q134, E198) to confer resistance to carbendazim are shown. Below these, die region corresponding to the double-stranded homology directed repair donors (dsHDRD) is shown as a long arrow. Likewise, the regions corresponding to die single-stranded homology directed repair donors (ssHDRD) are shown as short arrows and labeled as uRJPOOOl and uRJP0002. As shown below these ssHDRD, the regions corresponding to the target sites for synthetic guide RNAs (sgRNAs), tbbl-c and tbbl-d, are illustrated. The vertical box spanning across these tracks in FIG. 2 has been “zoomed-in”, as shown in FIG. 3.

[0015] FIG. 3 shows a “zoomed-in” view of the tbbl locus corresponding to the open vertical box in FIG. 2, wherein the DNA and protein sequence of the two tbbl alleles used to interconvert between carbendazim and diethofencarb resistances / sensitivities are shown. To convert the wild-type tbbl allele (FIG. 3, TBB1-WT) to the carbendazim resistant tbblE198G allele (FIG. 3, TBB1E198G), the last two positions of the 198E codon were changed from AG to GC and the PAM site for sgRNA tbbl-c was removed by a synonymous change in the codon for 206 A. These nucleotide changes are boxed. To convert back to wild-type from the tbblE198G allele for diethofencarb selection, these changes were simply reversed.

[0016] Figure 4 shows altered sensitivity of Trichoderma strains containing substitutions at the 198E position. As presented in FIG. 4, strains containing either the parental / wild-type glutamic acid (E) residue at position 198 (labeled “P”), an E198G substitution (labeled “Gl” and “G3”) or an E198K substitution (labeled “K9”) were plated on Vogel’s minimal media containing differing amounts of diethofencarb (dfc) or carbendazim (cdz): no inhibitor (0 dfc), 1.2 ug / ml carbendazim (1.2 cdz), 0.6 ug / ml dicthofcncarb (0.6 dfc) and 1.2 ug / ml dicthofcncarb (1.2 dfc). As shown in FIG. 4, all strains grew when no beta-tubulin inhibitor was present. The parental strain (P) failed to grow on carbendazim but was resistant to diethofencarb. The strain with the tbblE198K allele was resistant to both carbendazim and die tho fencarb. The strains with the tbblE198G allele were resistant to carbendazim but were now sensitive to dietho fencarb, even at the lower concentration of 0.6 ug / ml.

[0017] Figure 5 shows characterizations of secreted protein production from industrial Trichoderma strains carrying different alleles at tbbl. Strains were fermented in slow release microti ter plates (srMTPs) under conditions for production of native Trichoderma cellulases. Fermentation broth filtrates were analyzed for enzyme activity (FIG. 5 A) and protein profile (FIG. 5B). More particularly. FIG. 5A presents a box plot for enzymatic activity of fermentation broth filtrates on an artificial substrate for xylanase (FIG. 5A’, Rel_pNPX. dark grey) and beta-glucosidase (FIG. 5A Rel_pNPG, light grey) normalized to activity of the parent / WT (relative activity). No statistically significant reduction in activity (alpha < 0.05) was observed from filtrates of the tbbl mutants carrying either the tbblE198G (tbbl / 198G) or tbblE198K (tbbl / 198K) alleles on either of the substrates relative to their parent. FIG. 5B shows SDS-PAGE analysis of tire fermentation broth filtrates from the parent (RL-P37nikl) strain containing the tbbl-WT allele and derivative strains containing either the tbblE198G or tbblE198K alleles. No substantial difference in total protein or banding pattern was observed in the tbbl mutants relative to the parent.

[0018] Figure 6 presents a diagram of the bovine chymosin expression cassette (bipl -chymosin) used to generate a Trichoderma strain expressing a heterologous polypeptide. As indicated in FIG. 6, coordinates are shown for the full expression cassette. The cassette starts with the bipl gene (SEQ ID NO: 33) with the structure of the coding sequence (bipl CDS) and mRNA (bipl mRNA) shown as series of arrows representing exons interconnected by thin lines corresponding to introns. This is followed by the cbhl promoter (SEQ ID NO: 34) driving tire expression of a translational fusion between tire CBH1 catalytic core and linker (SEQ ID NO: 35) with a codon optimized version of bovine pro-chymosin (SEQ ID NO: 36). The coding sequences (CDS) are shown as a series of arrows representing exons interconnected by thin lines representing introns. These coding sequences are followed by the cbhl terminator sequence (SEQ ID NO: 37) and then a pyr2 gene for transformant selection (SEQ ID NO: 38).

[0019] Figure 7 shows altered sensitivity of Trichoderma strains. In particular, as presented in FIG. 7 (i.e., panels 1-8) transformants were spread on selective agar plates having 100 gg / ml fluconazole + / -2.4 g / L uridine for ergll selection, or on minimal medium agar plates for pyr2 selection. For tire control experiment (FIG. 7, “Ctrl”) expression cassette was omitted. As shown in FIG. 7. expression of the ergll K148Q (SEQ ID NO: 82) mutation (panel 1), ergll K148R (SEQ ID NO: 83) mutation (panel 2), ergll Y137F (SEQ ID NO: 84) mutation (panel 3), C. tropicalis ergll Y137F (SEQ ID NO: 81) mutation (panel 4), C. albicans ergll Y132H_G464S_R467K (SEQ ID NO: 80) mutation (panel 5), C. albicans ergll Y132F (SEQ ID NO: 79) mutation (panel 6) results in Trichoderma strains resistant to fluconazole. When expression cassette was omitted no transformants were observed. Similarily, C. parapsilosis ergll Y132F_R398I mutation (panel 7) and C. albicans ergll K143Q mutation (panel 8) also did not yield transformants able to grow on fluconazole, while selection for pyr2 yielded high number of colonies. This indicates that expression of theses versions of ergll do not result in fluconazloe resistant Trichoderma strains.

[0020] Figure 8 presents the amino acid sequence of the native T. reesei ergl 1 protein (FIG. 8A, SEQ ID NO: 85), native Candida albicans ergll protein (FIG. 8B, SEQ ID NO: 86) and native Candida tropicalis ergl 1 protein (FIG. 8C, SEQ ID NO: 87). As shown in FIG. 8A, amino acid positions Y137, K148, G464, F467 of SEQ ID NO: 85 are indicated with grey shading. Likewsie, FIG. 8B and FIG.8C, show the native C. albicans and native C. tropicalis ergll proteins of SEQ ID NO: 86 and 87, respectively, wherein positions Y132, G464 and R467 are indicated with grey shading.BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0021] SEQ ID NO: 1 is a Trichoderma reesei polynucleotide (DNA) comprising a tbbl gene locus.

[0022] SEQ ID NO: 2 is a T. reesei polynucleotide comprising a tbbl gene coding sequence (CDS).

[0023] SEQ ID NO: 3 is the amino acid sequence of tbbl protein encoded by the tbbl gene CDS of SEQ ID NO: 2.

[0024] SEQ ID NO: 4 is a T. reesei polynucleotide comprising a tbb2 gene locus.

[0025] SEQ ID NO: 5 is a T. reesei polynucleotide comprising a tbb2 gene CDS.

[0026] SEQ ID NO: 6 is the amino acid sequence of tbb2 protein encoded by the tbb2 gene CDS of SEQ ID NO: 5.

[0027] SEQ ID NO: 7 is a synthetic DNA primer named “RPG2786”.

[0028] SEQ ID NO: 8 is a synthetic DNA primer named “RPG2787”.

[0029] SEQ ID NO: 9 is a synthetic DNA primer named “RPG2788”.

[0030] SEQ ID NO: 10 is a synthetic DNA primer named “RPG2789”.

[0031] SEQ ID NO: 11 is a synthetic DNA primer named “RPG2790”.

[0032] SEQ ID NO: 12 is a synthetic DNA primer named “RPG2791”.

[0033] SEQ ID NO: 13 is a synthetic DNA primer named “RPG2792”.

[0034] SEQ ID NO: 14 is a synthetic DNA primer named “RPG2793”.

[0035] SEQ ID NO: 15 is a polynucleotide comprising a T. reesei homology directed repair donor (HDRD) named “E198K” (HDRD-E198K).

[0036] SEQ ID NO: 16 is a dsDNA comprising a T. reesei HDRD named “E198G” (HDRD-E198G).

[0037] SEQ ID NO: 17 is a Cas9 synthetic guide RNA (sgRNA) named “tbbl-c”

[0038] SEQ ID NO: 18 is a synthetic DNA primer named “RPG2966”.

[0039] SEQ ID NO: 19 is a Cas9 sgRNA named “tbbl-d”.

[0040] SEQ ID NO: 20 is a Cas9 sgRNA named “TR3018684” targeting the als gene.

[0041] SEQ ID NO: 21 is a Cas9 sgRNA named “TR2118762” targeting the sdil gene.

[0042] SEQ ID NO: 22 is a Cas9 sgRNA named “TR1818971” targeting the pyr2 gene.

[0043] SEQ ID NO: 23 is a polynucleotide comprising a T. reesei HDRD named “G198E” (HDRD-G198E).

[0044] SEQ ID NO: 24 is a polynucleotide comprising a T. reesei HDRD named “als” (HDRD-als).

[0045] SEQ IDNO: 25is a polynucleotide comprising a 7. reesei HDRD named “sdil” (HDRD-sdil).

[0046] SEQ ID NO: 26 is a polynucleotide comprising a T. reesei HDRD named “pyr2” (HDRD-pyr2).

[0047] SEQ ID NO: 27 is a synthetic DNA primer named “RPG1829”.

[0048] SEQ ID NO: 28 is a synthetic DNA primer named “RPG23”.

[0049] SEQ ID NO: 29 is a synthetic DNA primer named “RPG25”

[0050] SEQ ID NO: 30 is a synthetic DNA primer named “SK3999”.

[0051] SEQ ID NO: 31 is a synthetic DNA primer named “RPG120”.

[0052] SEQ ID NO: 32 is a synthetic DNA primer named “SK3469”.

[0053] SEQ ID NO: 33 is a polynucleotide comprising a T. reesei bipl gene.

[0054] SEQ ID NO: 34 is a polynucleotide comprising a T. reesei cbhl promoter

[0055] SEQ ID NO: 35 is a polynucleotide encoding a T. reesei Cbhl protein catalytic core and linker.

[0056] SEQ ID NO: 36 is a polynucleotide encoding a Bos taurus (bovine) chymosin proenzyme, wherein the polynucleotide sequence has been codon optimized for expression in T. reesei.

[0057] SEQ ID NO: 37 is a polynucleotide comprising a T. reesei cbhl transcriptional terminator.

[0058] SEQ ID NO: 38 is a polynucleotide comprising a T. reesei pyr2 gene selection marker.

[0059] SEQ ID NO: 39 is a Cas9 sgRNA named “TR2094476” targeting the egl3 gene.

[0060] SEQ ID NO: 40 is a Cas9 sgRNA named “TR2094578” targeting the egl3 gene.

[0061] SEQ ID NO: 41 is a Cas9 sgRNA named “TR3081209” targeting the egl5 gene.

[0062] SEQ ID NO: 42 is a Cas9 sgRNA named “TR3081286” targeting the egl5 gene.

[0063] SEQ ID NO: 43 is a Cas9 sgRNA named “TR0303570” targeting the egl6 gene.

[0064] SEQ ID NO: 44 is a Cas9 sgRNA named “TR0303619” targeting the egl6 gene.

[0065] SEQ ID NO: 45 is a Cas9 sgRNA named “TR3938895” targeting the manl gene.

[0066] SEQ ID NO: 46 is a Cas9 sgRNA named “TR3938987” targeting the manl gene.

[0067] SEQ ID NO: 47 is a synthetic DNA primer named “RPG2958”.

[0068] SEQ ID NO: 48 is a synthetic DNA primer named “SK3961”.

[0069] SEQ ID NO: 49 is a synthetic DNA primer named “RPG2959”.

[0070] SEQ ID NO: 50 is a synthetic DNA primer named “RPG2960”.

[0071] SEQ ID NO: 51 is a synthetic DNA primer named “RPG296I”.

[0072] SEQ ID NO: 52 is a synthetic DNA primer named “RPG2962”.

[0073] SEQ ID NO: 53 is a synthetic DNA primer named “SK3970”.

[0074] SEQ ID NO: 54 is a synthetic DNA primer named “RPG2963”.

[0075] SEQ ID NO: 55 is a polynucleotide comprising a T. reesei HDRD named “uRJPOOOl” (HDRD-uRJPOOOl).

[0076] SEQ ID NO: 56 is a polynucleotide comprising a T. reesei HDRD named “uRJPOOlO” (HDRD-uRJPOOlO).

[0077] SEQ ID NO: 57 is a polynucleotide comprising a T. reesei HDRD named “uRJPOOl 1” (HDRD-uRJPOOll).

[0078] SEQ ID NO: 58 is a synthetic DNA primer named “SK3960”.

[0079] SEQ ID NO: 59 is a synthetic DNA primer named “SK3962”.

[0080] SEQ ID NO: 60 is a synthetic DNA primer named “RPG2861”

[0081] SEQ ID NO: 61 is a Cas9 sgRNA named “gSK2”.

[0082] SEQ ID NO: 62 is a polynucleotide comprising a T. reesei HDRD named “uRJJP0002” (HDRD-uRJJP0002).

[0083] SEQ ID NO: 63 is a polynucleotide comprising a T. reesei HDRD named “uRJP0013” (HDRD-URJP0013).

[0084] SEQ ID NO: 64 is a polynucleotide comprising a T. reesei HDRD named “uRJP0014” (HDRD-URJP0014).

[0085] SEQ ID NO: 65 is a synthetic DNA primer named “SK3967”.

[0086] SEQ ID NO: 66 is a synthetic DNA primer named “SK3966”.

[0087] SEQ ID NO: 67 is a synthetic DNA primer named “RPG2437”.

[0088] SEQ ID NO: 68 is a synthetic DNA primer named “SK3834”.

[0089] SEQ ID NO: 69 is a synthetic DNA primer named “SK3833”.

[0090] SEQ ID NO: 70 is a Cas9 sgRNA named “gRJP0051”.

[0091] SEQ ID NO: 71 is a Cas9 sgRNA named “gRJP0049”.

[0092] SEQ ID NO: 72 is a polynucleotide comprising a T. reesei HDRD named “uRJP0017” (HDRD-uRJP0017).

[0093] SEQ ID NO: 73 is a polynucleotide comprising a T. reesei HDRD named “uRJP0015” (HDRD-uRJP0015).

[0094] SEQ ID NO: 74 is a synthetic DNA primer named “RPG3161”.

[0095] SEQ ID NO: 75 is a synthetic DNA primer named “RPG3162”.

[0096] SEQ ID NO: 76 is a synthetic DNA primer named “RPG3163”.

[0097] SEQ ID NO: 77 is a synthetic DNA primer named “RPG3164”.

[0098] SEQ ID NO: 78 is a synthetic DNA primer named “RPG3167”.

[0099] SEQ ID NO: 79 is a polynucleotide comprising a Candida albicans erg 11 gene having a mutation named “Y132F’.

[0100] SEQ ID NO: 80 is a polynucleotide comprising a Candida albicans erg 11 gene having a mutation named “ Y132H_G464S_R467K”.

[0101] SEQ ID NO: 81 is a polynucleotide comprising a Candida tropicalis ergll gene having a mutation named “Y 132F’.

[0102] SEQ ID NO: 82 is a polynucleotide comprising T. reesei ergl 1 gene having a mutation named “K148Q”.

[0103] SEQ ID NO: 83 is a polynucleotide comprising T. reesei ergll gene having a mutation named “K148R”.

[0104] SEQ ID NO: 84 is a polynucleotide comprising T. reesei ergll gene having a mutation named “Y137F”.

[0105] SEQ ID NO: 85 is the amino acid sequence of the native T. reesei ergl 1 protein.

[0106] SEQ ID NO: 86 is the amino acid sequence of the native C. albicans ergll protein.

[0107] SEQ ID NO: 87 is the amino acid sequence of the native C. tropicalis ergl 1 protein.DETAILED DESCRIPTION

[0108] As described herein, certain embodiments of the disclosure are related to recombinant (genetically modified) filamentous fungal cells (strains) for use in the commercial scale production of proteins (polypeptides). Certain embodiments of the disclosure therefore provide, inter alia, nucleic acids, polynucleotides, plasmids, vectors, expression constructs, recombinant filamentous fungal cells and methods thereof for constructing, engineering, transforming, selecting, growing, fermenting, and the like filamentous fungal cells described herein.I. DEFINITIONS

[0109] Prior to describing the present compositions and methods in detail, die following terms are defined for clarity. Terms not defined should be accorded their ordinary meanings as used in die relevant art. Unless defined odierwise, all technical and scientific terms used herein have die same meaning as commonly understood by one of ordinary skill in the art to which the present compositions and metiiods apply.

[0110] All publications and patents cited in this specification are herein incorporated by reference.

[0111] Where a range of values is provided, it is understood that each intervening value, to die tenth of die unit of die lower limit unless the context clearly dictates odierwise, between the upper and lower limit of that range and any otiier stated or intervening value in that stated range, is encompassed within the present compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present compositions and methods.

[0112] Certain ranges are presented herein with numerical values being preceded by the term “about”. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantialequivalent of the specifically recited number. For example, in connection with a numerical value, tire term “about” refers to a range of 10% to+10% of the numerical value, unless the term is otherwise specifically defined in context. In another example, the phrase a “pH value of about 6” refers to pH values of from 5.4 to 6.6, unless tire pH value is specifically defined otherwise.

[0113] The headings provided herein are not limitations of tire various aspects or embodiments of the present compositions and methods which can be had by reference to tire specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0114] In accordance with this Detailed Description, the following abbreviations and definitions apply. Note that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an enzyme” includes a plurality of such enzymes, and reference to “the dosage” includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0115] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only”, “excluding”, “not including” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0116] It is further noted that the term “comprising”, as used herein, means “including, but not limited to”, the component(s) after the term “comprising”. The component(s) after the term “comprising” are required or mandatory, but the composition comprising the component(s) may further include other non-mandatory or optional component(s).

[0117] It is also noted that the term “consisting of,” as used herein, means “including and limited to”, the componcnt(s) after the term "consisting of’. The componcnt(s) after the term “consisting of’ arc therefore required or mandatory, and no other component(s) are present in the composition.

[0118] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the oilier several embodiments without departing from the scope or spirit of the present 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.

[0119] As used herein, the terms “wild-type” and “native” are used interchangeably and refer to genes, proteins, fungal cells or strains as found in nature.

[0120] As used herein, die terms “recombinant” or “non-natural” refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has at least one engineered genetic alteration, or has been modified by the introduction of a heterologous nucleic acid molecule, or refer to a cell (e.g. , a microbial cell) that has been altered such that the expression of a heterologous or endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from a non-naturalcell or is progeny of a non-natural cell having one or more such modifications. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, or other nucleic acid molecule additions, deletions, substitutions or other functional alteration of a cell’ s genetic material. For example, recombinant cells may express genes or other nucleic acid molecules that are not found in identical or homologous form within a native (wild-type) cell, or may provide an altered expression pattern of endogenous genes, such as being over-expressed, under-expressed, minimally expressed, or not expressed at all.

[0121] In certain aspects, reference to an amino acid sequence “position” may be presented as a single letter amino acid (residue) followed by the position number (e.g.. alanine at position 1 presented as “Al”, glutamic acid at position 2 presented as “E2”. etc.). In related embodiments, reference to a variant (mutant) amino acid sequence may be presented as a single letter amino acid (residue), wherein the amino acid position of the parent (reference or control) sequence is numbered followed by the mutated amino acid (residue) at the same position (e.g., alanine (A) at position 1 substituted with a glycine (G) presented as “A1G”, glutamic acid (E) at position 30 substituted with a histidine (H) presented as “E30H”, etc.).

[0122] As used herein, the terms “beta-tubulin 1” gene and “beta-tubulin 2” gene are abbreviated as “tbbl” and “tbb2”, respectively.

[0123] As used herein, a “wild-type Trichoderma reesei tbbl gene locus” comprises a DNA sequence set forth in SEQ ID NO: 1, a “wild-type Trichoderma reesei tbbl gene coding sequence” (abbreviated, “tbbl CDS”) comprises a DNA sequence set forth in SEQ ID NO: 2, and a “native tbbl protein” comprises an amino acid sequence set forth in SEQ ID NO: 3.

[0124] As used herein, a “wild-type Trichoderma reesei tbb2 gene locus” comprises a polynucleotide (DNA) sequence set forth in SEQ ID NO: 4, a “wild-type Trichoderma reesei tbb2 gene coding sequence” (tbb2 CDS) comprises a DNA sequence set forth in SEQ ID NO: 5, and a “native tbb2 protein” comprises an amino acid sequence set forth in SEQ ID NO: 6.

[0125] As used herein, a polypeptide referred to as a “Cas endonuclease” or having “Cas endonuclease activity” relates to a CRISPR associated (Cas) polypeptide encoded by a Cas gene where the Cas (endonuclease) protein is capable of cutting a target DNA sequence when functionally coupled with one or more guide polynucleotides (see, e.g., U.S. Patent No. 8,697,359 and PCT Publication No. W02016 / 100272). Variants of Cas endonucleases that retain guide polynucleotide directed endonuclease activity are also included in this definition, including Cas variants that have nicking endonuclease activity (i.e.. they introduce single strand nick at a double-stranded DNA target site). A Cas endonuclease is guided by the guide polynucleotide to recognize and cleave a specific target site in double stranded DNA (e.g., at a target site in the genome of a cell). The Cas9 endonuclease may be any convenient Cas9 endonuclease, including but not limited to Cas9 endonucleases, and functional fragments thereof, from the following bacterial species: Streptococcus sp. (e.g., S. pyogenes, S. mutans,and S. thermophilus), Campylobacter sp. (e.g., C. jejuni), Neisseria sp. (e.g., N. meningitides), Francisella sp. (e.g., F. novicida), and Pasteurella sp. (e.g., P. multocida).

[0126] As used herein, the term “guide polynucleotide” relates to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize and cleave a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be a RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNA combination sequence).

[0127] As used herein, a “Cas synthetic guide RNA” (abbreviated. “sgRNA”) is a RNA sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize and cleave a DNA target site. For example, in certain embodiments, a sgRNA of the disclosure enables the Cas endonuclease to recognize and cleave a Trichoderma sp. beta-tubulin 1 (tbbl) gene locus. In certain embodiments, the sgRNA targets and cleaves a wild-type tbbl gene locus having a tbbl gene coding sequence (tbbl CDS) encoding a native tbbl protein comprising a glutamic acid (E) at amino acid position 198 (E198), wherein the positions are numbered according to SEQ ID NO: 3. In another embodiment, a sgRNA of the disclosure comprises at least about 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9%7, 98%, 99% or 100% identity to the RNA sequence of SEQ ID NO: 17.

[0128] As used herein, the term “synthetic guide RNA / Cas endonuclease system” (abbreviated “sgRNA / Cas”) includes a complex of a Cas endonuclease and a guide RNA that is capable of introducing a double strand break at a DNA target site. The Cas endonuclease unwinds the DNA duplex in close proximity of the DNA target site and cleaves both DNA strands upon recognition of a target sequence by a guide RNA, but only if the correct protospacer-adjacent motif (PAM) is appropriately oriented at the 3' end of the target sequence.

[0129] As used herein, cells comprising “more than one edited gene locus” may be referred to a “coedited”.

[0130] As used herein, an “acetolactate synthase (als) gene” encodes an ALS protein that renders fungal strains resistance to die herbicide chlorimuron ethyl, as described in U.S. Patent No. 8,138,321.

[0131] As used herein, a “succinate dehydrogenase iron-sulfur protein encoding (sdil) gene” encodes a Sdil protein that renders fungal strains resistance to fungicide carboxin, as described in (Kilaru el al., 2020).

[0132] As used herein, a “pyr2 gene” encodes an orotate phosphoribosyl transferase protein (pyr2), wherein loss of function of the pyr2 protein (e.g., via mutation) renders the strain a uridine auxotroph, as well as conferring resistance to 5-fluoroorotic acid.

[0133] As used herein, a “Candida albicans ergll gene encoding a ‘Y132F’ mutation” comprises a DNA sequence set forth in SEQ ID NO: 79, a “Candida albicans ergll gene encoding a ‘ Y132H_G464S_R467K’ mutation” comprises a DNA sequence set forth in SEQ ID NO: 80, a “Candida tropicalis ergll gene encoding a ‘Y132F’mutation” comprises a DNA sequence set forth in SEQ ID NO: 81, a “Trichoderma reesei ergll gene encoding a ‘K148Q’ mutation” comprises a DNAsequence set forth in SEQ ID NO: 82, a “Trichoderma reesei ergll gene encoding a ‘K148R’ mutation” comprises a DNA sequence set forth in SEQ ID NO: 83, and a “Trichoderma reesei ergll gene encoding a ‘Y137F’ mutation” comprises a DNA sequence set forth in SEQ ID NO: 84.

[0134] As used herein with regard to amino acid residue positions, “corresponding to” or “corresponds to” or “correspond to” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide. As used herein, “corresponding region” generally refers to an analogous position in a related protein or a reference protein.

[0135] In certain embodiments, the position of an amino acid residue in a specified ergll protein sequence may be numbered by correspondence with the native T. reesei ergl 1 amino acid sequence set forth as SEQ ID NO: 85, numbered by correspondence with die native C. albicans ergll amino acid sequence set forth as SEQ ID NO: 86, or the native C. tropicalis ergll amino acid sequence set forth as SEQ ID NO: 87. That is, the native amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 86 and / or SEQ ID NO: 87 serve as a reference sequences. For example, the amino acid sequence of one or more ergll variants (mutants) described herein may be aligned with the amino acid sequence using an alignment algorithm as described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with an amino acid residue (e.g., SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87) is conveniently numbered by reference to the numerical position of that corresponding amino acid residue. Sequence alignment algoridims, such as, for example, described herein will identify the location or locations where insertions or deletions occur in a subject sequence when compared to a query sequence (also sometimes referred to as a “reference sequence”). Sequence alignment with oilier ergl 1 amino acid sequences can be determined using an amino acid alignment.

[0136] As used herein, the term “gene” is synonymous with the term “allele” in referring to a nucleic acid that encodes and directs the expression of a protein or RNA. Vegetative forms of filamentous fungi are generally haploid, therefore a single copy of a specified gene (i.e., a single allele) is sufficient to confer a specified phenotype.

[0137] As used herein, the term “gene” means the segment of DNA involved in producing a polypeptide (protein) chain, that may or may not include regions preceding and following the coding region (e.g.. 5' untranslated (5' UTR) or “leader” sequences, 3' UTR or “trailer” sequences, promoter sequences, terminator sequences and the like) as well as intervening sequences (introns) between individual coding segments (exons). For example, a gene (DNA) sequence of interest (GOI) may encode a protein of interest (e.g., commercially important industrial proteins or peptides, such as enzymes (e.g., proteases, mannanases, xylanases, amylases, glucoamylases, cellulases, oxidases, phytases, lipases, etc.). The gene of interest may be a naturally occurring gene, a mutated (modified) gene or a synthetic gene.

[0138] As used herein, the term “promoter” refers to a nucleic acid sequence that functions to direct transcription of a downstream gene, or an open reading frame (ORF) thereof. The promoter willgenerally be appropriate to the host cell (e.g., a filamentous fungal cell) in which the target gene is being expressed. The promoter together with other transcriptional and translational regulatory nucleic acid sequences (also termed “control sequences”) is necessary to express a given gene. In general, tire transcriptional and translational regulatory sequences include, but are not limited to, promoter and terminator sequences including a core promoter and enhancer or activator or repressor sequences, transcriptional and translational start and stop sequences. In certain embodiments, the promoter is an inducible promoter, or a constitutive promoter. In certain embodiments, the inducible promoter is an inducible cellulase gene promoter.

[0139] As used herein, the term “promoter activity” is the ability of a nucleic acid to direct transcription of a downstream (3') polynucleotide in a host cell. To test promoter activity, the (promoter) nucleic acid may be operably linked to a downstream polynucleotide to produce a recombinant nucleic acid. The recombinant nucleic acid may be introduced into a cell, and transcription of the polynucleotide may be evaluated. In certain cases, the polynucleotide may encode a protein, and transcription of the polynucleotide can be evaluated by assessing production of the protein in the cell.

[0140] As used herein, the term “operably linked” refers to a functional linkage between two or more nucleic acid sequences. Thus, a nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter sequence or a terminator sequence is operably linked to a coding sequence if it affects tire transcription of the coding sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation; a nucleic acid sequence encoding a secretory leader (i.e., a signal peptide) is operably linked to a nucleic acid sequence (e.g., an ORF) encoding a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide. Generally, “operably linked” means that the DNA (nucleic acid) sequences being linked arc contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking two or more nucleic acid sequences (i.e., operably linking) is accomplished using any of the methods to one of skill in the art.

[0141] As used herein, a “functional gene” is a gene capable of being used by cellular components to produce an active gene product, typically a protein. In contrast, a “non-functional gene” cannot be used by cellular components to produce an active gene product (i.e., a functional protein), or has a reduced ability to be used by cellular components to produce an active gene product (i.e., a functional protein).

[0142] As used herein, a “functional protein” is a protein that possesses a function or activity, such as an enzymatic function / activity, a binding function / activity (e.g., DNA binding), a surface-active property, and the like, and which has not been mutagenized, truncated, or otherwise modified to abolish or reduce that function / activity.

[0143] As used herein, phrases such as “modified filamentous fungal cell”, “mutant or variant filamentous fungal cell”, “recombinant fungal cell”, “modified filamentous fungal strain”, and the like may be used interchangeably and refer to filamentous fungal cells that are derived (obtained) from acontrol or parental filamentous fungal cell belonging to the Pezizomycotina subphylum. For example, a “modified” filamentous fungal cell may be derived (obtained or constructed) from a control or parental (isogenic) filamentous fungal cell, wherein the modified cell comprises at least one genetic modification which is not found in tire control or parental cell.

[0144] As used herein, the term “Ascomycete fungal cell” refers to any organism in the Division Ascomycota in the Kingdom Fungi. Examples of Ascomycetes fungal cells include, but are not limited to, filamentous fungi in the subphylum Pezizomycotina, such as Trichoderma sp., Aspergillus sp., Myceliophthora sp. and Penicillium sp.

[0145] As used herein, the term “filamentous fungus” refers to all filamentous forms of the subdivision Eumycota and Oomycota. For example, filamentous fungi include, without limitation. Acremonium, Aspergillus. Emericella. Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium. Talaromyces. Thielavia. Tolypocladium, or Trichoderma species. In some embodiments, the filamentous fungus may be an Aspergillus aculeatus. Aspergillus awamori, Aspergillus foetidus. Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae.

[0146] In some embodiments, the filamentous fungus is a Fusarium sp. such as Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseuni, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum. Fusarium trichothecioides, Fusarium venenatum, and the like. In other embodiments, the filamentous fungus is Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Scytalidium thermophilum, Thielavia terrestris and the like. In certain other embodiments, a filamentous fungus is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride and the like.

[0147] As used herein, exemplary parental Trichoderma reesei strains include, but are not limited to, T. reesei strain QM6a (ATCC Deposit No. 13631). T. reesei strain RL-P37 (NRRL Deposit No. 15709) and T. reesei strain RUT-C30 (ATCC Deposit No. 56765); exemplary parental Aspergillus niger strains include, but are not limited to, A. niger strain designated as ATCC Deposit No. 1015; exemplary parental Aspergillus oryzae strains include, but are not limited to A. oryzae strain RIB40 (ATCC Deposit No. 42149); and exemplary parental Myceliophthora thermophila strains include, but are not limited to, M. thermophila strain designated as ATCC Deposit No.42464. For example, Trichoderma strains RUT-C30 and RL-P37 are mutagenized (cellulase overproducing) derivatives of Trichoderma natural isolate QM6a (Sheir-Neiss and Montenecourt, 1984). with strain NG14 being the last common ancestor.

[0148] As used herein, the terms “polypeptide” and “protein” (and / or their respective plural forms) are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. The conventional one-letter or three-letter codes for amino acid residues are used herein.The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non- amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0149] As used herein, the phrases “substantially similar” and “substantially identical”, in the context of at least two nucleic acids or polypeptides, typically means that a polynucleotide or polypeptide comprises a sequence that has at least about 40% to 100% sequence identity. Thus, in one or more embodiments, a substantially similar or substantially identical nucleic acid or polypeptide of the disclosure comprises at least about 40%, 50%, 60%. 70%. 80%, 90% or 100% identity to one or more sequences set forth herein. In certain related embodiments, one or more nucleic acid sequences and / or one or more protein sequences of the disclosure comprise at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 50%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to one or more sequences set fortlr herein.

[0150] Sequence identity can be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. Software for performing BLAST analyses is publicly available through die National Center for Biotechnology Information. Also, databases can be searched using FAST A. One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides diat differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each oilier under stringent conditions (e.g., witliin a range of medium to high stringency).

[0151] As used herein, a “variant” or “mutant” protein refers to a polypeptide sequence having homology to a native or reference protein sequence. For instance, variant protein (amino acid) sequences may differ from a native (or reference) protein sequence by a small number of amino acid residues and may be defined by their level of primary amino acid sequence homology / identity with the native (or reference) protein sequence.

[0152] As used herein, the term “identical” in the context of two polynucleotide or polypeptide sequences refers to the nucleotides or amino acids in the two sequences that are the same when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms described below and known in the art. The phrase “percent (%) identity” (abbreviated, “PID”) refersto polynucleotide (nucleic acid) or polypeptide (amino acid) sequence identity. Percent identity may be determined using standard techniques known in the art. In certain aspects, the percent amino acid identity shared by sequences of interest can be determined by aligning the sequences to directly compare the sequence information, e.g., by using an alignment program / algorithm such as BLAST, MUSCLE, or CLUSTAL. For example, the BLAST algorithm has been described in Altschul et al. (1990) and Karlin et al. (1993). In particular, a percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “reference” sequence including any gaps created by the program for optimal / maximum alignment. BLAST algorithms refer to the “reference” sequence as the “query” sequence.

[0153] A “homologous” protein refers to polypeptides having a distinct similarity in primary, secondary, and / or tertiary structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins are aligned. Homology can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, MUSCLE, or CLUSTAL. Homologous search of protein sequences can be done using BLASTP and PSLBLAST from NCBI BLAST with threshold (E-value cut-off) at 0.001 (e.g., see Altschul et al., 1997).

[0154] The BLAST program uses several search parameters, most of which are set to tire default values. The NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity but is not recommended for query sequences of less than 20 residues (Altschul et al., 1997 and Schaffer et al., 2001). Exemplary default BLAST parameters for a nucleic acid sequence searches include: Neighboring words threshold^ 11 ; E-value cutoffs 10; Scoring Matrix=NUC.3.1 (match=l, mismatch=-3);Gap Opening=5; and Gap Extension=2. Exemplary default BLAST parameters for amino acid sequence searches include: Word size = 3; E-value cutoff=10; Scoring Matrix=BLOSUM62; Gap Opcning=ll; and Gap cxtcnsion=l. Using this information, protein sequences can be grouped and / or a phylogenetic tree built therefrom. Amino acid sequences can be entered in a program such as the Vector NTI Advance suite and a Guide Tree can be created using tire Neighbor Joining (NJ) method (Saitou and Nei, 1987). The tree construction can be calculated using Kimura’s correction for sequence distance and ignoring positions with gaps. A program such as AlignX can display tire calculated distance values in parenthesis following tire molecule name displayed on the phylogenetic tree.

[0155] Understanding tire homology between molecules can reveal the evolutionary history of the molecules as well as information about their function; if a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the new protein’s biochemical function. Two molecules are said to be homologous if they have been derived from a common ancestor. Homologous molecules, or homologs, can be divided into two classes, paralogs and orthologs. Paralogs are homologs that are present within one species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs that are present within different species and have very similar or identical functions. A protein superfamily is the largest grouping (clade) of proteins for which commonancestry can be inferred. Usually, this common ancestry is based on sequence alignment and mechanistic similarity.

[0156] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (Thompson el al., 1994). Default parameters for tire CLUSTAL W algorithm include: Gap opening penalty=10.0; Gap extension penalty=0.05 ; Protein weight matrix=BLOSUM series ; DNA weight matrix=IUB ; Delay divergent sequences %=40; Gap separation distance=8; DNA transitions weight=0.50; List hydrophilic residues=GPSNDQEKR; Use negative matrix=OFF; Toggle Residue specific penalties=ON; Toggle hydrophilic penalties=ON; and Toggle end gap separation penalty=OFF. In CLUSTAL algorithms, deletions occurring at either terminus are included. For example, a variant protein with a five amino acid deletion at either terminus (or within the polypeptide) of a polypeptide of 500 amino acids would have a percent sequence identity of 99% (495 / 500 identical residues x 100) relative to the “reference” polypeptide. Such a variant protein would be encompassed by a variant having “at least 99% sequence identity” to the polypeptide.

[0157] For instance, in certain embodiments, variant (mutant) tbbl protein sequences have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to about 100% amino acid sequence identity with the native (reference) tbbl protein set forth in SEQ ID NO: 3. In particular embodiments, at least 99% to about 100% amino acid sequence identity with the native (reference) tbbl protein includes at least about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% and 99.9% amino acid sequence identity with the native tbbl protein set forth in SEQ ID NO: 3. In other embodiments, variant (mutant) ergl 1 protein sequences have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% to about 100% amino acid sequence identity with tire native (reference) ergll protein set forth in SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87. In particular embodiments, at least 99% to about 100% amino acid sequence identity with the native (reference) ergll protein includes at least about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% and 99.9% amino acid sequence identity with the native ergll protein set forth in SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87.

[0158] As used herein, “nucleic acid” refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, as well as to DNA, cDNA, and RNA of genomic or synthetic origin, which may be double- stranded or single-stranded, whether representing the sense or antisense strand.

[0159] As used herein, the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA, derived from a nucleic acid molecule of the disclosure. Expression may also refer to translation of mRNA into a polypeptide. Thus, the term “expression” may include any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, secretion and the like.

[0160] As used herein, the terms “modification” and “genetic modification” are used interchangeably and include, but are not limited to, the introduction, substitution, or removal of one or more nucleotides in a gene, or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of tire gene, gene disruption, gene conversion, gene deletion, the down-regulation of a gene (e.g., antisense RNA, siRNA, miRNA, and the like), specific mutagenesis (including, but not limited to, CRISPR / Cas9 based mutagenesis) and / or random mutagenesis of any one or more the genes disclosed herein.

[0161] As used herein, “the introduction, substitution, or removal of one or more nucleotides in a gene encoding a protein”, such genetic modifications include the gene’s coding sequence (i.e., exons) and non-coding intervening (introns) sequences.

[0162] As used herein, “disruption of a gene”, “gene disruption”, “inactivation of a gene” and “gene inactivation” are used interchangeably and refer broadly to any genetic modification that substantially disrupts / inactivates a target gene. Exemplary methods of gene disruptions include, but are not limited to, the complete or partial deletion of any portion of a gene, including a polypeptide coding sequence (CDS), a promoter, an enhancer, or another regulatory element, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and any combinations and variations thereof which disrupt / inacd vale the target gene(s) and substantially reduce or prevent the expression / production of the functional gene product.

[0163] In other embodiments, a protein of interest (POI) expressed / produced by fungal cells of the disclosure may be detected, measured, assayed and the like, by protein quantification methods, gene transcription methods, mRNA translation methods and the like, including, but not limited to, protein migration / mobility (SDS-PAGE), mass spectrometry, HPLC, size exclusion, ultracentrifugation sedimentation velocity analysis, transcriptomics, proteomics, fluorescent tags, epitope tags, fluorescent protein (GFP, RFP, etc.) chimeras / hybrids and the like.

[0164] As used herein, functionally and / or structurally similar proteins are considered to be “related proteins”. Such related proteins can be derived from organisms of different genera and / or species, or even different classes of organisms (e.g., bacteria and fungi). Related proteins also encompass homologues and / or orthologues determined by primary sequence analysis, determined by secondary or tertiary structure analysis, or determined by immunological cross-reactivity.

[0165] As defined herein, the term “introducing”, as used in phrases such as “introducing into a fungal cell” at least one polynucleotide open reading frame (ORF), or a gene thereof, or a vector thereof etc., includes methods known in the art for introducing polynucleotides into a cell, including, but not limited to protoplast fusion, natural or artificial transformation (e.g.. calcium chloride, electroporation), transduction, transfection and the like.

[0166] As used herein, “transformed” or “transformation” mean a cell has been transformed by use of recombinant DNA techniques. Transformation typically occurs by insertion of one or more nucleotide sequences (e.g., a polynucleotide, an ORF or gene) into a cell. The inserted nucleotide sequence maybe a heterologous nucleotide sequence (i.e., a sequence that is not naturally occurring in the cell that is to be transformed).

[0167] As used herein, “transformation” refers to introducing an exogenous DNA into a host cell so that the DNA is maintained as a chromosomal integrant or a self-replicating extra-chromosomal vector. As used herein, “transforming DNA”, “transforming sequence”, and “DNA construct” refer to DNA that is used to introduce sequences into a host cell. The DNA may be generated in vitro by PCR or any other suitable techniques. In some embodiments, the transforming DNA comprises an incoming sequence, while in other embodiments it further comprises an incoming sequence flanked by homology boxes. In yet a further embodiment, the transforming DNA comprises other non-homologous sequences, added to the ends (i.e., stuffer sequences or flanks). The ends can be closed such that the transforming DNA forms a closed circle, such as, for example, insertion into a vector.

[0168] As used herein “an incoming sequence” refers to a DNA sequence that is introduced into the fungal cell chromosome. In some embodiments, the incoming sequence is part of a DNA construct. In other embodiments, the incoming sequence encodes one or more proteins of interest. In some embodiments, the incoming sequence comprises a sequence that may or may not already be present in the genome of the cell to be transformed (i.e., it may be either a homologous or heterologous sequence). In some embodiments, the incoming sequence encodes one or more proteins of interest, a gene, and / or a mutated or modified gene. In alternative embodiments, the incoming sequence encodes a functional wild-type gene or operon, a functional mutant gene or operon, or a nonfunctional gene or operon. In some embodiments, an incoming sequence is a non-functional sequence inserted into a gene to disrupt function of the gene. In another embodiment, the incoming sequence includes a selective marker. In a further embodiment the incoming sequence includes two homology boxes.

[0169] As used herein, “homology box” refers to a nucleic acid sequence, which is homologous to a sequence in the fungal cell chromosome. More specifically, a homology box is an upstream or downstream region having between about 80 and 100% sequence identity, between about 90 and 100% sequence identity, or between about 95 and 100% sequence identity with die immediate flanking coding region of a gene or part of a gene to be deleted, disrupted, inactivated, down-regulated and die like, according to die invention. These sequences direct where in die fungal cell chromosome a DNA construct is integrated and directs what part of the fungal cell chromosome is replaced by die incoming sequence. While not meant to limit the present disclosure, a homology box may include about between 1 base pair (bp) to 200 kilobases (kb). Preferably, a homology box includes about between 1 bp and 10.0 kb; between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb, and between 0.25 kb and 2.5 kb. A homology box may also include about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb and 0.1 kb. In some embodiments, the 5' and 3' ends of a selective marker are flanked by a homology box wherein the homology box comprises nucleic acid sequences immediately flanking the coding region of the gene.

[0170] As defined herein, a host cell “genome”, a fungal cell “genome”, or a filamentous fungus cell “genome” includes chromosomal and extrachromosomal genes.

[0171] As used herein, the terms “plasmid”, “vector” and “cassette” refer to extrachromosomal elements, often carrying genes which are typically not part of the central metabolism of tire cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- stranded or double -stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0172] As used herein, the term “vector” refers to any nucleic acid that can be replicated (propagated) in cells and can carry new genes or DNA segments (e.g., an “incoming sequence”) into cells. Thus, the term refers to a nucleic acid construct designed for transfer between different host cells. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), PLACs (plant artificial chromosomes), and the like, that are “episomes” (i.e., replicate autonomously) or can integrate into the chromosome of a host cell.

[0173] A used herein, a “transformation cassette” refers to a specific vector comprising a gene and having elements in addition to the gene that facilitate transformation of a particular host cell.

[0174] As used herein, “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA in a cell. Many prokaryotic and eukaryotic expression vectors are commercially available and know to one skilled in the art. Selection of appropriate expression vectors is within the knowledge of one skilled in the art.

[0175] As used herein, the terms “expression cassette” and “expression construct” refer to a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements drat permit transcription of a particular nucleic acid in a target cell (e.g., vectors or vector elements described above). The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, DNA constructs also include a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. In certain embodiments, a DNA construct of the disclosure comprises a selective marker and an inactivating chromosomal or gene or DNA segment as defined herein.

[0176] As used herein, a “targeting vector” is a vector that includes polynucleotide sequences that are homologous to a region in the chromosome of a host cell into which the targeting vector is transformed and that can drive homologous recombination at that region. For example, targeting vectors find use in introducing genetic modifications into the chromosome of a host cell through homologousrecombination. In some embodiments, a targeting vector comprises other non-homologous sequences, e.g., added to the ends (i.e., staffer sequences or flanking sequences). The ends can be closed such that tire targeting vector forms a closed circle, such as, for example, insertion into a vector.III. TRANSFORMATION AND SELECTION OF TRICHODERMA CELLS HAVING MODIFED GENE LOCI

[0177] As briefly described above, certain embodiments of the disclosure are related to methods and compositions for constructing, engineering, transforming, selecting, growing, fermenting, and the like filamentous fungal strains / cells. More specifically, as set forth in the Examples below and described herein, Applicant has surprisingly observed that Trichoderma strains comprising a mutation of a betatubulin (tbbl) gene are particularly resistant to the fungicide carbendazim, whereas parental (control) Trichoderma strains were sensitive (not resistant) to the same concentration of carbendazim (Example 1; FIG. 1). Likewise, as set forth in (lie Examples, Applicant further observed that Trichoderma strains comprising a mutation of a beta-tubulin (tbbl) gene are sensitive (not resistant) to the fungicide diethofencarb, whereas parental (control) Trichoderma strains were resistant to the same concentration of diethofencarb (Example 1; FIG. 1).

[0178] Example 2 demonstrates that targeted editing of the tbbl gene locus to introduce a mutation can be selected for by carbendazim resistance using Cas9-RNP and DNA targeting the locus. For instance, Applicant constructed two plasmids carrying homology directed repair donor (HDRD) fragments for directing edits of the DNA encoding glutamate (E) at amino acid position 198 (E198) of the tbbl gene, wherein one plasmid (dsHDRD-E198K) changes E198 to a lysine (E198K) and the other plasmid (dsHDRD-E198G) changes E198 to glycine (E198G), as illustrated in FIG. 2 and FIG. 3. Independent transformations of dsHDRD-E198K and dsHDRD-E198K fragments were used to cotransform an industrial Trichoderma strain, along with assembled Cas9 nuclease with synthetic guide RNA tbbl -c that targets a cut site in the tbbl locus, and transformants selected on minimal media agar plates containing 1.2 pg / ml carbendazim. After about 3-5 days of growth, large and small colonies developed only on the transformation plates treated with HDRD and Cas9-RNP. Only the large colonies grew when isolated onto new minimal media agar plates containing 1.2 pg / ml carbendazim. After a few days growth, colony PCR was performed on the parent and five random carbendazim resistant transformants from each HDR fragment, and then PCR products were Sanger sequenced, wherein all five transformants of the dsHDRD-E198K and dsHDRD-E198G fragments contained the expected edits at the tbbl locus.

[0179] To test sensitivity of the Trichoderma tbblE198 mutants to dicthofcncarb, conidiospores of tire novel tbblE198G and tbblE198K Trichoderma mutant strains and their parent were spotted onto minimal media agar plates with and without 1.2 pg / ml carbendazim, 1.2 pg / ml dietho fencarb and 0.6 pg / nil dietho fencarb, wherein the plates were incubated for two days at 33°C. As shown in FIG. 4, all strains grew on media lacking beta-tubulin inhibitors (i.e., 0 pg / ml dfc). While the parental strain, RL-P37nikl (P), failed to grow on 1.2 pg / ml carbendazim (cdz), both E198G isolates (Gl, G3) and an E198K isolate (K9) were resistant to 1.2 pg / ml carbendazim and grew (EIG. 4). On the other hand, die parental strain RL-P37nikl was resistant to diethofencarb (dfc) even at the higher 1.2 pg / ml concentration, as was tire E198K isolate (FIG.4). Surprisingly, it was further observed that both of the E198G isolates were sensitive to diethofencarb, even at the lower concentration of 0.6 pg / ml, demonstrating that manipulation of tbbl alone at position E198 is sufficient to interconvert Trichoderma resistance and sensitivity to die beta-tubulin inhibitors carbendazim and diethofencarb. To verify that modified Trichoderma strains comprising a novel tbbl mutant of the disclosure retain their ability to produce (express / secrete) proteins of interest, Applicant fermented die three E198G derivatives, and two E198K derivatives in slow-release microtiter plates and assayed for protein production enzymatically and by SDS-PAGE (FIG. 5), wherein the beta-glucosidase and beta-xylosidase activities for the E198G mutants were at least as high as diose of the parental strain RL-P37nikl (FIG. 5A), confirming the modified strains have retained the ability to produce (express / secrete) proteins of interest. Consistent with this (FIG. 5B), there were no obvious reductions in total protein production, or changes in banding pattern by SDS-PAGE analysis.

[0180] Example 3 demonstrates that targeted editing of die tbbl locus to revert the E198G mutation can be selected for by diethofencarb resistance. Furthermore, among diethofencarb resistant transformants, tins example demonstrates that transformants with co-editing of a second locus can be isolated when Cas9-RNP and DNA targeting the second locus are co-transformed with those for tbbl editing. More specifically, four plasmids were developed carrying HDRD fragments for directing edits of the DNA at four different loci, wherein each HDRD was designed to be paired witii a specific Cas9 synthetic guide RNA (sgRNA), as generally summarized in TABLE 3 (Example 3). As summarized in this example, the co-editing of at least one other unsclcctcd locus can be achieved at relatively high frequency among transformants where modification of the tbblE198G allele is selected for by diethofencarb resistance.

[0181] Example 4 of the disclosure demonstrates the utility of selectable tbbl editing (e.g., by conversion between carbendazim and dielliofencarb resistances and sensitivities) to engineer recombinant Trichoderma strains having reduced background (enzyme) side activities and expressing a heterologous protein of interest. In particular, a DNA fragment containing a bovine chymosin expression cassette was used to co-transform Trichoderma strain ETD4, along witii assembled Cas9 nuclease with synthetic guide RNAs set forth in SEQ ID NOS: 39-46 that targeted the egl3, egl5, egl6 and manl loci, wherein the ETD4 strain comprises deletions of the cellulases cbhl. cbh2, egll and eg!2 and mutation of the pyr2 gene to enable its use as a selection marker. Transformants were selected for by pyrimidine prototrophy, and then isolated and outgrown on minimal agar plates before screening for bovine chymosin expression. Transformants were fermented in slow release microtiter plates and then fermentation filtrates were assayed for chymosin activity, wherein transformants with relatively high chymosin activity were then screened by PCR for mutational events at the targeted loci. A selectedtransformant named “BFA65” showed mutational events at egl6 and manl loci that were predicted to inactivate these enzymes. In other embodiments of Example 4, Applicant attempted to co-edit two genes, egl3 and egl5, while selecting for edit of wild-type tbbll98E to tbblE198G by carbendazim resistance. For instance, Trichoderma protoplasts for chymosin expression strain (BFA65) were cotransformed with all three HDRD / sgRNA pairs (TABLE 7) and transformants selected on minimal media agar plates containing 1.2 pg / ml carbendazim. After 3-5 days of growth, numerous colonies developed only on the transformation plates treated with HDRD and Cas9-RNP pairs. A selected transformant named “BML88” showed co-edits by design at tbbl and egl3 loci. In another embodiment of Example 4, Applicant attempted to co-edit two genes, tppl and egl5, while selecting for edit of the mutant tbblE198G back to the wildtype by diethofencarb resistance. In particular, Trichoderma protoplasts for chymosin expression strain (BML88) were co-transformed with all three HDRD / sgRNA pairs (TABLE 9) and transformants selected on minimal media agar plates containing 1.2 itg / ml diethofencarb. After 3-5 days of growth, numerous colonies developed only on the transformation plates treated with HDRD and Cas9-RNP pairs. As described, four out of fourteen transformants were identified with co-edits at both loci, and had tbbl alleles that can be used for another round of co-editing transformation again with carbendazim selection. Thus, as demonstrated in the above examples, collectively, through two rounds of transformation, a Trichoderma strain expressing a heterologous protein of interest can be engineered to remove three background proteins without ever having to introduce DNA encoding a selection marker. Additionally, by switching back and forth between the two alleles at tbbl , the strain could principally continue to be engineered indefinitely without having to use additional selection markers, or introduce additional steps to remove selection markers from the genome.

[0182] In other embodiments, Applicant has observed that Trichoderma strains sensitive to tire fungicide fluconazole can be modified herein for resistance. More particularly, as presented in Example 5 , Applicant has identified certain mutations in filamentous fungal erg 11 genes (i.e., T. reesei, C. tropicalis, C. albicans, C. parapsilosis ergll mutants; TABLE 13) capable of rendering strains expressing one of these mutant erg I 1 genes resistant to fluconazole. For instance, mutations producing the highest levels of resistance to fluconazole were introduced into Trichoderma strains as ergll expression constructs comprising a pyr2 marker for a second selection of transformants, and transformants spread on selective agar plates of modified medium comprising 100 pg / ml fluconazole and 2.4 g / L uridine. More particularly, as shown in FIG. 7, expression of the ergll gene from certain Candida species or Trichoderma containing one of the selected mutations results in transformants able to grow on agar media containing 100 jrg / ml fluconazole, whereas the control strain (no ergll expression cassette) had no observable colonies, as Trichoderma cannot grow on fluconazole at concentrations of 50 pg / ml and higher. Thus, as presented in Example 5, different ergll mutations result in varying transformation efficiencies (z'.e., yielding different numbers of colonies), with the Trichoderma ergll mutant comprising the Y137F mutation (SEQ ID NO: 82) exhibiting the highestnumber of transformants, confirming that Trichoderma growth observed on fluconazole is solely attributed to expression of ergll mutants.IV. RECOMBINANT NUCLEIC ACIDS AND MOLECULAR BIOLOGY

[0183] As briefly described above, certain embodiments of the disclosure are related to methods and compositions for constructing, engineering, transforming, selecting, growing, fermenting, and the like filamentous fungal cells. More particularly, certain embodiments of the disclosure provide modified Trichoderma sp. cells comprising introduced mutations at a beta-tubulin 1 (tbbl) gene locus. In other embodiments, the disclosure provides modified Trichoderma sp. cells comprising introduced polynucleotides overexpressing a mutant ergll protein. Thus, certain embodiments of the disclosure are related to molecular biology, genetic modifications, polynucleotides, genes, gene coding sequences (CDS), vectors, expression constructs (gene cassettes) and the like.

[0184] In certain embodiments, a polynucleotide may comprise 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. a bleomycin resistance marker, a blasticidin resistance marker, a pyri thiamine resistance marker, a chlorimuron ethyl resistance marker, a neomycin resistance marker, an adenine pathway gene, a tryptophan pathway gene, a thymidine kinase marker and the like. In a particular embodiment, the selectable marker is pyr2. which compositions and methods of use are generally set forth in PCT Publication No. WO2011 / 153449.

[0185] Standard techniques for transformation of filamentous fungi and culturing the fungi (which are well known to one skilled in the art) are used to transform a fungal host cell of the disclosure. Thus, the introduction of a DNA construct or vector into a fungal host cell includes techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated and DEAE- Dextrin mediated transfection), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated micro-projectiles, gene gun or biolistic transformation, protoplast fusion and the like. General transformation techniques are known in the art.

[0186] Often, transformation of Trichoderma sp. fungal cells uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 105to 107per mL, particularly about 2xlO6 / mL. A volume of 100 pL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 m CaCl2) is mixed with the desired DNA. Generally, a high concentration of polyethylene glycol (PEG) is added to the uptake solution. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells (e.g., see US6,022,725 and US6,268,328, both of which are incorporated by reference).

[0187] Thus, the methods and compositions of instant disclosure generally rely on routine techniques in the field of recombinant genetics. For example, in certain embodiments, a gene of interest is introduced into a filamentous fungal (host) cell. In certain embodiments, the gene (or gene CDS) iscloned into an intermediate vector, before being transformed into a filamentous fungal (host) cell for replication and / or expression. These intermediate vectors can be prokaryotic vectors, such as, e.g., plasmids, or shuttle vectors. In certain embodiments, the expression of die gene or gene CDS is under the control of a heterologous promoter, which can be a heterologous constitutive promoter or a heterologous inducible promoter.

[0188] The expression vector typically contains a transcription unit or “expression cassette” that contains all the additional elements required for the expression of the heterologous sequence. For example, a typical expression cassette contains an upstream (5') promoter operably linked to a nucleic acid sequence encoding a protein of interest and may further comprise nucleic acid sequences encoding protein (signal) secretion sequences, nucleic acid sequences required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination sequences. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

[0189] 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 die promoter sequence or may be obtained from different genes. Although any fungal terminator is likely to be functional in the present invention, preferred terminators include: the terminator from Trichoderma cbhl gene, the terminator from Aspergillus nidulans trpC gene and the Aspergillus awamori or Aspergillus niger glucoamylase genes.

[0190] The particular expression vector used to transport die genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include bacteriophages X and Ml 3, as well as plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc.

[0191] The elements that can be included in expression vectors may also be a replicon, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, or unique restriction sites in nonessential regions of die plasmid to allow insertion of heterologous sequences. The particular antibiotic resistance gene chosen is not dispositive either, as any of the many resistance genes known in the art may be suitable. The prokaryotic sequences are preferably chosen such tiiat titey do not interfere with the replication or integration of the DNA in the filamentous fungal host.

[0192] The methods of transformation of the present invention may result in the stable integration of all or part of the transformation vector into die genome of the filamentous fungus. However, transformation resulting in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated. Many standard transfection methods can be used to produce filamentous fungal cell lines that express large quantities of the heterologous protein, and as such, any of the known procedures for introducing foreign nucleotide sequences into fungal host cells may be used. Theseinclude the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other known mediods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell. Also of use is die Agrobacterium-mediated transfection method such as the one described in U.S. Patent No. 6.255,115. After the expression vector is introduced into the cells, the transformed cells are cultured under conditions favoring expression of gene. Large batches of transformed cells can be cultured as described herein. Finally, the protein product is recovered from die culture using standard techniques. Thus, the disclosure provides for the expression and enhanced production of desired proteins of interest, as described herein.

[0193] In certain one or more embodiments or aspects of the disclosure, filamentous fungal cells (strains) may comprise one or more genetic modifications, including, but is not limited to, the introduction, substitution, or removal of one or more nucleotides in a gene, or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of die gene, a gene disruption, a gene conversion, a gene deletion, a gene down-regulation, specific mutagenesis and / or random mutagenesis of a gene.

[0194] As generally set forth above and described hereinafter, one skilled in the art may readily perform one or more genetic modifications and construct recombinant / modified filamentous fungal strains thereof, by reference to one or more nucleic acid sequences and / or protein sequence disclosed herein. For example, gene deletion techniques enable the partial or complete removal of the gene, thereby eliminating or reducing expression / production of die protein, and / or thereby eliminating or reducing expression / production the encoded protein. In such methods, the deletion of the gene may be accomplished by homologous recombination using an integration plasmid / vector that has been constructed to contiguously contain die 5' and 3' regions flanking die gene. The contiguous 5' and 3' regions may be introduced into a filamentous fungal cell, for example, on an integrative plasmid / vector in association with a selectable marker to allow die plasmid to become integrated in die cell.

[0195] In other embodiments, a modified strain of filamentous fungus comprises genetic modifications which disrupt or inactivate a gene of interest. Exemplary mediods of gene disruption / inactivation include disrupting any portion of die gene, including die polypeptide coding sequence (CDS), promoter, enhancer, or another regulatory element, which disruption includes substitutions, insertions, deletions, inversions, and combinations thereof and variations diereof. A non-limiting example of a gene disruption technique includes inserting (integrating) into one or more of the genes of the disclosure an integrative plasmid containing a nucleic acid fragment homologous to the gene of interest, which will create a duplication of the region of homology and incorporate (insert) vector DNA between the duplicated regions. In certain other non-limiting examples, a gene disruption technique includes inserting into a gene of interest an integrative plasmid containing a nucleic acid fragment homologous to the gene of interest, which will create a duplication of the region of homology and incorporate (insert) vector DNA between the duplicated regions, wherein the vector DNA inserted separates, e.g., the promoter of the gene from the protein codingregion, or interrupts (disrupts) the coding, or non-coding, sequence of the gene, resulting in an enhanced protein productivity phenotype. A disrupting construct may be a selectable marker gene (e.g., pyr2) accompanied by 5' and 3' regions homologous to the gene of interest. The selectable marker enables identification of transformants containing the disrupted gene. Thus, in certain embodiments, gene disruption includes modification of control elements of the gene, such as the promoter, ribosomal binding site (RBS), untranslated regions (UTRs), codon changes, and the like.

[0196] In other embodiments, a modified strain of filamentous fungus is constructed (i.e., genetically modified) by introducing, substituting, or removing one or more nucleotides in the gene, or a regulatory element required for the transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a pre-mature stop codon, tine removal of the start codon, or a frame-shift of the open reading frame (ORF).

[0197] In certain other embodiments, a modified strain of filamentous fungus is constructed by means of CRISPR / Cas9 editing. More specifically, compositions and methods for fungal genome modification by CRISPR / Cas9 systems are described and well known in the art (e.g., see, PCT Publication Nos: W02016 / 100571, W02016 / 100568, W02016 / 100272, WQ2016 / 100562 and the like). Thus, a gene of interest can be disrupted, deleted, mutated or otherwise genetically modified by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g., Cas9) or a guide DNA (e.g., Ng Ago), which recruits the endonuclease to the target sequence on the DNA, wherein the endonuclease can generate a single or double stranded break in the DNA. This targeted DNA break becomes a substrate for DNA repair and can recombine with a provided editing template to disrupt or delete the gene. For example, the gene encoding the nucleic acid guided endonuclease (e.g., a Cas9 from S. pyogenes, or a codon optimized gene encoding the Cas9 nuclease) is operably lurked to a promoter active in the filamentous fungal cell and a terminator active in filamentous fungal cell, thereby creating a filamentous fungal Cas9 expression cassette. Likewise, one or more target sites unique to tire gene of interest are readily identified by a person skilled in tire art. For example, to build a DNA construct encoding a gRNA-directed to a target site within tire gene of interest, the variable targeting domain (VT) will comprise nucleotides of tire target site which are 5' of tire (PAM) proto-spacer adjacent motif (TGG), which nucleotides are fused to DNA encoding tire Cas9 endonuclease recognition domain for S. pyogenes Cas9 (CER). The combination of the DNA encoding a VT domain and the DNA encoding the CER domain thereby generate a DNA encoding a gRNA. Thus, a filamentous fungal expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in filamentous fungal cells and a terminator active in filamentous fungal cells.

[0198] In certain embodiments, the DNA break induced by the endonuclease is repaired / replaced with an incoming sequence. For example, to precisely repair the DNA break generated by the Cas9 expression cassette and the gRNA expression cassette described above, a nucleotide editing template is provided, such that the DNA repair machinery of the cell can utilize the editing template. For example, about 500bp 5' of targeted gene can be fused to about 500bp 3' of the targeted gene to generate an editing template, whichtemplate is used by the filamentous fungal host’s machinery to repair tire DNA break generated by tire RGEN (RNA-guided endonuclease).

[0199] The Cas9 expression cassette, tire gRNA expression cassette and tire editing template can be codelivered to filamentous fungal cells using marry different methods (e.g., protoplast fusion, electroporation, natural competence, or induced competence). The transformed cells are screened by PCR, by amplifying the target locus with a forward and reverse primer. These primers can amplify tire wild-type locus or the modified locus that has been edited by the RGEN. These fragments are then sequenced using a sequencing primer to identify edited colonies.V. PROTEENS OF INTEREST

[0200] As stated above, certain embodiments are related to modified fungal cells expressing / producing proteins of interest. In certain embodiments, proteins of interest are encoded by endogenous filamentous fungal genes, e.g., endogenous genes encoding cellulases, endoglucanases, xylanases, and the like. In other embodiments, proteins of interest are encoded by introduced (heterologous) polynucleotides (e.g., expression constructs). In certain one or more embodiments, a protein of interest (POI) is an enzyme selected from the group consisting of cellulases, hemicellulases, xylanases, peroxidases, proteases, lipases, phospholipases, esterases, cutinases, polyesterases, phytases, pectinases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, mannanases, a-glucanases, |3-glucanases, hyaluronidases, chondroitinases, laccases, amylases, glucoamylases, acetyl esterases, aminopeptidase, arabinases, arabinosidases, arabinofuranosidases, carboxypeptidases, catalases, nucleases, deoxyribonucleases, ribonucleases, epimerases, a-galactosidases, -galactosidases, glucan lysases, endo-P-glucanases, glucose oxidases, glucuronidases, invertases, trehalases, and isomerases. In certain other embodiments, a POI is selected from an Enzyme Commission (EC) Number selected from the group consisting of EC 1, EC 2, EC 3, EC 4, EC 5, and EC 6.

[0201] Optimal conditions for the production of the proteins will vary with the choice of the host cell, and with the choice of the protein(s) to be expressed. Such conditions may be readily ascertained by one skilled in the art through routine experimentation and / or optimization. The POI can be purified or isolated after expression. The POI may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include, but are not limited to, electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the POI may be purified using a standard antiprotein of interest antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. The degree of purification necessary will vary depending on the intended use of the protein of interest. In certain instances, no purification of the protein will be necessary.

[0202] In certain other embodiments, to confirm that a genetically modified fungal cell of tire disclosure produces a POI, various methods of screening may be performed. In certain embodiments, tire POI may be detected by its activity (e.g., enzymatic activity, binding activity, etc.) or chromatographic profile. In other embodiments, air expression vector may encode a polypeptide fusion to tire target protein which serves as a detectable label or tire target protein itself may serve as the selectable or screenable marker. The labeled protein may be detected via western blotting, dot blotting (methods available at tire Cold Spring Harbor Protocols website), ELISA, or, if the label is GFP, whole cell fluorescence and / or FACS. For example, a 6-histidine tag would be included as a fusion to the target protein, and this tag would be detected by western blotting. If the target protein expresses at sufficiently high levels, SDS-PAGE combined with Coomassie / silver staining, may be performed to detect increases in variant host cell expression over parental (control) cell, in which case no label is necessary. In addition, other methods may be used to confirm the improved level of a POI, such as, the detection of the increase of protein amount per cell or protein amount per milliliter of fermentation medium using HPLC methods of protein separation or standard total protein measurements based on Coomassie Blue or BCA Reagents.

[0203] The detection of specific productivity is another method to evaluate the protein production. Specific productivity (Qp) can be determined by the following equation:Qp = gP / gDCW«hrwherein “gP” is grams of protein produced in the tank, “gDCW” is grams of dry cell weight (DCW) in the tank, “hr” is fermentation time in hours from tire time of inoculation, which include the time of production as well as growth time. Ultimately, if a protein of interest has enzymatic activity, its level of expression can be calculated from enzymatic assay.

[0204] In certain other embodiments, total protein yield may be determined, wherein total protein yield is defined as the amount of protein produced (g) per gram of carbohydrate fed, relative to tire (unmodified) parental strain. Thus, as used herein, total protein yield (g / g) may be calculated using tire following equation:Yf = Tp / Tcwherein “Yf ’ is total protein yield (g / g), “Tp” is the total protein produced during the fermentation (g) and “Tc” is the total carbohydrate (g) fed during the fermentation (bioreactor) run.

[0205] Total protein yield may also be described as carbon conversion efficiency / carbon yield, for example, as in the percentage (%) of carbon fed that is incorporated into total protein. Tirus, in certain embodiments, a modified filamentous fungal cell comprises an increased carbon conversion efficiency (e.g., an increase in the percentage (%) of carbon fed that is incorporated into total protein), relative to the (unmodified) parental strain. In certain embodiments, the increase in carbon conversion efficiency of tire modified strain (i.e.. relative to the control strain) is an increase of at least about 0.1 %, at least about 1%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% or more as compared to the unmodified (parental) cell.VI. FERMENTATION

[0206] Certain embodiments are related to compositions and methods for producing proteins of interest comprising fermenting a modified filamentous fungal cell of die disclosure. In general, fermentation methods well known in the art are used to ferment the fungal cells. In some embodiments, the fungal cells are grown under batch, fed batch or continuous fermentation conditions. A classical batch fermentation is a closed system, where the composition of the medium is set at the beginning of the fermentation and is not altered during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism(s). In this method, fermentation occurs without the addition of any components to the system. Typically, a batch fermentation qualifies as a “batch” with respect to the addition of the nutrients, while factors such as pH and oxygen concentration are controlled. The broth and culture compositions of the batch system change constantly up to the time the fermentation is stopped. Within batch cultures, cells progress through a static lag phase to a high growth log phase and finally to a stationary phase, where growth rate is diminished or halted. If untreated, cells proceed to apoptosis and eventually die. In general, in the batch phase, the bulk of the production of product occurs during the log phase.

[0207] A suitable variation on die standard batch system is the “fed-batch fermentation” system. In this variation of a typical batch system, after the log phase is finished, die substrate is added in increments as the fermentation progresses. Fed-batch systems are often used to avoid catabolite repression. Continuous feeding of the substrate allows the process to keep its concentration below critical level diat could lead to inhibition of cellular metabolism and protein production. Batch and fed-batch fermentations are common and well known in the art.

[0208] Continuous fermentation is a system where a defined fermentation medium is added continuously to a biorcactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant (high) density, where cells are primarily kept in log phase growth. In other systems, a number of factors affecting growth can be altered continuously while die cell concentration, measured by media turbidity (or dry cell weight), is kept constant. Continuous systems strive to maintain steady state growtii conditions. Thus, cell loss due to medium being drawn off should be balanced against the cell growtii rate in the fermentation. Methods of modulating nutrients and growtii factors for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[0209] Certain embodiments of the instant disclosure are related to fermentation procedures for culturing fungi. Fermentation procedures for production of cellulase enzymes are known in the art. For example, cellulase enzymes can be produced either by solid or submerged culture, including batch, fed batch and continuous-flow processes. Culturing is generally accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, a carbon and energy sourcematerial, molecular oxygen, and, of course, a starting inoculum of the filamentous fungal host to be employed.

[0210] In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the fermentation media.

[0211] The composition of the aqueous mineral medium can vary over a wide range, depending in part on the microorganism and substrate employed, as is known in the art. The mineral media should include, in addition to nitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium, in suitable soluble assimilable ionic and combined forms, and certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art.

[0212] The fermentation process can be an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion.

[0213] The fermentation tempera lure can vary somewhat, but for filamentous fungi such as Trichoderma reesei, the temperature generally will be within the range of about 20°C to 40°C, generally preferably in the range of about 25°C to 34°C.

[0214] The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap 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 oilier ammonium compounds can be utilized. Ammonia gas itself is convenient for large scale operations and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control.

[0215] The pH range in the aqueous microbial ferment should be in the exemplary range of about 2.0 to 10.0. With filamentous fungi, the pH normally is within the range of about 2.5 to 8.0; with Trichoderma reesei, the pH normally is within the range of about 3.0 to 7.0. Preferences for pH range of microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus can be somewhat adjusted as can be readily determined by those skilled in the art.

[0216] Preferably, the fermentation is conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon- containingsubstrate to products and avoiding contamination of the cells with a substantial amount of unconverted substrate. The latter is not a problem with water-soluble substrates since any remaining traces are readily washed off. It may be a problem, however, in the case of non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps.

[0217] As described above, the time to reach this level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved.

[0218] The fermentation can be conducted as a batch or continuous operation, fed batch operation is much to be preferred for ease of control, production of uniform quantities of products, and most economical uses of all equipment.

[0219] If desired, part or all of the carbon and energy source material and / or part of die assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermenter.

[0220] Each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in response to a need determinable by monitoring such as concentrarion of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gases from the fermenter, cell density measurable by dry cell weights, light transmittancy, or tire like. The feed rates of tire various materials can be varied so as to obtain maximal production rates and / or maximum yields.

[0221] In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling devices, and the like, are initially sterilized, usually by employing steam such as at about 121°C for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of tire selected microorganism in the presence of all tire required nutrients, including oxygen, and tire carbon-containing substrate. The type of fermenter employed is not critical.VII. EXEMPLARY EMBODIMENTS

[0222] Non- limiting embodiments of compositions and methods disclosed herein are as follows:

[0223] 1. A modified Trichoderma sp. cell comprising an introduced mutation at a beta-tubulin 1 (tbbl) gene locus, wherein the mutated tbbl gene locus encodes a variant tbbl protein comprising an amino acid substitution at position 198, wherein the amino acid positions of the variant tbbl protein are numbered according to SEQ ID NO: 3.

[0224] 2. The modified cell of embodiment 1, wherein the modified cell can be selected by carbendazim resistance or diethofencarb sensitivity.

[0225] 3. The modified cell of embodiment 1, wherein the substitution at position 198 is a glutamic acid (E) to glycine (G) substitution (E198G).

[0226] 4. The modified cell of embodiment 1, wherein the substitution at position 198 is a glutamic acid (E) to lysine (G) substitution (E198K)

[0227] 5. The modified cell of embodiment 1, wherein the substitution at position 198 is a glutamic acid (E) to lysine (G) substitution (E198D).

[0228] 6. The modified cell of embodiment 1, constructed from a parental (isogenic) Trichoderma sp. cell comprising a bela-lubulin 1 (tbbl) gene locus encoding a native tbbl protein comprising a glutamic acid (E) at amino acid position 198 (E 198) , wherein the amino acid positions of the native tbbl protein are numbered according to SEQ ID NO: 3.

[0229] 7. The modified cell of embodiment 6, wherein the parental cell expresses an endogenous protein of interest (POI) and / or a heterologous POI.

[0230] 8. The modified cell of embodiment 6. wherein the parental cell can be selected by carbendazim sensitivity or diethofencarb resistance.

[0231] 9. The modified cell of embodiment 7. wherein the modified cell produces at least an equivalent amount of the endogenous POI and / or heterologous POI as compared to the parental cell fermented under the same conditions.

[0232] 10. The modified cell of embodiment 7, wherein the POI is an enzyme.

[0233] 11. The modified cell of embodiment 1, comprising at least one introduced polynucleotide expression construct encoding a heterologous protein on interest (POI).

[0234] 12. A mutant tbbl gene comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to about 100% identity to SEQ ID NO: 1.

[0235] 13. The mutant tbbl gene of embodiment 12, encoding a variant tbbl protein having an amino acid substitution at position 198, wherein the amino acid positions of the variant tbbl protein are numbered according to SEQ ID NO: 3.

[0236] 14. A mutant tbbl gene coding sequence (CDS) comprising at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to about 100% identity to SEQ ID NO: 2.

[0237] 15. The mutant tbbl gene CDS of embodiment 14, encoding a variant tbbl protein having an amino acid substitution at position 198, wherein the amino acid positions of the variant tbbl protein are numbered according to SEQ ID NO: 3.

[0238] 16. The mutant tbbl gene of embodiment 12, or the mutant tbbl gene CDS of embodiment 14, encoding a variant tbbl protein comprising at least about 90%, 91%, 92%.93%, 94%, 95%, 96%, 97%, 98%, 99% to about 100% identity to SEQ ID NO: 3 and having an amino acid substitution at position 198.

[0239] 17. 1’he mutant tbbl gene of embodiment 12, or the mutant tbbl gene CDS of embodiment 14, wherein the substitution at position 198 is selected from a glutamic acid (E) to glycine (G) substitution (E198G), a glutamic acid (E) to lysine (G) substitution (E198K). and a glutamic acid (E) to lysine (G) substitution (E198D).

[0240] 18. A method for co-editing gene loci in a Trichoderma sp. cell comprising co-transforming a plurality of Trichoderma sp. cells with at least two homology directed repair donor (HDRD) fragments, wherein tire first HDRD fragment edits a beta-tubulin 1 (tbbl ) gene locus resulting in a mutant tbbl gene locus encoding a variant tbbl protein comprising a substitution of the glutamic acid (E) residue at amino acid position corresponding to position 198 of SEQ ID NO: 3, and the second HDRD fragment edits a first gene locus of interest (GLOI), culturing the plurality of cells in a media comprising carbendazim and selecting transformants thereof resistant to carbendazim, isolating and analyzing the selected transformants for the targeted edit at the first GLOI, and selecting and isolating at least one transformant having the targeted edit at the first GLOI, wherein the isolated cell comprises the mutant tbbl gene locus and targeted edit at the first GLOI.

[0241] 19. The method of embodiment 18, wherein editing is stimulated by at least two guide RNA / Cas (RNA / Cas) complexes, wherein one targets the tbbl gene locus and the other targets the first gene locus of interest (GLOI).

[0242] 20. The method of embodiment 18, wherein the plurality of Trichoderma sp. cells are cotransformed with at least three HDRD fragments and at least three sgRNA / Cas complexes, wherein the first HDRD fragment and sgRNA / Cas complex target and edit the WT tbbl gene locus resulting in a mutant tbbl gene locus encoding a variant tbbl protein comprising a substitution of the glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, the second HDRD fragment and sgRNA / Cas complex target and edit a first GLOI, and the third HDRD fragment and sgRNA / Cas complex target and edit a second GLOI, culturing the plurality of cells in a media comprising carbendazim and selecting transformants thereof resistant to carbendazim, isolating and analyzing the selected transformants for the targeted edits at the first and second GLOI, and selecting and isolating at least one transformant having the targeted edits at the first and second GLOI, wherein the isolated cell comprises the mutant tbbl gene locus and targeted edit at the first and second GLOI.

[0243] 21. A method for iterative editing of multiple gene loci in a Trichoderma sp. cell comprising co-transforming a plurality of the Trichoderma sp. cells of embodiment 18 comprising the mutant tbbl gene locus and targeted edit at the first GLOI with al least two HDRD fragments and at least two sgRNA / Cas complexes, wherein the first HDRD fragment and sgRNA / Cas complex target and edit the mutant tbbl gene locus resulting in a WT tbbl gene locus encoding a native tbbl protein comprising a glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, and the second HDRD fragment and sgRNA / Cas complex target and edit a second GLOI, culturing the plurality of cells in a media comprising diethofencarb and selecting transformants thereof resistant to diethofencarb, isolating and analyzing the selected transformants for the targeted edit at the second GLOI, selecting and isolating at least one transformant having the targeted edit at the second locus, wherein the isolated cell comprises the WT tbbl gene locus and the targeted edits at the first and second GLOI.

[0244] 22. The method of embodiment 21, wherein the isolated cell comprising the WT tbbl gene locus and targeted edits at the first and second GLOI is subjected to another round of co-transformationwith at least two HDRD fragments and at least two sgRNA / Cas complexes, wherein tire first HDRD fragment and sgRNA / Cas complex target and edit the WT tbbl gene locus resulting in a mutant tbbl gene locus encoding a variant tbbl protein comprising a substitution of the glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, and the second HDRD fragment and sgRNA / Cas complex target and edit a third GLOI, culturing the plurality of cells in a media comprising carbendazim and selecting transformants thereof resistant to carbendazim, isolating and analyzing the selected transformants for the targeted edit at the third GLOI, and selecting and isolating at least one transformant having the targeted edit at the third GLOI, wherein the isolated cell comprises the mutant tbbl gene locus and the targeted edits at the first, second and third GLOI.

[0245] 23. The method of embodiment 18, wherein the at least one transformant having the targeted edit at the first GLOI is selected by sequencing the gene locus of the transformant and confirming the targeted edit at the first GLOI.

[0246] 24. The method of any one of embodiments 18-23, further comprising co-transforming the plurality of the Trichoderma sp. cells with at least one expression construct encoding a heterologous protein of interest.

[0247] 25. The method of any one of embodiments 18-23, wherein Trichoderma sp. cells express one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.

[0248] 26. A modified Trichoderma sp. cell comprising an introduced polynucleotide expressing a mutant ergl 1 protein, wherein the modified cell can be selected by fluconazole resistance.

[0249] 27. The modified cell of embodiment 26, wherein the mutant ergl 1 protein comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% to about 100% identity to the native erg 11 protein of SEQ ID NO: 85 and comprises at least one amino acid substitution at a position corresponding to position 137, 148, 464 or 467 of SEQ ID NO: 85.

[0250] 28. The modified cell of embodiment 27, wherein the substitution at position 137 is a tyrosine (Y) to phenylalanine (F) substitution (Y137F), or tire substitution at position 148 is lysine (K) to arginine (R) substitution (K148R), or a lysine (K) to glutamine (Q) substitution (K148Q), or tire substitution at position 464 is a glycine (G) to serine(S) substitution, or tire substitution at position 467 is a arginine (R) to lysine (K) substitution.

[0251] 29. The modified cell of embodiment 26, wherein the mutant ergl 1 protein comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% to about 100% identity to the native ergl 1 protein of SEQ ID NO: 86 or SEQ ID NO: 87, and comprises at least one amino acid substitution at a position corresponding to position 132, 464 or 467 of SEQ ID NO: 86 or SEQ ID NO: 87.

[0252] 30. 1’he modified cell of embodiment 29, wherein the substitution at position 132 is a tyrosine (Y) to phenylalanine (F) substitution (Y 132F), or a tyrosine (Y) to histidine (H) substitution (Y132H), or tire substitution at position 464 is glycine (G) to serine (S) substitution (G464S), or the substitution at position 467 is arginine (R) to lysine (K) substitution (R467K).

[0253] 31. The modified cell of embodiment 26, wherein the polynucleotide expressing the mutant ergll protein comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% to about 100% identity to a polynucleotide of any one of SEQ ID NO: 79-84.

[0254] 32. The modified cell of embodiment 26, constructed from a parental (isogenic) Trichoderma sp. cell which does not comprise an introduced polynucleotide expressing a mutant ergll protein, wherein parental cell can be selected by fluconazole sensitivity.

[0255] 33. The modified cell of embodiment 26, expressing one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.

[0256] 34. The modified cell of embodiment 26, wherein the introduced polynucleotide expressing the mutant ergll protein further comprises an operably linked polynucleotide encoding a protein of interest.

[0257] 35. An isolated polynucleotide encoding a mutant ergl 1 protein comprising at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% to about 100% identity to SEQ ID NO: 85 and having at least one substitution at an amino acid position selected from 137, 148, 464 and 467 of SEQ ID NO: 85.

[0258] 36. The polynucleotide of embodiment 35, wherein the substitution at position 137 is a tyrosine (Y) to phenylalanine (F) substitution (Y137F), the substitution at position 148 is lysine (K) to arginine (R) substitution (K148R), or a lysine (K) to glutamine (Q) substitution (K148Q), the substitution at position 464 is a glycine (G) to serine (S) substitution, or the substitution at position 467 is a arginine (R) to lysine (K) substitution.

[0259] 37. The polynucleotide of embodiment 35, comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% to about 100% identity to any one of SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84.

[0260] 38. A method for selecting a transformed Trichoderma sp. cell comprising transforming a plurality of Trichoderma sp. cells with a polynucleotide comprising a gene coding sequence (CDS) encoding a mutant ergll protein, wherein die gene CDS comprises upstream and downstream homologous recombination (HR) regions flanking the gene CDS to enable integration at a selected gene locus, culturing the plurality of cells in a media comprising fluconazole, selecting and isolating at least one transformed cell thereof resistant to fluconazole, wherein the isolated cell comprises the gene CDS cassette integrated the selected gene locus.

[0261] 39. The method of embodiment 38, wherein the transformed polynucleotide further comprises an operably linked polynucleotide encoding a protein of interest.

[0262] 40. A method for selecting a transformed Trichoderma sp. cell comprising transforming a plurality of Trichoderma sp. cells with a polynucleotide expression construct encoding a mutant ergl 1 protein, wherein the construct comprises an upstream promoter operably linked to a downstream mutant ergll gene coding sequence (CDS) and upstream and downstream homologous recombination (HR)regions flanking the construct to enable integration at a selected gene locus, culturing the plurality of cells in a media comprising fluconazole, selecting and isolating at least one transformed cell thereof resistant to fluconazole, wherein the isolated cell comprises the ergl 1 construct integrated the selected gene locus.

[0263] 41. The method of embodiment 40, wherein the transformed polynucleotide further comprises an operably linked polynucleotide encoding a protein of interest.

[0264] 42. The method of embodiment 40, wherein the plurality of cells are co-transformed with a sgRNA / Cas complex that generates a double strand break at the selected gene locus.

[0265] 43. The method of embodiment 38 or 40, wherein the plurality of cells express one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.EXAMPLES

[0266] It should be understood drat the following Examples, while indicating embodiments of die disclosure, are given by way of illustration only. From die above discussion and these Examples, one of skill in die art can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Such modifications are also intended to fall within the scope of the claimed invention. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).EXAMPLE 1CARBENDAZIM RESISTANCE CONFERRED BY MUTATION OF TBB1 LOCUS

[0267] A. Overview

[0268] In die present example, spontaneous carbendazim mutants of Trichoderma were isolated. More particularly, Applicant surprisingly observed dial only mutants in die tbbl gene arose under selection, suggesting tiiat mutation of tbbl is sufficient to confer resistance to carbendazim.

[0269] B. Expression analysis of Trichoderma beta-tubulins

[0270] If only one tubulin were expressed during asexual growth, then it would be more likely that inhibition of only this tubulin would be responsible for sensitivity. Applicant therefore looked at internal and external transcriptional data for the two beta-tubulins, tbbl (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) and tbb2 (SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6 ). For instance, T. reesei transcript data from RNA-Seq experiments conducted under excess glucose conditions were compared during fermentation. Total RNA was isolated using TriZOL (Thermo Fisher Scientific). A TruSeq RNA library kit (Illumina, USA) was used to construct sequencing libraries for two (2) biological replicates. The libraries were sequenced on an Illumina HiSeq 2000 platform. Using the GeneData Analysis vXY platform (GeneData, Switzerland), reads were mapped to the T. reesei JGI v2 genome assembly (IGI Trichoderma reesei genome database v. 2.0 (genome.jgi.doe.gov; Martinez et al., 2008) using Bowtie2 (Langmead and Salzberg, 2012). Read counts were quantified using featureCounts (Liao etal., 2014). These results are compared with those from a related strain, QM9414 (Dos Santos Castro et al., 2014).TABLE 1RNA-SEQ DATA FOR TRICHODERMA REESEI BETA- AND ALPHA-TUBULINS*Dos Santos Castro et al., 2014; QM9414 RNAseq

[0271] As shown in TABLE 1 above, RNA-Seq data shows that both tbbl and tbb2 genes are expressed at similar levels during Trichoderma growth, offering no insight to predict what combinationof beta-tubulin mutations would confer resistance to carbendazim. To normalize between the two data sources, FPKM values are also given normalized to alpha-tubulin gene (tubl , JGI PID 120789).

[0272] C. Sensitivity of Trichoderma to carbendazim and diethofencarb

[0273] Media: Vogel’s minimal media (Vogel, 1956).

[0274] Carbendazim (MilliporeSigma 378674) and diethofencarb (MilliporeSigma 34087) were dissolved to 2.5 mg / ml in DMF (N, N-Dimethylformamide).

[0275] Spores of an industrial Trichoderma strain were collected in sterile water, quantified, and then IxlO7conidia spores were plated on Vogel’s Minimal Medium plates containing 0, 0.5, and 1 itg / ml carbendazim. Plates were incubated at 33°C for four (4) days. As shown in FIG. 1, carbendazim (cdz) was selective at 1 pg / ml with a few presumably spontaneous mutants per plate. At 0.5 jrg / ml cdz, there was growth at one end of plate where spore density appeared to be higher. Diethofencarb (dfc) was non-selective at 1 iig / ml.

[0276] D. Characterization of spontaneous carbendazim mutants of Trichoderma

[0277] Applicant isolated spontaneous mutants of Trichoderma resistant to carbendazim. Both tbbl and thb2 loci were sequenced from resistant strains. Only mutations in tbbl were recovered.

[0278] Colony PCR was performed on five (5) spontaneous carbendazim resistant colonies to amplify the tbbl and tbb2 loci from these strains, using a Phire Plant Direct PCR Master Mix (ThermoFisher F160L) as described by manufacturer using methods known to one skilled in the art. PCR products were then fully sequenced using Sanger sequencing. The primers used to amplify (“amplification” column) and sequence (“sequencing” column) the tbbl and tbb2 loci as set forth below in TABLE 2. Sequencing reads as .abi files were dien aligned to tbbl and tbb2 loci of the reference QM6a genome in the DNA analytical software Geneious Prime (Biomatters Ltd).TABLE 2PRIMERS USED TO AMPLIFY AND SEQUENCE BETA-TUBULIN LOCI

[0279] No mutations were identified in the tbb2 loci. However, all five (5) strains had a mutation in die tbbl coding sequence representing drree (3) alleles: E198K, E198D and Q134L. For instance, FIG. 2 and FIG. 3 show schematic maps of the tbbl locus with relative positions of the Q 134 and E198 amino acid positions.EXAMPLE 2ISOLATION OF CARBENDAZIM RESISTANT TRICHODERMA TRANSFORMANTS BY TARGETED EDIT AT THE TBB1 LOCUS

[0280] A. Overview

[0281] This example demonstrates that targeted editing of tire tbbl locus to introduce the E198G mutation can be selected for by carbendazim resistance using Cas9-RNP and DNA targeting the locus. In particular, presence of the tbblE198G allele made transformants sensitive to beta-tubulin inhibitor diethofencarb, but had no significant impact on secreted protein production.

[0282] B. Targeted editing of tbbl gene to tbblE198G allele was selectable by carbendazim resistance

[0283] In the present example, two (2) plasmids were developed carrying homology directed repair donor fragments for directing edits of the DNA encoding glutamate (E) at amino acid position 198 (E198) of the tbbl gene using methods known to one skilled in the art. For instance, each homology directed repair donor (HDRD) fragment corresponds to roughly 1.6 kb of the tbbl locus centered on the position of the edits. One, dsHDRD-E198K (SEQ ID NO: 15), changes E198 to a lysine (K, i.e., E198K) and the other, dsHDRD-E198G (SEQ ID NO: 16), changes E198 to glycine (G; i.e., E198G). In addition, each HDRD contained a synonymous codon change for alanine (A) at amino acid position 206 to inactivate the recognition site for the Cas9 synthetic guide RNA (sgRNA) tbbl-c (SEQ ID NO: 17). The tbbl locus and relative positions of the HDRD fragments and sgRNA target sites are illustrated in FIG. 2. These HDRDs could be constructed and / or commercially synthesized (e.g., Gene Art, IDT) by one skilled in the art from the disclosed sequences.

[0284] In independent transformations, dsHDRD-E198K and dsHDRD-E198K fragments was used to co-transform an industrial Trichodcrma strain, along with assembled Cas9 nuclease with synthetic guide RNA tbbl-c (SEQ ID NO: 17) that targets a cut site in the tbbl locus. This Cas9-sgRNA complex was assembled in vitro according to tire manufacturer’s protocol (Synthego) and used to transform Trichoderma as generally set forth in PCT Publication No. W02016 / 100568. More specifically, tire following components were mixed and then incubated al 37°C for 10-20 minutes: 22 pl of RNase-free water, 3 pl of 10X NEB buffer 3.1, 3pl of EnGen® Spy Cas9 NLS (New England Biolabs M0646) and 2 pl of Synthego sgRNA at 100 pM. Approximately three (3) pg of plasmid DNA carrying the HDRDs and 10 pl of the assembled Cas9-sgRNA complex were used to transform roughly 4xl07protoplasts of a Trichoderma strain, RL-P37nikl (U.S. Patent No. 10,907,187). For instance, RL-P37nikl is a mutagenized derivative of wild-type isolate QM6a with improved secretion of tire native Trichoderma cellulases. The transformation was performed using the polyethylene glycol (PEG) mediated protoplast transformation protocol (Ouedraogo et al., 2015; Penttila et al., 1987).

[0285] Transformants were selected on Vogel’s minimal media agar plates containing 1.2 pg / ml carbendazim. After 3-5 days of growth, large and small colonies developed only on the transformation plates treated with HDRD and Cas9-RNP. Only the large colonies grew when isolated onto newVogel’s minimal media agar plates containing 1.2 tg / ml carbendazim. After a few days growth, colony PCR was performed on the parent and five (5) random carbendazim resistant transformants from each HDRD fragment using primers RPG2790 (SEQ ID NO: 11) and RPG2966 (SEQ ID NO: 18) and then PCR products were Sanger sequenced with primer RPG2793 (SEQ ID NO: 14), essentially as described above in Example 1.

[0286] All five (5) transformants of the dsHDRD-E198K and dsHDRD-E198G fragments contained the expected edits at the tbbl locus, at both the E198 codon and A206 codon positions, that were provided by the HDRD fragments.

[0287] C. Transformants with the tbblE198G mutation acquired sensitivity to diethofencarb

[0288] In Neurospora crassa. tire E198G substitution in its sole beta-tubulin encoding gene, Bml, confers resistance to carbendazim, but also confers sensitivity to the beta-tubulin inhibitor diethofencarb (Fujimura et al., 1992). Since Trichoderma has a second tubulin encoding gene, tbb2. it was unclear if the E198G substitution in only tbbl would confer sensitivity to diethofencarb or if, for instance, a resistant wild-type tbb2 gene still present in the genome would be sufficient to maintain resistance.

[0289] To test sensitivity of Trichoderma tbblE198G mutants to diethofencarb, conidiospores of the novel RL-P37nikl tbblE198G and tbblE198K mutant strains and their parent were spotted with a toothpick onto Vogel’s minimal media agar plates widi and without 1.2 |ig / ml carbendazim, 1.2 )ig / ml diethofencarb and 0.6 pg / nil diethofencarb. Plates were incubated for two (2) days at 33°C. As shown in FIG. 4, all strains grew on media lacking beta-tubulin inhibitors (0 dfc). While parental strain RL-P37nikl (P) failed to grow on 1.2 pg / ml carbendazim (1.2 cdz), both E198G isolates (Gl, G3) and an E198K isolate (K9) were resistant and grew. On the other hand, parental strain RL-P37nikl was resistant to dicthofcncarb even at the higher 1.2 pg / ml concentration (1.2 dfc), as was the E198K isolate, but both E198G isolates were sensitive to diethofencarb even at die lower concentration of 0.6 |ig / ml (0.6 dfc). This demonstrates that manipulation of tbbl alone at position E198 is sufficient to interconvert Trichoderma resistance and sensitivity to tire beta-tubulin inhibitors carbendazim and dietho fencarb.

[0290] D. Protein production in carbendazim-resistant tbblE198G mutant

[0291] Filamentous fungi like Trichoderma are of industrial importance due to their ability to produce and secrete large quantities of protein into die fermentation broth during submerged culture fermentations. For diis application, it would be important that engineered strains carrying the tbblE198G mutation did not have significantly reduced protein secretion. To test the protein secretion of diese novel beta-tubulin mutants, RL-P37nikl , three E198G derivatives, and two E198K derivatives were fermented in slow-release microtiter plates then assayed for protein production enzymatically and by SDS-PAGE.

[0292] Media Composition: 400x T. reesei trace elements: citric Acid (anhydrous), 175 g / L; FeSO4.7 H2O, 200 g / L, ZnSO4.7 H2O, 16 g / L, CuSO4.5 H2O, 3.2 g / L; MnS04.H20, 1.4 g / L; H3BO3, 0.8 g / L.

[0293] Citrate minimal medium 5 g / L (NH4)2S04, 4.5 g / L KH2PO4, 1 g / L MgS04.7 H20, and 14.4 g / L citric acid, adjusted to pH 5.5 with 5% NaOH. After autoclaving for 30 minutes, sterile 50% glucose was added to a final concentration of 0.5 %, along with 2.5 mL / L of 400x trace element solution.

[0294] Liquid defined (LD) culture medium contained the following components. Casamino acids, 12 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; 400x 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.

[0295] Fermentation: Transformants were grown in citrate minimal media for 36-48 hours at 32°C in 96 well plates with shaking. After incubation, 0.11 mL of seed culture were added to 0.99 ml of LD medium per well of a 24-well 20% Lactose slow-release micro-titer plate (srMTP). srMTPs have been described for example, in US Patent No. 10,030,221. These production cultures were then fermented for 4 to 5 days at 25°C and 250 RPM. Following fermentation, secreted proteins were separated from the cell mass by filtration through a filter-bottom 96-well plate into a 96-well non-binding assay plate.

[0296] pNPG and pNPX Assays: Secreted protein production was measured using artificial substrates for beta-glucosidase (pNPG) and beta-xylosidase (pNPX). The pNPG and pNPX substrates were prepared by dissolving to 3mM in 0.25M Acetate buffer at pH 5.5. Fermentation broth supernatants were diluted as appropriate in 0.25M Acetate buffer at pH 5.5. Fifty (50) microliters of substrate were added to fifty (50) microliters of diluted fermentation broths in wells of a 96 well microtiter assay plate. These were incubated at 48°C for several minutes and then reactions were stopped with 120ul of stop buffer (200mM boric acid, pH 10.2). Absorbance was read at 405 nm in a plate-reading spectrophotometer.

[0297] SDS-PAGE Analysis: To observe the proteins secreted by Trichoderma into the fermentation broth, one (1) to five (5) microliters of filtrate was diluted in 4x LDS loading buffer (Invitrogcn, NP0008), denatured and run on NuPAGE 4-12% SDS-PAGE gels (Invitrogen, NP0323) along with tire See Blue Plus 2 molecular weight standard (Invitrogen, LC5925) in IX NuPAGE MOPS buffer (Invitrogen, NP0001). Gels were then stained with Simply Blue Safe Stain (Invitrogen, LC6065) and de-stained in water using standard molecular biology procedures.

[0298] As shown in FIG. 5A, pNPG and pNPX activities for the 198G mutants were at least as high as those of the parental strain RL-P37nikl. Consistent with this, as shown in FIG. 5B, there were no obvious reductions in total protein production or changes in banding pattern by SDS-PAGE analysis.EXAMPLE 3ISOLATION OF CO-EDITING AT AN UNSELECTED LOCUS BY SELECTION OF TARGETED REVERSION TO DIETHOFENCARB RESISTANCE AT THE TBB1 LOCUS

[0299] A. Overview

[0300] Here we demonstrate that targeted editing of the tbbl locus to revert the E198G mutation can be selected for by diethofencarb resistance using Cas9-RNP and DNA targeting the locus. Furthermore,we demonstrate that, among diethofencarb resistant transformants, transformants with co-editing of a second locus could be isolated when Cas9-RNP and DNA targeting to that second locus were cotransformed with those for tbbl editing.

[0301] B. Isolation of co-editing at an unselected locus by selection of targeted reversion to diethofencarb resistance at the tbbl locus

[0302] Four (4) plasmids were developed carrying homology directed repair donor (HDRD) fragments of approximately 1.3 kb for directing edits (nucleotide changes) of the DNA at four different loci. Each HDRD was designed to be paired with a specific Cas9 synthetic guide RNA (sgRNA). HDRDs and the sgRNA pairs are summarized below in TABLE 3. By design, each HDRD fragment would alter the coding of an amino position in the target locus and mutate the PAM site corresponding to its paired sgRNA. HDRDs were designed to be roughly centered on the edited positions. These double stranded DNA HDRDs could be constructed and / or commercially synthesized (e.g.. GeneArt, IDT) by one skilled in the art from the disclosed sequences.

[0303] Co-transformation can be high in Trichoderma as in other filamentous fungi, so when a mix of HDRD / sgRNAs are present in the protoplast transformation reaction, protoplasts frequently take up multiple HDRDs and sgRNAs. Consequently, cells derived from protoplasts that have an edit at one locus, could also have taken up HDRD / sgRNA to other loci and edited those as well, termed “coediting”.

[0304] For co-editing, isolation of transformants is greatly facilitated when one of the cas9-mediated edits at one of the loci generates a phenotypic change that is selectable. In the case of tbbl, this selection is by either conferring carbendazim or diethofencarb resistance, depending on the nature of the tbbl allele. This selection allows for cells derived from the protoplasts that took up the HDRD / sgRNA pairs to be purified from those that did not transform, i.e., did not take up HDRD / sgRNAs.

[0305] One IIDRD fragment, for the “selected edit”, contained an 1.3 kb portion of the wild-type tbbl gene spanning the El 98 coding position and designed to direct the reversion of the E198G mutation back to die wild-type 198E. Reversion also removes the PAM site for sgRNA tbbl-d (SEQ ID: 19). Applicant hypothesized that this could be selectable by diethofencarb resistance.

[0306] The other three (3) HDRD / sgRNA pairs were designed as unselected “co-edits”. All co-edits would result in an amino acid change in the protein encoded by the target locus that would have an easily observable phenotype. One HDRD fragment targeted an amino acid and PAM site change in the acetolactate synthase (als) gene known to confer resistance to the herbicide chlorimuron ethyl (U.S. Patent No. 8,138,321). Another HDRD fragment targeted an amino acid and PAM site change in the succinate dehydrogenase iron-sulfur protein encoding gene (sdil) that confers resistance to fungicide carboxin (Kilaru et al..2020). The third co-edit HDRD fragment introduced a stop codon at the coding position for amino acid 35 in the pyrimidine biosynthesis enzyme orotate phosphoribosyl transferase encoded by the gene pyr2. Loss of pyr2 function renders the strain a uridine auxotroph as well as conferring resistance to 5 -fluorooro tic acid.TABLE 3sgRNA / IIDRD PAIRS FOR CO-EDITING

[0307] Trichoderma protoplasts for one of the carbendazim resistant transformants with the tbblE198G allele was co-transformed with all four HDRD / sgRNA pairs listed in TABLE 3. These Cas9-sgRNA complexes were assembled in vitro according to the manufacturer’s protocol (Synthego) and used to transform Trichoderma as generally set forth in PCT Publication No. W02016 / 100568. More specifically, the following components were mixed and then incubated at 37°C for 10-20 minutes: 22 p.1 of RNase-free water, 3 pl of 10X NEB buffer 3.1, 3 pl of EnGen® Spy Cas9 NLS (New England Biolabs M0646) and 2 pl of Synthego sgRNA at 100 pM. Approximately three (3) pg of plasmid DNA carrying the HDRDs and 10 pl of the assembled Cas9-sgRNA complex were used to transform roughly 4xl07protoplasts of a Trichoderma strain “G3” from Example 2. The transformation was performed using the polyethylene glycol (PEG) mediated protoplast transformation protocol (Ouedraogo el al., 2015; Penttila etal., 1987). An additional transformation was done in parallel with only the tbblE198G HDRD / sgRNA pair.

[0308] Transformants were selected on Vogel’s minimal media agar plates containing 1.2 pg / ml diethofencarb and 1 mg / ml uridine. After 3-5 days of growth, numerous colonies developed only on tire transformation plates treated with HDRD and Cas9-RNP pairs, although transformation efficiency was much lower for the co-edit transformations. Sixteen (16) transformants for tire co-edit transformations and six (6) transformants for tire tbblE198G-only transformation were isolated onto Vogel’s minimal media agar plates containing 1.2 pg / ml diethofencarb and 1 mg / ml uridine.

[0309] Following several days growth at 28°C, each transformant was spotted onto tester media for the different target loci using a toothpick tip of spores. Four Vogel’s minimal media agar plates were used: (1) plates with no supplements (transformants with edits at pyr2 would not grow), (2) plates with uridine supplementation and 80 pg / ml chlorimuron ethyl (only transformants with edits at als locus would grow), (3) plates with uridine supplementation and 150 pg / ml carboxin (only transformants with edits at sdil locus would grow), and (4) plates with only uridine supplementation (all transformants should grow). Strain RL-P37 was also inoculated as a phenotypic reference. After point inoculation, plates were left to incubate at 33°C for a few days before observing die phenotypes. No co-edits wereisolated among the six transformants tested where only targeting edit of tbblE198G. Of the sixteen transformants isolated by co-transformation of all four HDRD / sgRNA pairs (co-editing transformation), six were phenotypically pyr2 mutant, four were phenotypically als mutants, but none were phenotypically sdil mutants. Four of these transformants were phenotypical mutants for both pyr2 and als. This represents a 38% phenotypic efficiency of co-editing at a second unselected locus when selecting for reversion to diethofencarb resistance.

[0310] Co-edited transformants were further analyzed by attempting to PCR amplify the edited loci and then Sanger sequencing of the PCR products. PCR primer pairs (screen primer 1 and screen primer 2) used for amplification of the edited loci, the expected size of the edited locus (size) and the primer used for Sanger sequencing of the PCR product (sequence primer) are given in TABLE 4.

[0311] The als locus was readily recovered and sequenced from all four chlorimuron ethyl sensitive transformants and all contained the mutations as designed and provided by the HDRD. The pyr2 locus could only be amplified cleanly from two of the six uridine auxotrophs. Of these only one contained the mutations as designed and provided by the HDRD. The other had a small portion of the tbbl gene, probably from its HDRD, inserted into the cut site for the cas9-RNP targeting pyr2. Furthermore, at tbbl, PCR of the locus was only clean for three of the six transformants and, of these, only two contained the mutations as designed and provided by the HDRD that revert E198G back to the wildtype G198E.TABLE 4PRIMERS USED FOR ANALYSIS OF TRANSFORMANTS

[0312] This demonstrates that co-editing of at least one other unselected locus can be achieved at relatively high frequency among transformants where modification of die tbblE198G allele is selected for by diethofencarb resistance. However, not all edits at the target loci, even at tbbl. will be repaired by homologous recombination as guided by the HDRDs. For some applications, any co-edit that modifies and inactivates the target locus would be acceptable; however, for those applications where a specific modification is required, a larger set of transformants may need to be screened to find those that only underwent homologous recombination at all loci.

[0313] Efficiency of repair by homologous recombination with tire HDRD in principle could be further improved by host modifications influencing which DNA damage repair pathways are utilized, such asmutations in the non-homologous end-joining (NHEJ) pathway, although these changes may not be desirable for some applications. Additionally, modification of the HDRD physical properties could also influence which DNA damage repair pathways are utilized, for instance by use of linear double stranded DNA (dsDNA) or single-stranded (ssDNA) as tire HDRD.EXAMPLE 4ISOLATION OF CO-EDITING AT AN UNSELECTED LOCUS BY SELECTION OF EDITING AT TBB1 LOCUS IN A TRICHODERMA STRAIN EXPRESSING A HETEROLOGOUS PROTEIN

[0314] A. Overview

[0315] The instant example demonstrates the utility of selectable tbbl editing by conversion between carbendazim and diethofencarb resistances and sensitivities, to engineer reduced background side activities in a Trichoderma strain expressing a heterologous protein.

[0316] B. Construction of Trichoderma strain expressing bovine chymosin

[0317] An expression cassette for bovine chymosin proenzyme was developed to overexpress bovine chymosin in Trichoderma as a translational fusion with a catalytically inactive Cbhl_core with linker, using methods known to one skilled in the art (U.S, Patent No. 8,546,126). Expression of the fusion protein was placed under regulation of the Trichoderma cbhl promoter and the Trichoderma cbhl terminator. The Trichoderma reesei pyr2 gene was placed at one end of the cassette to facilitate isolation of Trichoderma transformants carrying tire expression cassette. At the other end of tire cassette, we placed tire native Trichoderma bipl chaperone under tire control of its native promoter and terminator sequences. A schematic map of tire expression cassette is given in FIG. 6, showing the relative position and orientation of the DNA parts in the cassettes. Pertinent biological parts of the cassette are listed in below TABLE 5 in their order found in the cassette, along with corresponding SEQ ID NOS. These cassettes could be constructed and / or commercially synthesized (e.g.. GeneArt, IDT) by one skilled in the art from the disclosed information.TABLE 5PARTS OF THE BIP1 AND BOVINE CHYMOSIN EXPRESSION CASSETTE

[0318] A DNA fragment containing the bipl -bovine chymosin-pyr2 expression cassette was used to co-transform strain ETD4, along with assembled Cas9 nuclease with synthetic guide RNAs (SEQ ID NOS: 39-46) that targeted the egl3 (JGI PID 123232), egl5 (JGI PID 49976), egl6 (JGI PID 49081) and manl (JGI PID 56996) loci. Strain ETD4 is a Trichoderma reesei strain derived from RL-P37 (described by Sheir-Neiss and Montenecourt, 1984) that has been genetically engineered for industrial enzyme production, with modifications including deletion of tine cellulases cbhl, cbh2, egll and egl2 to reduce background protein expression and mutation of tine pyr2 gene to enable its use as a selection marker. These Cas9-sgRNA complexes were assembled in vitro according to the manufacturer’s protocol (Synthego) and used to transform Trichoderma as generally set forth in PCT Publication No. W02016 / 100568. More specifically, the following components were mixed and then incubated at 37°C for 10-20 minutes: 22 pl of RNase-free water, 3 pl of 10X NEB buffer 3.1 , 3 pl of EnGen® Spy Cas9 NLS (New England Biolabs M0646) and 2 pl of Synthego sgRNA at 100 pM. Approximately ten (10) pg of purified bipl and bovine chymosin expression cassette and 20ul of an equal blend of assembled Cas9 -sgRNA complexes were used to transform approximately 4xl07protoplasts of strain ETD4. The transformation was performed using a standard polyethylene glycol (PEG) mediated protoplast transformation protocol.

[0319] Transformants were selected for by pyrimidine prototrophy. Transformants were then isolated and outgrown on minimal agar plates before screening for bovine chymosin expression. Transformants were fermented in srMTPs as generally described in Example 2D and then fermentation filtrates were assayed for chymosin activity using essentially the same methods as previously described (Dunn-Coleman et al., 1991). Transformants with relatively high chymosin activity were then screened by PCR for mutational events at die targeted loci by PCR amplification of amplicons spanning the paired Cas9-RNP target sites. PCR products widi the similar molecular weight as die parental strain were Sanger sequenced using the primers used for amplification. TABLE 6 lists die primers used to screen transformants. A selected transformant, named BFA65, showed mutational events at egl6 and manl diat were predicted to inactivate these enzymes.TABLE 6PRIMERS FOR SCREENING TRANSFORMANTS

[0320] C. Endoglucanase 3 (egl3) inactivation by co-editing oftbbl to carbendazim resistance

[0321] In the present example, Applicant attempted to co-edit two genes, egl3 and egl5, while selecting for edit of tbbl to tbblE198G by carbendazim resistance. Rather than die double stranded DNA fragments used as HDRDs in Examples 2 and 3, here we used unmodified single stranded DNA oligonucleotides as HDRDs. Each HDRD was designed to be paired with a specific Cas9 synthetic guide RNA (sgRNA). HDRDs and the sgRNA pairs are summarized in TABLE 7. By design, HDRDs were designed to be roughly centered on the engineered edit site which removed the PAM site for the paired sgRNA and introduced a stop codon into the open reading frame of the target gene. These singlestranded DNA HDRDs could be constructed and / or commercially synthesized (e.g., IDT) by one skilled in the art from the disclosed sequences.

[0322] For the egl3 gene, the HDRD replaces the whole R109 codon with a TA, introducing an inframe stop and a frameshift. For the egl5 gene, the HDRD replaces the last two positions of the S6 codon with an A, introducing and in-frame stop and a frameshift.TABLE 7sgRNA / HDRD PAIRS FOR CO-EDITING

[0323] Trichoderma protoplasts for chymosin expression strain BFA65 were co-transformed with all three HDRD / sgRNA pairs listed in TABLE 7. These Cas9-sgRNA complexes were assembled in vitro according to the manufacturer’s protocol (Synthego) and used to transform Trichoderma as generally set forth in PCT Publication No. W02016 / 100568. More specifically, the following components were mixed and then incubated at 37°C for 10-20 minutes: 22 pl of RNase-free water, 3 pl of 10X NEB buffer 3.1 , 3 pl of EnGen® Spy Cas9 NLS (New England Biolabs M0646) and 2 pl of Synthego sgRNA at 100 pM. For the selected locus, approximately 6 pl of single stranded HDRDs at 200 pM and approximately 3pl of the assembled Cas9-sgRNA complex were used to transform approximately 500 pl protoplasts at approximately 4xl07protoplasts / ml. For the co-edit loci, approximately 3 pl of single stranded HDRDs at 400 pM and 9 pl of an equal blend of the assembled Cas9-sgRNA complexes were used to transform protoplasts. The transformation was performed using the polyethylene glycol (PEG) mediated protoplast transformation protocol (Ouedraogo et al., 2015; Penttila et al., 1987).

[0324] Transformants were selected on Vogel’s minimal media agar plates containing 1.2 pg / ml carbendazim. After 3-5 days of growth, numerous colonies developed only on die transformation plates treated with HDRD and Cas9-RNP pairs. Twenty-nine (29) transformants for the co-edittransformations were isolated onto minimal media agar plates containing 1.2 pg / ml carbendazim. Following a few days growth at 28°C, DNA was extracted from transformants and the tbbl, egl3 and egl5 loci were PCR amplified and Sanger sequenced.

[0325] PCR primer pairs (screen primer 1 and screen primer 2) used for amplification of the edited loci, the expected size of the edited locus (size) and the primer used for Sanger sequencing of the PCR product (sequence primer) are given in TABLE 8.TABLE 8PRIMERS FOR ANALYSIS OF EDITING IN TRANSFORMANTS

[0326] All 29 transformants had the designed edits at tbbl encoding the tbblE198G mutation. Of these, two (2) contained co-edits in the egl3 gene, but only one was as designed in the HDRD. The other egl3 co-edit allele had a single nucleotide deletion at the cut site consistent with repair by non-homologous end joining. None of the screened transformants had co-edits at egl5. The one strain with co-edits by design at tbbl and egl3 was named BML88.

[0327] D. Tripeptidyl-peptidase 1 (tppl) and endoglucanase 5 (eglS) by co-editing of tbbl to diethofencarb resistance

[0328] In this example, Applicant attempted to co-edit two genes, tppl (JGI PID 82623) and egl5, while selecting for edit of tbblE198G back to the wildtype tbb!198E by diethofencarb resistance. Here again, unmodified single stranded DNA oligonucleotides were used as HDRDs, but as longer “ultramers” (IDT) for the co-edit loci. Each HDRD was designed to be paired with a specific Cas9 synthetic guide RNA (sgRNA). HDRDs and the sgRNA pairs are sunmiarized in TABLE 9. For instance, HDRDs were designed to be roughly centered on the engineered edit site which removed tire PAM site for the paired sgRNA and introduced a stop codon into the open reading frame of the target gene. These single-stranded DNA HDRDs could be constructed and / or commercially synthesized (e.g., IDT) by one skilled in the art from die disclosed sequences.

[0329] For die egl5 gene, a new sgRNA / HDRD pair were designed. The HDRD inserts a T between the codons for T124 and N 125 introducing an in-frame stop codon and a frame shift. For the tppl gene, the HDRD converts the first codon position for E225 to a T introducing an in-frame stop codon and replaces all of the T232 codon with TAA, introducing a second in-frame stop.TABLE 9sgRNA / IIDRI) PAIRS

[0330] Trichoderma protoplasts for chymosin expression strain BML88 were co- transformed with all three (3) HDRD / sgRNA pairs listed in TABLE 9. These Cas9-sgRNA complexes were assembled in vitro according to the manufacturer’s protocol (Syndiego) and used to transform Trichoderma as generally set forth in PCT Publication No. W02016 / 100568. More specifically, the following components were mixed and then incubated at 37°C for 10-20 minutes: 22 pl of RNase-free water, 3 pl of 10X NEB buffer 3.1, 3 pl of EnGen® Spy Cas9 NLS (New England Biolabs M0646) and 2 pl of Synthego sgRNA at 100 pM. For the selected locus, approximately 2 pl of 400uM single stranded HDRDs and assembled Cas9-sgRNA complex, ranging from 0.6 to 2.4 pl, were used to transform 250 pl of protoplasts at approximately 4 xlO7protoplast per mL. For the co-edit loci, approximately 2ul of 400 pM of single stranded HDRDs and 4.8ul of the assembled Cas9-sgRNA complex were used to transform protoplasts. The transformation was performed using die polyethylene glycol (PEG) mediated protoplast transformation protocol (Ouedraogo et al., 2015: Penttila et al., 1987).

[0331] Transformants were selected on Vogel’s minimal media agar plates containing 1.2 pg / ml di ethofencarb. After 3-5 days of growth, numerous colonies developed only on the transformation plates treated with HDRD and cas9-RNP pairs. Colony PCR amplifying die tbbl, tppl and egl5 loci were done from fourteen (14) colonies developing on primary transformation plates and the PCR products were Sanger sequenced. PCR primer pairs (screen primer 1 and screen primer 2) used for amplification of the edited loci, the expected size of the edited locus (size) and the primer used for Sanger sequencing of the PCR product (sequence primer) are given in TABLE 10.TABLE 10PRIMERS FOR ANALYSIS OF TRANSFORMANTS

[0332] As seen above, following diethofencarb selection, tire tbbl locus could not be amplified from some of the transformants. This seems to indicate that diethofencarb resistance can also be conferred by tbbl alleles other than wildtype tbbl. Nine of fourteen transformants had the expected edits at tbbl reverting tire lbblE198G allele to wildtype. Eight (8) of fourteen transformants had co-edits at tppl locus, with four of these being as designed by the HDRD. Again, co-editing efficiency at egl5 was low, but four of fourteen had co-edits with one of these being as designed by the HDRD. Of these fourteen, four were identified with co-edits at both loci and had tbbl alleles that could be used for another round of co-editing transformation again with carbendazim selection.

[0333] Collectively, through two rounds of transformation, a Trichoderma strain expressing a heterologous polypeptide was engineered to remove three background proteins without ever having to introduce DNA encoding a selection marker. Furthermore, by switching back and forth between the two alleles at tbbl. the strain could principally continue to be engineered indefinitely without having to use additional selection markers or introduce an additional step to remove a selection marker from the genome.EXAMPLE 5AZOLE RESISTANCE CONFERRED BY EXPRESSION OF ERG11 MUTANTS

[0334] A. Selection and Design of Marker Genes

[0335] A set of three ergll genes (SEQ ID NO:79; SEQ ID NO:80, SEQ ID NO:81 from two species Candida albicans and Candida tropicalis carrying mutations, resulting in resistance to azole antifungals, were selected from literature (Choi et al.. 2018; Berkow et al.. 2017). More particularly, as presented below, three mutations producing highest level of resistance to azole antifungals were introduced into the wild-type (endogenous) T. reesei ergll gene (i.e., mutations K148Q, K148R and Y137F).

[0336] B. Construction Expression Vectors

[0337] Genes originating from Candida species were ordered synthetically and cloned into a pB KT030 vector under control of the gpdA promoter from Aspergillus nidulans using Seamless Cloning and Assembly Kit (Invitrogen), according to the manufacturer’s protocol. The T. reesei ergll genes containing selected mutations (K148Q (SEQ ID NO: 82), K148R (SEQ ID NO: 83) and Y137F (SEQ ID NO: 84) were ordered synthetically and cloned into a pBKT030 vector under control of the (a) native erg / 1 promoter from 7. reesei or (b) the A. niger gpdA promoter using Seamless Cloning and Assembly Kit (Invitrogen), according to the manufacturer’s protocol. The pBKT030 vector further comprised a cbhl terminator from T. reesei, a pyr2 marker with its native promoter and terminator for second selection of T. reesei transformants, and upstream and downstream homologous recombination (HR) regions to enable targeted integration of the expression cassette at a selected gene locus in tire T. reesei reporter host.

[0338] C. Transformation of T. reesei with ergll selection Marker Cassette

[0339] The T. reesei reporter strains described herein were derived from parental 7. reesei strain RL-P37 (NRRL Deposit No. 15709), wherein the 7. reesei pyr2 gene has been deleted, as generally described by Sheir-Neiss and Montenecourt, 1984. More specifically, linear cassettes comprising one of the above ergll gene coding sequences, the gpdA or ergll promoter, the cbhl terminator, the pyr2 marker with its native promoter and terminator and approximately 1 kb of upstream and downstream HR region flanks, were PCR amplified from the set of pBKT030 based plasmids and transformed into the reporter strain. Preparation of protoplasts and transformation were carried out as described in PCT Publication No. WO2013 / 102674.

[0340] Integration of the expression cassettes into genome of the reporter strain was mediated using a Crispr-Cas9 technology to generate double strand breaks (DSB) at a specific genomic position that stimulated DNA repair. In this example, the DSBs were generated in the fungal cells using in vitro assembled RNP complexes that can specifically cut T. reesei genome in the defined locus. For instance, the ergl 1 selection marker was integrated in the selected gene locus together with the pyr2 marker, used as a secondary selection for process control. RNP complexes were formed in a 1:1 molar ratio between Cas9 nuclease EnGenRSpy Cas9 NLS (New England BioLabs, Inc) and synthetic crRNAs annealed with tracrRNA according to recommendations of the supplier (Synthego, USA). For transformation, four (4) pl of RNP complex were mixed with approximately 3 pg expression cassette amplified from the appropriate plasmid together with HR region flanks for targeted integration in selected locus.

[0341] Transformants were spread on selective agar plates of modified Vogel’s medium (3 g / L Na3Citrate*2H2O, 5.0 g / L KLLPCty 2.0 g / L NFtyNCty, 0.2 g / L MgSO 7H2O, 0.1 g / L CaCL*2H2O, 0.25% of 0.1 g / L D-biotin solution and 0.5 % T. reesei trace elements solution: 50 g / L Citric Acid, 50 g / L ZnSO4*7H2O, 10 g / L Fe(NH4)2SO4*6H20, 2.5 g / L CuSO4*5H2O, 0.5 g / L MnSO4*4H20, 0.5 g / L H3BO3, 0.5 g / L NaMoO4*2H2O; Davis and de Serres, 1970) with 2.0% (w / w) glucose as a carbon source, 100 pg / ml fluconazole and 2.4 g / L uridine. In tire control experiment, fluconazole and uridine were omitted. Positive transformants were counted after seven (7) days of outgrowth at 28°C. Growing clones were screened for correct integration by PCR.

[0342] For instance, as presented in FIG. 7, expression of the ergll mutants from Candida species (SEQ ID NO: 79; panel 6, SEQ ID NO: 80; panel 5 and SEQ ID NO: 81; panel 4), or the Trichoderma ergll mutants (i.e., containing one of the selected mutations (K148Q, K148R and Y137F) results in transformants able to grow on agar media containing 100 pg / ml fluconazole. In the control experiment (FIG. 7, Ctrl), where the expression cassette was omitted, no colonies were observed, as Trichoderma cannot grow on fluconazole at concentrations of 50 pg / ml and higher. Similarity, the C. parapsilosis ergll Y132F_R398I mutation (FIG. 7, panel 7) and C. albicans ergll K143Q mutation (FIG. 7, panel 8) also did not yield transformants able to grow on fluconazole, while selection for pyr2 yielded high number of colonies.

[0343] These results confirm that growth observed on fluconazole containing plates is solely attributed to expression of ergll variants. 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Claims

CLAIMS1. A modified Trichoderma sp. cell comprising an introduced mutation al a beta-tubulin 1 bbl) gene locus, wherein die mutated tbbl gene locus encodes a variant tbbl protein comprising an amino acid substitution at position 198, wherein the amino acid positions of the variant tbbl protein are numbered according to SEQ ID NO: 3.

2. The modified cell of claim 1, wherein the modified cell can be selected by carbendazim resistance or diethofencarb sensitivity.

3. The modified cell of claim 1, wherein the substitution at position 198 is a glutamic acid (E) to glycine (G) substitution (E198G).

4. The modified cell of claim 1. wherein the substitution at position 198 is a glutamic acid (E) to lysine (K) substitution (E198K).

5. The modified cell of claim 1, wherein the substitution at position 198 is a glutamic acid (E) to aspartic acid (D) substitution (E198D).

6. The modified cell of claim 1, constructed from a parental (isogenic) Trichoderma sp. cell comprising a beta-tubulin 1 (tbbl) gene locus encoding a native tbbl protein comprising a glutamic acid (E) at amino acid position 198 (E198) of SEQ ID NO: 3.

7. The modified cell of claim 6, wherein the parental cell expresses one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.

8. The modified cell of claim 6, wherein the parental cell can be selected by carbendazim sensitivity or diethofencarb resistance.

9. The modified cell of claim 7, wherein die modified cell produces at least an equivalent amount of die endogenous and / or heterologous proteins of interest as compared to die parental cell fermented under the same conditions.

10. The modified cell of claim 1, comprising at least one introduced polynucleotide expression construct encoding a heterologous protein on interest (POI).

11. The modified cell of claim 1 , wherein the variant tbbl protein comprises at least about 90% identity to SEQ ID NO: 3.

12. A mutant tbbl gene comprising at least about 80% identity to SEQ ID NO: 1 and encoding a variant tbbl protein having an amino acid substitution at position 198, wherein the amino acid positions of the variant tbbl protein are numbered according to SEQ ID NO:

313. The mutant tbbl gene of claim 12, wherein the substitution at position 198 is selected from a glutamic acid (E) to glycine (G) substitution (E198G), a glutamic acid (E) to lysine (K) substitution (E198K), or a glutamic acid (E) to aspartic acid (D) substitution (E198D).

14. The mutant tbbl gene of claim 12, wherein the tbbl gene coding sequence (CDS) comprises at least about 85% identity to SEQ ID NO: 2.

15. The mutant tbbl gene of claim 12, wherein the variant tbbl protein comprises at least about 90% identity to SEQ ID NO: 3.

16. A method for co-editing gene loci in a Trichoderma sp. cell comprising:co-transforming a plurality of Trichoderma sp. cells with at least two homology directed repair donor (HDRD) fragments, wherein the first HDRD fragment edits a beta-tubulin 1 (tbbl) gene locus resulting in a mutant tbbl gene locus encoding a variant tbbl protein comprising a substitution of the glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, and the second HDRD fragment edits a first gene locus of interest (GLOI), culturing the plurality of cells in a media comprising carbendazim and selecting transformants thereof resistant to carbendazim, isolating and analyzing the selected transformants for the targeted edit at the first GLOI, and selecting and isolating at least one transformant having the targeted edit at the first GLOI, wherein the isolated cell comprises the mutant tbbl gene locus and targeted edit at the first GLOI.

17. The method of claim 16, wherein editing is stimulated by at least two guide RNA / Cas (RNA / Cas) complexes, wherein one targets the tbbl gene locus and the other targets the first gene locus of interest (GLOI).

18. The method of claim 16, wherein die plurality of Trichoderma sp. cells are co-transformed with at least three HDRD fragments and at least three sgRNA / Cas complexes, wherein the first HDRD fragment and sgRNA / Cas complex target and edit the WT tbbl gene locus resulting in a mutant tbbl gene locus encoding a variant tbbl protein comprising a substitution of the glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, the second HDRD fragment and sgRNA / Cas complex target and edit a first GLOI, and die third HDRD fragment and sgRNA / Cas complex target and edit a second GLOI, culturing the plurality of cells in a media comprising carbendazim and selecting transformants thereof resistant to carbendazim, isolating and analyzing the selected transformants for the targeted edits at the first and second GLOI, and selecting and isolating at least one transformant having the targeted edits at the first and second GLOI, wherein the isolated cell comprises the mutant tbbl gene locus and targeted edit at the first and second GLOI.

19. A method for iterative editing of multiple gene loci in a Trichoderma sp. cell comprising: co-transforming a plurality of the Trichoderma sp. cells of claim 16 comprising the mutant tbbl gene locus and targeted edit at the first GLOI with at least two HDRD fragments and at least two sgRNA / Cas complexes, wherein the first HDRD fragment and sgRNA / Cas complex target and edit the mutant tbbl gene locus resulting in a WT tbbl gene locus encoding a native tbbl protein comprising a glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, and the second HDRD fragment and sgRNA / Cas complex target and edit a second GLOI, culturing the plurality of cells in a media comprising diethofencarb and selecting transformants thereof resistant to diethofencarb, isolating and analyzing the selected transformants for the targeted edit at the second GLOI, selecting and isolating at least one transformant having the targeted edit at the second locus, wherein the isolated cell comprises the WT tbbl gene locus and the targeted edits at the first and second GLOI.

20. The method of claim 19, wherein the isolated cell comprising the WT tbbl gene locus and targeted edits at the first and second GLOI is subjected to another round of co-transformation with at least two HDRD fragments and at least two sgRNA / Cas complexes, wherein the first HDRD fragment and sgRNA / Cas complex target and edit the WT tbbl gene locus resulting in a mutant tbbl gene locus encoding a variant tbbl protein comprising a substitution of the glutamic acid (E) residue at amino acid position 198 of SEQ ID NO: 3, and the second HDRD fragment and sgRNA / Cas complex target and edit a third GLOI, culturing the plurality of cells in a media comprising carbendazim and selecting transformants thereof resistant to carbendazim, isolating and analyzing the selected transformants for the targeted edit at the third GLOI, and selecting and isolating at least one transformant having the targeted edit at the third GLOI, wherein the isolated cell comprises the mutant tbbl gene locus and the targeted edits at the first, second and third GLOI.

21. The method of claim 16, wherein tire at least one transformant having the targeted edit at the first GLOI is selected by sequencing the gene locus of the transformant and confirming the targeted edit at the first GLOI.

22. The method of any one of claims 16-21, further comprising co-transforming the plurality of the Trichoderma sp. cells with at least one expression construct encoding a heterologous protein of interest.

23. The method of any one of claims 16-21, wherein Trichoderma sp. cells express one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.

24. A modified Trichodenna sp. cell comprising an introduced polynucleotide expressing a mutant ergl 1 protein, wherein die modified cell can be selected by fluconazole resistance.

25. The modified cell of claim 24, wherein die mutant ergl 1 protein comprises at least 90% identity to the native ergl 1 protein of SEQ ID NO: 85 and comprises at least one amino acid substitution at a position corresponding to position 137, 148, 464 or 467 of SEQ ID NO: 85.

26. The modified cell of claim 25, wherein the substitution at position 137 is a tyrosine (Y) to phenylalanine (F) substitution (Y137F), or the substitution at position 148 is lysine (K) to arginine (R) substitution (K148R), or a lysine (K) to glutamine (Q) substitution (K148Q), or the substitution at position 464 is a glycine (G) to serine(S) substitution, or the substitution at position 467 is a arginine (R) to lysine (K) substitution.

27. The modified cell of claim 24, wherein the mutant erg 11 protein comprises at least 90% identity to the native ergl 1 protein of SEQ ID NO: 86 or SEQ ID NO: 87, and comprises at least one amino acid substitution at a position corresponding to position 132, 464 or 467 of SEQ ID NO: 86 or SEQ ID NO: 87.

28. The modified cell of claim 27, wherein die substitution at position 132 is a tyrosine (Y) to phenylalanine (F) substitution (Y132F),or a tyrosine (Y) to histidine (H) substitution (Y132H), or die substitution at position 464 is glycine (G) to serine (S) substitution (G464S), or the substitution at position 467 is arginine (R) to lysine (K) substitution (R467K).

29. The modified cell of claim 24, wherein the polynucleotide expressing die mutant ergl 1 protein comprises at least 80% identity to a polynucleotide of any one of SEQ ID NO: 79-84.

30. The modified cell of claim 24, constructed from a parental (isogenic) Trichoderma sp. cell which does not comprise an introduced polynucleotide expressing a mutant erg 11 protein, wherein parental cell can be selected by fluconazole sensitivity.

31. The modified cell of claim 24, expressing one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.

32. The modified cell of claim 24, wherein the introduced polynucleotide expressing the mutant erg 11 protein further comprises an operably linked polynucleotide encoding a protein of interest.

33. An isolated polynucleotide encoding a mutant erg 11 protein comprising at least 90% identity to SEQ ID NO: 85 and having at least one substitution at an amino acid position selected from 137, 148, 464 and 467 of SEQ ID NO: 85.34 The polynucleotide of claim 33, wherein tire substitution at position 137 is a tyrosine (Y) to phenylalanine (F) substitution (Y137F), the substitution at position 148 is lysine (K) to arginine (R) substitution (K148R), or a lysine (K) to glutamine (Q) substitution (K148Q), the substitution at position 464 is a glycine (G) to serine (S) substitution, or the substitution at position 467 is a arginine (R) to lysine (K) substitution.

35. The polynucleotide of claim 33, comprising at least about 80% identity to any one of SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84.

36. A method for selecting a transformed Trichoderma sp. cell comprising:transforming a plurality of Trichoderma sp. cells with a polynucleotide comprising a gene coding sequence (CDS) encoding a mutant ergll protein, wherein the gene CDS comprises upstream and downstream homologous recombination (HR) regions flanking the gene CDS to enable integration at a selected gene locus, culturing the plurality of cells in a media comprising fluconazole, selecting and isolating at least one transformed cell thereof resistant to fluconazole, wherein the isolated cell comprises the gene CDS cassette integrated the selected gene locus.

37. The method of claim 36, wherein the transformed polynucleotide further comprises an operably linked polynucleotide encoding a protein of interest.

38. A method for selecting a transformed Trichoderma sp. cell comprising:transforming a plurality of Trichoderma sp. cells with a polynucleotide expression construct encoding a mutant ergll protein, wherein the construct comprises an upstream promoter operably linked to a downstream mutant ergll gene coding sequence (CDS) and upstream and downstream homologous recombination (HR) regions flanking the construct to enable integration at a selected gene locus, culturing the plurality of cells in a media comprising fluconazole, selecting and isolating at least one transformed cell thereof resistant to fluconazole, wherein the isolated cell comprises the ergll construct integrated the selected gene locus.

39. The method of claim 38, wherein the transformed polynucleotide further comprises an operably linked polynucleotide encoding a protein of interest.

40. The method of claim 38, wherein the plurality of cells are co-transformed with a sgRNA / Cas complex dial generates a double strand break al the selected gene locus.

41. The method of claim 36 or 38, wherein the plurality of cells express one or more endogenous proteins of interest and / or one or more heterologous proteins of interest.

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