Conditional regulation of protein function in filamentous fungal cells

Abstract

The present disclosure is generally related to filamentous fungal strains comprising genetic modifications that give rise to recombinant cells having altered phenotypes, wherein such recombinant cells are particularly well-suited for growth in submerged cultures (e.g., for the large-scale production of proteins of interest). Certain embodiments of the disclosure are related to, inter alia, compositions and methods for designing, constructing, testing, screening, growing, fermenting, and the like recombinant filamentous fungal strains expressing intein-modified fitness proteins.

Description

CONDITIONAL REGULATION OF PROTEIN FUNCTION IN FILAMENTOUS FUNGALCELLSTECHNICAL 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. In certain embodiments, the disclosure is related to filamentous fungal strains (cells) comprising genetic modifications that give rise to recombinant (modified) strains having altered phenotypes, wherein such recombinant strains are particularly well-suited for growth in submerged cultures (e.g., for the large-scale production of proteins of interest).CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit to U.S. Provisional Patent Application No. 63 / 627,481, filed January 31, 2024, which is incorporated herein by referenced in its entirety.REFERENCE TO A SEQUENCE LISTING

[0003] The contents of the electronic submission of the text file Sequence Listing, named “NB41668- US-PSP_SequenceListing.xml” was created on January 31, 2024 and is 64,793 bytes in size, which is hereby incorporated by reference in its entirety.BACKGROUND

[0004] Filamentous fungi (e.g.. Aspergillus sp., Penicillium sp., Talaromyces sp.. Fusarium sp., Myceliophthora sp.. Neurospora sp., Candida sp., Trichoderma sp., and the 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, receptors, peptides, etc. and / or metabolites for industrial and commercial applications such as pharmaceutical applications, animal health applications, food applications, beverage applications, laundry, and textile applications, and the like. Filamentous fungi are typically grown in mycelial submerged cultures in bioreactors (fermentors), which bioreactors are adapted to introduce and distribute oxygen and nutrients into the culture medium (i.e., culture broth). For example, the filamentous fungus Trichoderma reesei (T. reeser, an anamorph of the fungus Hypocrea jecorina) is known to be an efficient producer of cellulase enzymes.

[0005] As such, filamentous fungi have been utilized for their ability to produce proteins (e.g., enzymes) which are valuable in the production of commodities such as cellulosic (derived) ethanol, grain processing, detergents, fibers / pulp / paper, food additives, feed additives, textile processing and the like. Likewise, filamentous fungi are utilized for their ability to produce protein biologies (e.g., antibodies, antibody fragments, protein receptors, growth factors and the like). In particular, recombinant gene expression in fungal host strains is a common method for the production of proteins of interest, and as such, protein productivity improvements of fungal host strains are important economic factors of proteinproduction costs. In general, the large-scale expression and secretion of endogenous and / or heterologous proteins of interest in fungal strains can be problematic for countless reasons. For instance, cell propagation and / or cell integrity / survival may be compromised when redirecting the metabolic flux towards producing proteins of interest that are not beneficial for growth or maintenance. Furthermore, expression of some endogenous proteins (e.g., regulatory proteins) may be directly associated with detrimental or toxic effects in the fungal expression host.

[0006] Thus, as appreciated by one of skill in the art, novel methods and compositions for the enhanced production of recombinant proteins in filamentous fungal strains are of significant commercial interest, with the goal of redirecting the maximum metabolic flux towards producing proteins of interest exclusively during the production phase conditions of fermentation, but not during other fermentation conditions (e.g., lag or growth phase conditions). In particular, there remain unmet needs in the art for methods and compositions suitable for conditionally modulating (controlling) the function or activity of proteins (e.g., regulatory proteins) which can negatively influence the growth or survival of filamentous fungal cells.SUMMARY

[0007] As generally set forth and described hereinafter, certain embodiments of the disclosure are related to, inter alia, compositions and methods for designing, constructing, testing, screening, growing, fermenting, and the like recombinant filamentous fungal strains expressing intein-modifled fitness proteins. Certain one or more embodiments of the disclosure are therefore related to methods and compositions for the design and construction of intein-modified proteins. In other related embodiments, the disclosure provides polynucleotide constructs (e.g., expression cassettes) encoding intein-modified fitness proteins of interest. In other embodiments, polynucleotide constructs encoding intein-modified fitness proteins of interest are introduced into filamentous fungal strains of the disclosure. In certain one or more embodiments, it is highly desirable to modulate (control) a fitness protein function or activity (e.g., an enzymatic activity / function, a protein / DNA binding activity / function, etc.) until a suitable point of microbial cell growth, and / or until a suitable fermentation condition (e.g., a desired temperature, a desired cell density, etc.) and the like.

[0008] Thus, certain one or more embodiments of the disclosure provide methods for modulating the function of a fitness protein of interest (FPOI) in a filamentous fungal cell comprising (a) obtaining or constructing a filamentous fungal cell comprising gene encoding a FPOI, wherein the open reading frame (ORF) of the gene encoding the FPOI comprises at least one wild-type or engineered codon selected from the group consisting of TGT, TGC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA and ACG, modifying the ORF by inserting a polynucleotide encoding a functional (auto-splicing) intein immediately upstream of the at least one codon, (b) fermenting the modified cell for a period of time under a non-splicing intein temperature, wherein the FPOI is non-functional under the non-splicing intein temperature, and (c) fermenting the modified cell for a period of time under an intein splicing temperature, wherein the FPOI isfunctional under the intein splicing temperature. For instance, as described below and presented in the Examples section, the function (or activity) of such intein-modified fitness proteins may be modulated by means of one or more specified temperature conditions.

[0009] Certain other embodiments are directed to one or more polynucleotides encoding one or more intein-modified fitness proteins of interest. In certain one or more embodiments, the disclosure therefore provides polynucleotides encoding intein-modified fitness proteins of interest, wherein the intein- modified fitness proteins comprises at least one wild-type or genetically engineered cysteine (Cys), serine (Ser) or threonine (Thr) residue, and an intein inserted immediately N-terminal to the at least one Cys, Ser or Thr residue. In other embodiments the disclosure provides expression cassette comprising an upstream (5') promoter sequence operably linked to a downstream (3') polynucleotide encoding an intein- modified FPOI, optionally comprising a terminator sequence downstream and operably linked to the polynucleotide encoding the intein-modified FPOI. Thus, certain other embodiments are related to recombinant filamentous fungal strains expressing an intein-modified FPOI and one or more endogenous or heterologous proteins of interest.

[0010] As set forth and described hereinafter, recombinant filamentous fungal strains of the instant disclosure expressing an intein-modified FPOI are particularly well-suited for growth in submerged cultures for the large-scale production of commercially relevant proteins of interest (e.g., enzymes, antibodies, receptor proteins, food or flavor proteins, animal feed proteins, and the like).BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0011] SEQ ID NO: 1 is a S. cerevisiae open reading frame (ORF) polynucleotide sequence encoding a wild-type VMA intein (SEQ ID NO: 2). As set forth in SEQ ID NO: 1, the S. cerevisiae wild-type VMA sequence has been codon optimized for expression in T. reesei.

[0012] SEQ ID NO: 2 is the amino acid sequence of the wild-type VMA intein encoded by the ORF of SEQ ID NO: 1.

[0013] SEQ ID NO: 3 is a wild-type T. reesei ade2 polynucleotide sequence encoding the wild-type Ade2 protein of SEQ ID NO: 4.

[0014] SEQ ID NO: 4 is the wild-type Adc2 protein encoded by the ade2 polynucleotide of SEQ ID NO: 3.

[0015] SEQ ID NO: 5 is an ade2 guide RNA (gRNA).

[0016] SEQ ID NO: 6 is the amino acid sequence of a T. reesei variant Ace3-L regulatory protein.

[0017] SEQ ID NO: 7 is a T. reesei did promoter sequence.

[0018] SEQ ID NO: 8 is a wild-type T. reesei ace3 terminator sequence.

[0019] SEQ ID NO: 9 is the polynucleotide sequence of the Ace3L-C80-iWT expression cassette.

[0020] SEQ ID NO: 10 is the polynucleotide sequence of the Ace3L-C80-iF19 expression cassette.

[0021] SEQ ID NO: 11 is the polynucleotide sequence of the Ace3L-C80-iDead expression cassette.

[0022] SEQ ID NO: 12 is the polynucleotide sequence of the Ace3L-C376-iWT expression cassette.

[0023] SEQ ID NO: 13 is the polynucleotide sequence of the Ace3L-C376-iF19 expression cassette.

[0024] SEQ ID NO: 14 is the polynucleotide sequence of the Ace3L-C376- iDead expression cassette.

[0025] SEQ ID NO: 15 is the amino acid sequence of the T. reesei xylanase regulator 1 (Xyrl) protein.

[0026] SEQ ID NO: 16 is the amino acid sequence of the Xyrl binuclear zinc (Z^Cyse) DNA binding domain present at amino acid positions 97-126 of SEQ ID NO: 15.

[0027] SEQ ID NO: 17 is the amino acid sequence of the T. reesei activator of cellulase 2 (Ace2) regulatory protein.

[0028] SEQ ID NO: 18 is the amino acid sequence of the Ace2 binuclear zinc (Z^Cysg) DNA binding domain present at amino acid positions 7-36 of SEQ ID NO: 18.

[0029] SEQ ID NO: 19 is the amino acid sequence of the Ace3 binuclear zinc ('ZmCyse) DNA binding domain present at amino acid positions 76-108 of SEQ ID NO: 6.

[0030] SEQ ID NO: 20 is the amino acid sequence of a S. cerevisiae Gal4 binuclear zinc (Zn2Cys6) DNA binding domain.

[0031] SEQ ID NO: 21 is the amino acid sequence of the T. reesei carbon catabolite repressor 1 (Crel) regulatory protein.

[0032] SEQ ID NO: 22 is the amino acid sequence of the Crel zinc finger (C2H2) DNA binding domain 1 present at amino acid positions 57-84 of SEQ ID NO: 21.

[0033] SEQ ID NO: 23 is the amino acid sequence of the Cre 1 zinc finger (C2H2) DNA binding domain 2 present at amino acid positions 85-114 of SEQ ID NO: 21.

[0034] SEQ ID NO: 24 is an ORF sequence encoding the temperature-sensitive iF19 (VMA variant) intein of SEQ ID NO: 25. As set forth in SEQ ID NO: 24, the ORF sequence has been codon optimized for expression in T. reesei.

[0035] SEQ ID NO: 25 is the amino acid sequence of the temperature-sensitive iF19 (VMA variant) intein encoded by the ORF of SEQ ID NO: 24.

[0036] SEQ ID NO: 26 is a polynucleotide coding sequence (CDS) encoding the variant Nik 1M743Tprotein of SEQ ID NO: 31.

[0037] SEQ ID NO: 27 is the amino acid sequence of the variant NiklM743Tprotein.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 shows a polynucleotide (DNA) sequence (FIG. 1A; SEQ ID NO: 1) encoding the wildtype (WT) S. cerevisiae VMA intein (FIG. IB; SEQ ID NO: 2). More particularly, as presented in FIG. 1A (SEQ ID NO: 1) the DNA sequence encoding the WT VMA intein has been codon optimized for expression in T. reesei filamentous fungal cells. As described in the Examples, Applicant constructed certain variant VMA intein sequences derived from the WT VMA intein (“iWT”, FIG. IB SEQ ID NO: 2), such as the variant VMA intein sequences named “iF19” and “iDead”. In particular, as shown in FIG. IB, the iF19 variant comprises a W157R and a G219R substitution relative to the WT intein (which substituted positions W157 and G219 are indicated in with a bold underlined amino acid residue at positions W and G), and theiDead variant comprises a deletion of amino acid residue position N254 relative to the wild-type intein (which deleted N254 residue is indicated with a bold grey shadow amino acid residue position t§254).

[0039] Figure 2 presents a T. reesei ade2 polynucleotide coding sequence (SEQ ID NO: 3), wherein intron sequences are indicated with grey boxes and the labels “_intein#l_” and “_intein#2_” indicate the positions of intein insertion (z.e., position 1 at Cl 83 and position 2 at C242).

[0040] Figure 3 shows the amino acid sequence of Ade2 protein, wherein the labels “_intein#l_” and “_intein#2_” indicate the positions of intein insertion (i.e., SEQ ID NO: 4; position 1 at C183 and SEQ ID NO: 4; position 2 at C242).

[0041] Figure 4 shows complementation assays using ade2 as a marker (FIG. 4A) or as a reporter (FIG. 4B). As presented in FIG. 4A, radial outgrowth of mycelia on agar plates of strains harboring intein variants iWT, iF4, iF19 and iDead inserted into the coding sequence of ade2 at position 1 (codon encoding C183) and position 2 (codon encoding C242) are shown. FIG. 4B shows a temperature switching assay with ade2 as a reporter. Radial outgrowth of mycelia on agar plates supplemented with adenine, with intein variants inserted into the coding sequence of ade2 at position 1 (Cl 83) and position 2 (C242).

[0042] Figure 5 presents the amino acid sequence of the Ace3L protein (SEQ ID NO: 6). As shown in FIG. 5 and further described below in the Examples, the cysteine (C) residues at positions C80 and C376 (SEQ ID NO: 6) indicate the positions in which the intein sequences were inserted (i.e., immediately upstream (5') of the C80 or C376 residue).

[0043] Figure 6 presents a schematic representation of vector pYL63. As shown in FIG. 6, the pYL63 vector encodes the Ace3L-intein namedllAce3L-C80-iF19” , wherein the Ace3L protein comprises an insertion of the iF19 intein sequence immediately upstream (5') of the cysteine residue at position 80 of the Ace3L protein (see FIG. 5).

[0044] Figure 7 shows an SDS-PAGE of the modified LT329 (Acc3L-C80-iF19) strain supernatants, wherein the cells were grown in defined medium supplemented with either 2.5% glucose / sophorose (sop) or 2.5% glucose (glu) in microtiter plates at the indicated temperatures. Equal volumes of culture supernatant were loaded in each lane. “M” is molecular weight marker and “KD” is kilodalton.

[0045] Figure 8 shows the relative growth rates from a large-scale fermentation of the wild-type parental T4abc (Parental control), modified LT167 (T4abc Ace3L), and modified LT329 (T4abc Ace3L-C80- iF19) strains under a non-permissive splicing temperature of intein iF19. The growth rate of the parental stain was artificially set as 100.

[0046] Figure 9 shows the relative total protein yields from a large-scale fermentation of the wild-type parental T4abc (Control), modified LT 167 (T4abc Ace3L), and modified LT329 (T4abc Ace3L-C80-iF19) strains under inducing (glucose / sophorose feed) conditions. The total protein yield of the parental stain was artificially set as 100.

[0047] Figure 10 shows the relative total protein yields from a large-scale fermentation of the modified LT329 (T4abc Ace3L-C80-iF19) strains under inducing (glucose / sophorose feed) conditions or non-inducing (glucose feed) conditions. The total protein yield under inducing (glucose / sophorose feed) conditions was artificially set as 100.

[0048] Figure 11 is a schematic representation of vector pYL71 (sdil-ace3-C80-iF19).

[0049] Figure 12 shows an SDS-PAGE of supernatants from a parental T. reesei glucoamylase (GA) reporter strain (Control) and a modified LT351 (Ace3L-C80-iF19) GA reporter strain, wherein the control and modified strains were grown in defined medium supplemented with either 2.5% glucose / sophorose (“Sop”, inducing condition) or 2.5% glucose (“Glu”, non-inducing condition) in microtiter plate. Equal volumes of culture supernatant were loaded in each lane. M is molecular weight marker and KD is kilodalton. The relative total protein concentrations of the culture supernatants are listed at the bottom of each corresponding lane, the concentration from parental strain grown on glucose / sophorose was arbitrarily set at 100.

[0050] Figure 13 shows the relative growth rates from a large-scale fermentation of the parental T. reesei GA reporter strain, the modified LT 182 (Ace3L) GA reporter strain and the modified LT351 (Ace3L- C80-iF19) GA reporter strain under a non-permissive splicing temperature of intein iF19. The growth rate of the parental stain was artificially set as 100.

[0051] Figure 14 shows the relative protein yields from a large-scale fermentation of the parental T. reesei GA reporter strain, the modified LT182 (Ace3L) GA reporter strain and the modified LT351 (Ace3L-C80-iF19) GA reporter strain under inducing (glucose / sophorose feed). The total protein yield of the parental stain was artificially set as 100.

[0052] Figure 15 shows the relative protein yields from a large-scale fermentation of the modified LT351 (Ace3L-C80-iF19) GA reporter strain, under inducing (glucose / sophorose feed) or non-inducing (glucose feed) conditions. The total protein yield under inducing (glucose / sophorose feed) conditions was artificially set as 100.

[0053] Figure 16 shows the polynucleotide coding sequence of T. reesei niklM743T(SEQ ID NO: 26), wherein intron sequences are indicated with grey boxes, the codon encoding M743T is underlined in bold and the label “_intein_” indicates the position of intein insertion (i.e., upstream of the codon encoding C1065).

[0054] Figure 17 shows the amino acid sequence of the T. reesei NiklM743Tprotein (SEQ IDNO: 27), wherein M743T is underlined in bold and the label “_intein_” indicates the position of intein insertion (i.e., position Cl 065).DETAILED DESCRIPTION

[0055] As described herein, certain embodiments of the disclosure provide, inter alia, compositions and methods for designing, constructing, testing, screening, growing, fermenting, and the like recombinant filamentous fungal strains expressing intein-modified fitness proteins of interest. Certain one or more embodiments of the disclosure are therefore related to methods and compositions for the design and construction of intein-modified proteins. In related embodiments, the disclosure provides polynucleotideconstructs (e.g., expression cassettes) encoding intein-modified fitness proteins. In other embodiments, polynucleotide constructs encoding intein-modified proteins are introduced into filamentous fungal strains of the disclosure. Certain embodiments are particularly related to recombinant filamentous fungal strains expressing / producing temperature-sensitive intein-modified fitness proteins. For example, in certain one or more embodiments, it is highly desirable to modulate a fitness protein function or activity (e.g., an enzymatic activity, a protein / DNA binding activity, etc.) until a suitable point of microbial cell growth, and / or until a suitable fermentation condition (e.g., a desired temperature, a desired cell density, etc.) and the like.I. DEFINITIONS

[0056] Prior to describing the present strains, compositions and methods in further detail, the following terms and phrases are defined. Terms not defined should be accorded their ordinary meaning as used and known to one skilled in the art.

[0057] All publications and patents cited in this specification are herein incorporated by reference.

[0058] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other 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.

[0059] 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 substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the 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 the pH value is specifically defined otherwise.

[0060] The headings provided herein are not limitations of the various aspects or embodiments of the present compositions and methods which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0061] 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 dictatesotherwise. 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.

[0062] 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 or “proviso”. For example, in certain embodiments, the proviso “wherein the medium does not comprise an inducing substrate” may be used to exclude inducing substrates such as cellulose, lactose, gentibiose, sophorose and the like.

[0063] 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 nonmandatory or optional component(s).

[0064] It is also noted that the term “consisting of,” as used herein, means “including and limited to”, the component(s) after the term "consisting of’. The component(s) after the term “consisting of’ are therefore required or mandatory, and no other component(s) are present in the composition.

[0065] 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 other several embodiments without departing from the scope or spirit of the resent 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.

[0066] As used herein, the term “Ascomycete fungal cell” refers to any organism in the Division Ascomycota in the Kingdom Fungi. Examples of Ascomycctcs fungal cells include, but arc not limited to, filamentous fungi in the subphylum Pezizomycotina, such as Trichoderma sp., Aspergillus sp., Myceliophthora sp., Penicillium sp., and the like.

[0067] 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, and Trichoderma species.

[0068] In certain embodiments, a filamentous fungus is a Trichoderma sp. cell (strain) including, but not limited to, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei and Trichoderma viride. As known to one skilled in the art, Trichoderma reesei was previously classified as “ Hypocrea jecorina”.

[0069] In other embodiments, a filamentous fungus is an Aspergillus sp. cell (strain) such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus clavatus. Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Aspergillus terreus.

[0070] As used herein, exemplary parental Trichoderma reesei strains include, but are not limited to, T. reesei strain QM6a (ATCC® 13631), T. reesei strain RL-P37 (NRRL Deposit No. 15709) and T. reesei strain RUT-C30 (ATCC® 56765); exemplary parental Aspergillus niger strains include, but are not limited to, A. f tiger strain ATCC® 1015; exemplary parental Aspergillus oryzae strains include, but are not limited to A. oryzae strain RIB40 (ATCC® 42149); and exemplary parental Myceliophthora thermophila strains include, but are not limited to, M. thermophila strain ATCC® 42464.

[0071] For example, Trichoderma strains Rut-C30 and RL-P37 are mutagenized derivatives of Trichoderma natural isolate QM6a (Le Crom et al., 2009; Sheir-Neiss and Montenecourt, 1984), with strain NG14 being the last common ancestor. In certain aspects of the disclosure, an exemplary filamentous fungal strain is derived / obtained from T. reesei strain RL-P37 and comprises a deletion of the T. reesei pyr2 gene (URA5, UniProt Entry Identifier P21846) (abbreviated hereinafter, “Apyr2”), as generally described by Sheir-Neiss and Montenecourt (1984) and PCT Publication No. WO2011 / 153449.

[0072] 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.

[0073] As used herein, the 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-natural cell 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.

[0074] “Recombination”, “recombining” or generating a “recombined” nucleic acid is generally the assembly of two or more nucleic acid fragments wherein the assembly gives rise to a chimeric gene.

[0075] 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.

[0076] 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) coding sequence (CDS) may encode a regulatory protein,a structural protein, commercially important industrial proteins or peptides, such as enzymes (e.g., proteases, mannanases, xylanases, amylases, glucoamylases, cellulases, oxidases, phytases, lipases) and the like. The gene of interest may be a naturally occurring gene, a mutated (modified) gene or a synthetic gene.

[0077] 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 will generally 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, the 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.

[0078] 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.

[0079] 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 (CDS) if it affects the 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 (CDS, e.g., 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 are 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.

[0080] As used herein, the phrases “lignocellulosic degrading enzymes”, “cellulase enzymes”, and “cellulases” are used interchangeably, and include glycoside hydrolase (GH) enzymes such as cellobiohydrolases, xylanases, endoglucanases, and [3-glucosidases, that hydrolyze glycosidic bonds of cellulose (hemi-cellulose) to produce sugars (e.g., glucose, xylose, arabinose, etc.).

[0081] As used herein, “endoglucanase” proteins may be abbreviated as “EG”, “cellobiohydrolase” proteins may be abbreviated “CBH”, “ -glucosidase” proteins may be abbreviated “BG” and “xylanase” proteins may be abbreviated “XYL”. Thus, as used herein, a gene (or ORF) encoding a EG protein maybe abbreviated “eg”, a gene (or ORF) encoding a CBH protein may be abbreviated “cbh”, a gene (or ORF) encoding a BG protein may be abbreviated “bg”, and a gene (or ORF) encoding a XYL protein may be abbreviated “xyl”. In certain embodiments, cellobiohydrolases include enzymes classified under Enzyme Commission No. (EC 3.2.1.91), endoglucanases include enzymes classified under EC 3.2.1.4, endo- (3-1,4-xylanases include enzymes classified under EC 3.2.1.8, (3-xylosidases include enzymes classified under EC 3.2.1.37, and P-glucosidases include enzymes classified under EC 3.2.1.21.

[0082] As used herein, a “cellulase gene promoter” includes, but is not limited to, a cellobiohydrolase (cbh) gene promoter sequence, an endoglucanase (eg) gene promoter sequence, a P-glucosidase (bg) gene promoter sequence, a xylanase (xyl) gene promoter sequence, and the like.

[0083] The term “glucoamylase” (abbreviated, “GA”) refers to the amyloglucosidase class of enzymes (E.C. 3.2.1.3, glucoamylase, 1,4-alpha-D-glucan glucohydrolase). In certain aspects, the disclosure describes filamentous fungal strains expressing heterologous copies of a glucoamylase (GA) gene. More particularly, the heterologous GA protein set forth in Examples 4 and 5 (below), has been described in US Patent No. 7,413,879 (incorporated herein by reference in its entirety).

[0084] As used herein, the terms “modification” and “genetic modification” are used interchangeably and include: (a) the introduction, substitution, or removal of one or more nucleotides in a gene, or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the gene, (b) a gene disruption, (c) a gene conversion, (d) a gene deletion, (e) the down-regulation and / or up-regulation of a gene, (f) specific mutagenesis and / or (g) random mutagenesis of any one or more the genes / DNA sequences disclosed herein.

[0085] As used herein, the phrases “modified filamentous fungal cell(s)”, “mutant filamentous fungal cell(s)”, “recombinant fungal cell(s)”, “modified filamentous fungal strain(s)”, and the like may be used interchangeably and refer to filamentous fungal cells that arc derived (obtained) from a parental (or control) filamentous fungal cell belonging to the Pezizomycotina subphylum. For example, a “modified” filamentous fungal cell may be derived from a parental (or control) filamentous fungal cell, wherein the modified cell comprises at least one genetic modification which is not found in the parental (or control) cell.

[0086] As used herein, the term “intein” (intervening protein) has the same meaning as used in the art. In general, inteins carry out a process known as protein splicing, which is a multi-step biochemical reaction comprised of both the cleavage and formation of peptide bonds (Xu and Perler, 1996; Shah and Muir, 2014). More particularly, as described by Shah and Muir (2014), an intein carries out a unique auto-processing event known as protein splicing, in which the intein excises itself out from a larger precursor polypeptide through the cleavage of two (2) peptide bonds and, in the process, ligates the flanking extein (external protein) sequences through the formation of a new peptide bond.

[0087] As used herein, an intein with differential kinetics of protein splicing dependent on a desired temperature (condition) may be referred to herein as an “auto-splicing intein”. For example, in certain embodiments, a polynucleotide (DNA) sequence encoding a protein of interest (POI) is geneticallymodified by interesting a nucleic acid (DNA) sequence encoding an auto-splicing intein immediately upstream (5') of a TGT or TGC codon present (or engineered into) the DNA sequence encoding the POL

[0088] 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 function / activity, and the like, and which has not been mutagenized, truncated, or otherwise modified to abolish or reduce that function / activity.

[0089] As used herein, a “fitness protein” or “fitness protein of interest” (abbreviated, “fitness POI” or “FPOI”) refers to any endogenous protein or heterologous protein that provides a favorable trait or benefit to the host cell producing the FPOI under industrial or commercially relevant conditions, or refers to any endogenous protein or heterologous protein that avoids an unfavorable trait to the host cell expressing the FPOI under industrial or commercially relevant conditions. In certain embodiments, a FPOI refers to a regulatory protein as defined below. In other embodiments, a FPOI is selected from the group consisting of a xylanase regulator 1 (Xyrl) protein, an activator of cellulase 2 (Ace2) protein, an activator of cellulase 3 (Ace3) protein, a variant Ace3-L protein, an activator of cellulase 4 (Ace4) protein, a carbon catabolite repressor 1 (Crel) protein, a histidine kinase (Niki ) protein, a GTP:a-D-mannose-l-phosphate guanyltransferase (Mpgl) protein, a beta-glucosidase regulator (BglR) protein, a Trire2_82512 protein, a Trire2_62386 protein, a hydrophobin Hfblprotein and a hydrophobin Hfb2 protein.

[0090] As used herein, a “favorable host cell trait or benefit” includes, but is not limited to, high protein yields, high activity yields, low viscosity, and desired glycosylation patterns. An “unfavorable host cell trait or benefit” includes, but is not limited to, low growth rates, low protein yields, low activity yields, high viscosity, undesired glycosylation patterns, protein degradation and foaming.

[0091] As used herein, a “regulatory protein” includes, but is not limited to, transcription factor proteins such as transcriptional activator proteins and transcriptional repressor proteins, protein kinases, phosphatases, guanyltransferases, glycosyltransferase, glycosidase, proteins involved in foaming (e.g., hydrophobins), proteins involved in histone modification or chromatin remodeling, and similar functions or activities related to regulation of gene or protein activity.

[0092] As used herein, phrases such as “modulating a fitness protein of interest (FPOI) function” in a filamentous fungal cell, “modulating a FPOI activity” in a filamentous fungal cell, “modulating the function of a FPOI” in a filamentous fungal cell, “modulating the activity of a FPOI” in a filamentous fungal cell and the like, means modifying the gene coding sequence (CDS / ORF) of a gene encoding the FPOI in the fungal cell, wherein the gene CDS is modified by introduction (insertion) of a functional (i.e., auto-splicing) temperature-sensitive intein sequence therein. As generally set forth below in the Examples, the function or activity of intein-modified fitness proteins can be modulated (e.g., functional FPOI vs. non-functional FPOI) according to the fermentation (cultivation) conditions of the filamentous fungal strains described herein. More particularly, as described below, an intein (DNA) sequence may be introduced (inserted) into a gene CDS having at least one TGT, TGC, TCT, TCC, TCA, TCG,AGT, AGC, ACT, ACC, ACA or ACG codon, and screened for optimal intein splicing conditions. If the gene CDS encoding the FPOI does not comprise at least one TGT, TGC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA or ACG codon, one of skill in the art may readily design, engineer and introduce one or more TGT, TGC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA or ACG codons into the gene CDS.

[0093] For instance, as presented in the Examples section below, a filamentous fungal knock-out strain (Aat / e2) was constructed to test / screen candidate intein sequences for conditional (z.e., temperature) splicing activity. More specifically, the parental (control) knock-out strain (Aaz / e2) was assessed by complementation assays using intein-harboring ade2 expression cassettes. As generally described below (Example 1), a person of skill in the art may readily modify and adapt the intein screening to identity other candidate intein sequences. Likewise, as further described below (Section III), the temporal control of a gene CDS (e.g., Ace3-L) modified by insertion of a candidate intein sequence may be tested, screened and identified as generally described in Examples 2-5.

[0094] 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 prevents a host cell from producing a functional gene product (e.g., a functional protein). Exemplary methods of gene disruptions include complete or partial deletion of any portion of a gene, including a polypeptide-coding sequence, 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 / inactivate the target gene(s) and substantially reduce or prevent the production of the functional gene product (i.e., the functional protein).

[0095] As used herein, “deletion of a gene,” refers to its removal from the genome of a host cell. Where a gene includes control elements (e.g., enhancer elements) that are not located immediately adjacent to the coding sequence of a gene, deletion of a gene refers to the deletion of the coding sequence, and optionally adjacent enhancer elements, including but not limited to, for example, promoter and / or terminator sequences.

[0096] As used herein, a “heterologous gene” refers to polynucleotide (DNA) sequences having at least a portion of the sequence which is not native or existing in a native form to the cell in which it is introduced and / or expressed.

[0097] As used herein, a “heterologous nucleic acid construct” or “heterologous DNA sequence” has a portion of the sequence which is not native or existing in a native form to the cell in which it is expressed.

[0098] As used herein, a “heterologous protein” is encoded by a heterologous gene, a heterologous nucleic acid (polynucleotide) sequence, a heterologous DNA sequence, and the like.

[0099] Thus, in certain embodiments, a heterologous DNA sequence encoding a protein of interest (POI) is introduced (e.g., transformed) into a filamentous fungal cell (strain). For example, a heterologous geneconstruct encoding a POI may be introduced into the filamentous fungal cell (strain) before, during, or after performing other genetic modification described herein.

[0100] Heterologous, with respect to a control sequence refers to a control sequence (e.g., promoters, enhancers, terminators) that does not function in nature to regulate the same gene the expression of which it is currently regulating. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell, by infection, transfection, transformation, microinjection, electroporation, or the like. A “heterologous” nucleic acid construct may contain a control sequence / DNA coding sequence combination that is the same as, or different from a control sequence / DNA coding sequence combination found in the native cell.

[0101] As used herein, the term “coding sequence” or “CDS” refers to a polynucleotide sequence, which directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of the CDS are generally determined by an open reading frame (ORF), which usually begins with a start codon (ATG). The coding sequence typically includes DNA, cDNA, and recombinant nucleotide sequences. For example, an ORF generally refers to polynucleotide sequence (whether naturally occurring, non-naturally occurring, or synthetic) comprising an uninterrupted reading frame consisting of (i) an initiation codon, (ii) a series of codons representing amino acids of the encoded protein product, and (iii) a termination codon, the ORF being read (or translated) in the 5' to 3' direction.

[0102] As used herein, the term “DNA construct” or “expression construct” refers to a nucleic acid sequence, which comprises at least two DNA polynucleotide fragments. A DNA or expression construct can be used to introduce nucleic acid sequences into a fungal host cell. The DNA may be generated in vitro (e.g., by PCR) or any other suitable techniques. In some preferred embodiments, the DNA construct comprises a sequence of interest (e.g., encoding a protein of interest). In certain embodiments, a polynucleotide sequence of interest is operably linked to a promoter and / or terminator. In some embodiments, the DNA construct further comprises at least one selectable marker. In further embodiments, the DNA construct comprises sequences homologous to the host cell chromosome. In other embodiments, the DNA construct comprises non-homologous sequences to the host cell chromosome.

[0103] As used herein, a “flanking sequence” refers to any sequence that is either upstream or downstream of the sequence being discussed (e.g., for genes A-B-C, gene B is flanked by the A and C gene sequences). In certain embodiments, the incoming sequence is flanked by a homology box on each side. In another embodiment, the incoming sequence and the homology boxes comprise a unit that is flanked by stuffer sequence on each side. In some embodiments, a flanking sequence is present on only a single side (either 3' or 5'), but in preferred embodiments, it is on each side of the sequence being flanked. The sequence of each homology box is homologous to a sequence in the filamentous fungal chromosome. These sequences direct where in the filamentous fungal chromosome the new construct gets integrated and what part of the chromosome will be replaced by the incoming sequence.

[0104] As used herein, the term “down-regulation” of gene expression includes any methods that result in lower (down-regulated) expression of a gene.

[0105] The term “vector” is defined herein as a polynucleotide designed to carry nucleic acid sequences to be introduced into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage or virus particles, DNA constructs, cassettes and the like. Expression vectors may include regulatory sequences such as promoters, signal sequences, a coding sequences and transcription terminators.

[0106] An “expression vector” as used herein means a DNA construct comprising a coding sequence that is operably linked to suitable control sequences capable of effecting expression of a protein in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0107] As used herein, the term “secretory signal sequence” denotes a DNA sequence that encodes a polypeptide (i.e., a “secretory peptide”) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

[0108] As used herein, the term “isolated” or “purified” refers to a filamentous fungal cell, a nucleic acid or a polypeptide that is removed from at least one component with which it is naturally associated.

[0109] As used herein, the terms “protein of interest” or “POI” refer to a polypeptide that is desired to be expressed in a filamentous fungal cell. Such a protein can be an enzyme, a substrate -binding protein, a surface-active protein, a structural protein, and the like, and can be expressed at high levels, and can be for the purpose of commercialization.

[0110] As used herein, the term “increased productivity” and variations thereof mean an increase of at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% (e.g., greater than 20%) in the production of a protein by a modified (recombinant) filamentous fungal cell relative to a control (parental) filamentous fungal cell, when cultivated under the same conditions.

[0111] As used herein, the term “increased amount” when used in phrases such as a recombinant cell “produces an ‘increased amount’ of a protein”, and variations thereof mean an increase of at least o.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% (e.g., greater than 20%) in the amount of a protein produced by a modified (recombinant) filamentous fungal cell relative to a control (parental) filamentous fungal cell, when cultivated under the same conditions.

[0112] 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 bynon-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component). Also included within 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.

[0113] As used herein, functionally and / or structurally similar proteins are considered to be “related proteins.” Such 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 homologs determined by primary sequence analysis, determined by secondary or tertiary structure analysis, or determined by immunological cross-reactivity.

[0114] As used herein, the phrase “substantially free of an activity,” or similar phrases, means that a specified activity is either undetectable in an admixture or present in an amount that would not interfere with the intended purpose of the admixture.

[0115] As used herein, the term “derivative polypeptide” refers to a protein which is derived or derivable from a protein by addition of one or more amino acids to either or both the N- and C-terminal end(s), substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, deletion of one or more amino acids at either or both ends of the protein or at one or more sites in the amino acid sequence, and / or insertion of one or more amino acids at one or more sites in the amino acid sequence. The preparation of a protein derivative can be achieved by modifying a DNA sequence which encodes for the native protein, transformation of that DNA sequence into a suitable host, and expression of the modified DNA sequence to form the derivative protein.

[0116] Related (and derivative) proteins include “variant proteins.” Variant proteins differ from a rcfcrcncc / parcntal protein (e.g., a wild-type protein) by substitutions, deletions, and / or insertions at a small number of amino acid residues. The number of differing amino acid residues between the variant and parental protein can be one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more amino acid residues. Variant proteins can share at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or even at least about 99%, or more, amino acid sequence identity with a reference protein. A variant protein can also differ from a reference protein in selected motifs, domains, epitopes, conserved regions, and the like.

[0117] As used herein, the term “homologous” protein refers to a protein that has similar activity, function and / or structure to a reference protein. It is not intended that homologs necessarily be evolutionarily related. Thus, it is intended that the term encompass the same, similar, or corresponding protein(s) (i.e., in terms of structure and function) obtained from different organisms. In some embodiments, it is desirable to identify a homolog that has a quaternary, tertiary and / or primary structure similar to the reference protein.

[0118] The degree of homology between sequences can be determined using any suitable method known in the art (e.g., programs such as GAP, BESTFIT, FASTA, and TFASTA). For purposes of the present disclosure, the degree of identity between two amino acid sequences is determined using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1970) as implemented in the Needle program of the EMBOSS package, preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:(Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0119] For purposes of the present disclosure, the degree of identity between two deoxyribonucleotide (DNA) sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package, preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0120] 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% identity, at least about 50% identity, at least about 60% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or even at least about 99% identity, or more, compared to the reference (z.e., wild-type) sequence. Sequence identity can be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters.

[0121] As used herein, the terms “inducer”, “inducers”, or “inducing substrates” are used interchangeably and refer to any compounds that cause filamentous fungal cells to produce “increased amounts” of secreted protein. Examples of inducing substrates include, but are not limited to, sophorose, lactose, gentibiose and cellulose.

[0122] As used herein, the term “induction” refers to the increased transcription of a gene resulting in the synthesis of a protein in a filamentous fungal cell at a markedly increased rate in response to the presence of the “inducer” ( / .<?., inducing substrate). For example, to measure the “induction” of a gene encoding a protein, modified cells are treated with a candidate inducing substrate (inducer) and are compared vis-a-vis to parental (control) cells which are not treated with the inducing substrate (inducer). Thus, the untreated (control) cells are assigned a relative protein activity value of 100%, wherein induction of the gene encoding the protein in the modified cells is achieved when the activity value (i.e..relative to the control cells) is greater than 100%, (e.g., 100.5%), greater than 101% to 109%, greater than 110%, greater than 150%, greater than 200-500% (i.e.. relative to the control), or higher.

[0123] As used herein, “aerobic fermentation” refers to growth in the presence of oxygen.

[0124] As used herein, the term “cell broth” refers collectively to medium and cells in a liquid / sub merged culture.

[0125] As used herein, the term “cell mass” refers to the cell component (including intact and lysed cells) present in a liquid / submerged culture. Cell mass can be expressed in dry or wet weight.

[0126] It will be understood that the methods of the present disclosure are not limited to a particular order for obtaining the modified, mutant, or recombinant filamentous fungal cell (strain).II. INTEINS

[0127] As generally understood in the art, inteins are (protein) auto-processing domains found in organisms from all domains of life. Inteins carry out a process known as protein splicing, which is a multi-step biochemical reaction comprised of both the cleavage and formation of peptide bonds (Xu and Perler, 1996; Shah and Muir, 2014). For example, as described by Shah and Muir (2014), an intein (intervening protein) carries out a unique auto-processing event known as protein splicing, in which it excises itself out from a larger precursor polypeptide through the cleavage of two (2) peptide bonds and, in the process, ligates the flanking extein (external protein) sequences through the formation of a new peptide bond. This rearrangement occurs post-translationally (or possibly co-translationally), as intein genes are found embedded in frame within other protein-coding genes. More particularly, intein- mediated protein splicing is spontaneous, and requires no external factor or energy source, only the folding of the intein domain (Shah and Muir, 2014).

[0128] Protein splicing elements were first described in 1990 as in-frame insertions in the Saccharomyces cerevisiae VMA gene that were unrelated to the sequence of homologous ATPases (e.g., see Hirata et al., 1990). For instance, Perler et al. (1997) compiled a list of all inteins (protein splicing elements) whose sequences were published, or available from on-line sequence databases at the time (i.e., September 1996). As generally described in Perler et al. (1997; Table 1), suitable intein sequences may be derived from eukaryotic organisms, eubacterial organisms, and archaeal organisms. Likewise, suitable variant (mutant) intein sequences may be derived from known intein sequences, newly identified intein sequences, combinations thereof and the like.

[0129] In certain aspects, one of skill may also use site scanning mutagenesis, and other useful methods to screen and identify candidate (mutagenized) intein (variant) sequences with differential kinetics of protein splicing (herein, “auto-splicing”) depending on a desired condition (e.g., a “temperature condition”, a “light (wavelength) condition”, a “re-dox condition”, etc. . Temperature-sensitive alleles based on conditionally active inteins have been described by Zeidler et al., 2004 and Tan et al., 2009. For example, as generally summarized in Tan et al. (2009), an intein is a self-excising stretch of amino acids, which are removed during protein maturation. The intein (acting like a “switch”), splices itselfonly at permissive conditions, to generate an intact host protein. At non-permissive conditions, the intein fails to splice, and remains within the host protein, leading to the inactivation of that protein (Tan et al., 2009). In certain aspects, suitable variant (mutant) intein sequences may be derived from known intein sequences, wherein the intein sequences are mutagenized and screened for a desirable intein splicing condition as generally set forth in Zeidler et al. (2004), Tan et al. (2009), and / or using other suitable methods know in the art.

[0130] More recently, relevant concepts and mechanisms of intein-mediated protein splicing have been reviewed. For example, as described in Mills et al. (2014), protein splicing occurs when the intein and the first C-extein residue are embedded in a heterologous host protein, inefficient splicing can result in off-pathway single splice site cleavage, splicing can be controlled, and splicing proceeds through a slowly migrating branched intermediate with two N termini (see, Mills et al. (2014); Figure 1 and Figure 2). In particular, Mills et al. (2014) speculate that inteins have evolved to tightly regulate the steps of splicing, which is essential as inteins interrupt highly conserved domains of proteins important to their host organisms, including, e.g., DNA polymerases and helicases. Inteins can be engineered to be controlled or sensitive to changes in light, pH, temperature, or redox state, and to be responsive to the addition of small molecules (Mills et al., 2014). For instance, US Patent Publication No. US2004 / 0091966 generally describes methods for controlling a target polypeptide bioactivity in plants and animals by use of an intein. US Patent No. 8,420,387 generally describes methods related to intein modified enzymes (xylanase or a cellulase).

[0131] As described herein and set forth below in the Examples, Applicant has surprisingly observed that inteins can be used to modulate (control) the functionality (e.g., activity) of fitness proteins of interest in filamentous fungal cells. More particularly, as set forth in Example 1, Applicant screened inteins for a desired “conditional” (temperature) splicing activity in filamentous fungal cells during fermentation. For example, certain inteins were screened for a desired conditional splicing activity in a Trichodenna reesei (knock-out) strain (Aacfe2) using complementation assays, wherein expression cassettes of the adeZ gene having an intein insertion were introduced into the T. reesei adeZ strain (Example 1). For instance, intein (DNA) sequences obtained from DNA synthesis were codon-optimized for expression in Trichoderma and inserted immediately upstream (5') of a cysteine (C) residue encoded by the adeZ gene (e.g., see FIG. 2, “intein#!” and “inteintfZ”). In particular, as shown in FIG. 3, the Ade2 protein (SEQ ID NO: 4) encoded by the adeZ gene (SEQ ID NO: 3) has two (2) suitable cysteine amino acid positions for intein insertion, which are presented as cysteine positions C183 and C242.

[0132] As further described in Example 1, in assays using adeZ as an auxotrophic marker, the iWT colonies grew comparable to the complementation colonies, indicating the generation of functional Ade2 protein via intein (iWT) splicing (FIG. 4A), wherein none of the iDead colonies grew, confirming that the non-spliced (iDead) intein inserted in the Ade2 protein results in the disruption of Ade2 function (i.e., Ade2 enzymatic activity). The colonies expressing the variant intein iF4 also showed no growth, suggesting the iF4 mutations likely caused slow, partial, or even complete inhibition of intein splicing,whereas the iF19 colony growth was dependent on the incubation temperature (FIG. 4A). For example, at 25.0°C the iF19 colonies grew comparable to iWT and Complementation colonies, while at 32.5°C the iF19 colonies showed no growth, demonstrating temperature-sensitive splicing of the iF19 intein variant at the permissive temperature of 25°C, whereas the iF4 intein variant showed no growth at 25°C, indicating the iF4 intein does not splice at the permissive temperature (FIG. 4A).

[0133] In an additional reporter assay, color switching of a parental population of iF19 cells was demonstrated by following the radial growth of mycelia discs on agar plates (FIG. 4B). In particular, the plates were inoculated and incubated in quadruplicate, either at 25.0°C or at 32.5°C (as described in Example 1), with switching between the two (2) indicated temperatures after three (3) days as shown in FIG. 4B (e.g., either 25.0°C - 32.5°C - 25.0°C, or 32.5°C -«■ 25.0 °C - 32.5°C). For example, in iF19 assays, colonies with alternating white and dark (red pigment) rings appeared (FIG. 4B), demonstrating the temperature dependency of the conditional intein splicing activity. Interestingly, mycelium rings that formed at previous incubation temperatures, did not change in color retroactively, suggesting that high metabolic activity is required for changes in coloration of the ade2 reporter system.

[0134] In particular, based on the conditional temperature screening of the intein -modified ade2 gene constructs expressed in T. reesei cells (Example 1), it was observed that the strain expressing the iF4 intein-modified Ade2 protein does not auto-splice during the growth phase temperatures of 7. reesei cells (e.g., temperatures of about 30°C or higher) nor the production phase temperatures of the T. reesei cells (e.g., temperatures of about 29°C or lower), whereas the strain expressing the iF19 intein-modified Ade2 protein was surprisingly capable of auto-splicing during the protein production phase temperatures of cells (e.g., temperatures of about 29°C or lower).

[0135] Based on the foregoing, in other embodiments Applicant designed and constructed intein- modified fitness proteins of interest which were subsequently screened for suitable temperature-sensitive splicing activity during the protein production phase of filamentous fungal cells (e.g., temperatures of about 29°C or lower) and screened for the absence the temperature-sensitive splicing activity during the growth phase of filamentous fungal cells (e.g., temperatures of about 30.0°C or higher). For instance, in certain embodiments, it is desirable to modulate (control) a protein function (e.g., an enzymatic activity, a protein / DNA binding activity, etc.) until a suitable point of fungal cell growth, and / or desirable to regulate a protein function or activity until a suitable fermentation condition or state is achieved (e.g., a desired temperature, a desired cell density, etc.) and / or it may be desirable to regulate a protein function or activity until the target protein is recovered, purified and the like. More specifically, in certain embodiments, applicant has observed that expression of the transcriptional regulator Ace3L fitness protein can greatly improve protein yield, but it can be particularly toxic and detrimental to the growth of the Trichoderma cells. In particular, the constitutive expression of Ace3L has been demonstrated to reduce cell growth rate during the growth phase, wherein the usage of a glucose-starvation induced did promoter mitigated the growth defect (PCT Publication No. WO2018 / 067599 and Luo et cd., 2020). However, the growth rate of Ace3L strain remains lower as compared to the parental strain.

[0136] In the present disclosure, Applicant has surprisingly observed that the cell growth rate is significantly improved by using an Ace3L-intein, wherein the protein yield improvement from Ace3L is also maintained. Based on these observations, Example 2 of the disclosure describes compositions and methods for constructing and identifying an intein-modified fitness protein of interest (e.g., Ace3L) capable of auto-splicing during the protein production phase of a Trichoderma fermentation, but not capable of auto-splicing during the growth phase of a Trichoderma fermentation. More particularly, Example 2 describes the use of a temperature regulated VMA intein variant (iF19) to modulate the function of the Ace3L transcription factor (TF) protein (SEQ ID NO: 6; FIG. 5). As set forth in this example, Ace3L-intein expression vectors (FIG. 6) were constructed (iWT, iF19, and iDead), which intein sequences were inserted upstream of either one of the two cysteine (C) residues present in the Ace3L amino acid sequence (FIG. 5; SEQ ID NO: 6, positions C80 and C376). More particularly, the Ace3L protein C80 residue is one of six cysteine residues present in the ZmCyse DNA binding domain of Ace3L, whereas the C376 residue of Ace3L is located outside of the (Zn^Cysg) DNA binding domain. As described in Example 1, the iWT intein is cable of splicing at growth temperatures between 18°C and 37 °C, the iDead intein is a non-functional (dead) intein which is not capable of splicing, and the iF19 intein is a temperature-sensitive intein with a permissive splicing temperature below about 28°C.

[0137] Thus, the Ace3L-intein expression cassettes shown in TABLE 2 (Example 2) were introduced into T. reesei strains and total secreted protein production of the supernatant was assessed under the cultivation conditions specified. In particular, a temperature of 32.5°C (or higher) was a non-permissive temperature for intein (iF19) splicing, whereas lower temperatures of either 27.5° C or 25°C, were permissive temperatures for intein (iF19) splicing (e.g., see FIG. 7). As shown in TABLE 2, total secreted protein in the supernatants is presented as a ratio, which is relative to the total protein production levels produced by the parental (control) strain under the specified conditions. In particular, applicant has surprisingly observed that the modified LT329 strain (Ace3L-C80-iF19) with the iF19 intein inserted at the C80 position of Ace3L, was cable of splicing at the permissive temperature, while the modified LT320 with the iF19 intein inserted at the C376 position of Ace3L, was not cable of splicing. This result demonstrated C80 position of Ace3L is a suitable position for intein insertion. The strain growth rate and protein yield were evaluated in a large-scale fermentation. For example, the LT329 strain (Ace3L-C80- iF19) showed improved growth rate compared to both parental strain and strain LT167 (Ace3L) under a non-permissive temperature for intein (iF19) splicing (FIG. 8). The LT329 strain (Ace3L-C80-iF19) secreted a similar amount of total protein as strain LT167 (Ace3L) on glucose / sophorose, both of which are about 20% higher than the parental strain on glucose / sophorose (FIG. 9, inducing conditions). The LT329 strain (Ace3L-C80-iF19) was also able to produce protein on glucose (FIG. 10, non-inducing conditions), albeit of a lower yield than itself on glucose-sophorose (FIG. 10, inducing conditions). These results demonstrate that the C80 position of the Ace3L protein (SEQ ID NO: 6) is a particularly suitable cysteine residue position for intein insertion, and that Ace3L-C80-iF19 construct enables temperature-sensitive modulation of the Ace3L fitness protein when expressed in a filamentous fungal cell. The modified Ace3L-C80-iF19 strain was able to grow faster than the parental strain, and maintain a similar protein yield improvement as the Ace3L strain.

[0138] In other aspects, Applicant has designed, constructed, tested, and screened filamentous fungal strains expressing a reporter protein and an intein-modified Ace3L protein expression cassette (Ace3L- C80-iF19). In particular, a T. reesei (parent) strain expressing heterologous copies of a glucoamylase (GA) gene was modified as described in Examples 4 and 5 below. For example, the parental T. reesei (GA) strain comprises glucoamylase expression cassettes, e.g., cassettes expressing the glucoamylase coding sequence (CDS) under the control of an (5r) upstream ' / . reesei cellulase gene promoter (e.g., a cbhl promoter; 5'-[cbhl]-[GA_CDS]-3') and the introduced Ace3L-intein expression vector. As generally described in Example 4, the parental GA reporter strain was transformed with an expression cassette (Ace3L-C80-iF19), wherein the parental and modified (LT351) GA reporter strains were grown in microtiter plates (MTPs) at a permissive splicing temperature of 27.5°C, and supernatant from the cell cultures harvested and analyzed (FIG. 12). For example, the parental strain produced a large amount of the GA reporter protein in defined medium with glucose / sophorose (“sop”, inducing condition), and only a small amount of GA reporter in defined medium with glucose (“glu”, non-inducing condition). In contrast, as shown in FIG. 12, the modified LT351 strain (Ace3L-C80-iF19) produced 1.6-fold higher GA under the inducing (“sop”) conditions (relative to parent under inducing (“sop”) conditions), and produced similar amounts of GA under non-inducing (“glu”) conditions (relative to parent under inducing conditions), or 3-fold higher GA under non-inducing (“glu”) conditions (relative to parent under noninducing conditions). These results demonstrate that the intein-modified LT351 strain (Ace3L-C80-iF19) produces extracellular (secreted) proteins (GA reporter) in the absence of an inducer, and also produces more total protein than the parental strain under inducing (Sop) conditions. Likewise, the results further confirm that the expressed Ace3L-C80-iF19 protein successfully spliced out the iF19 intein, and correctly ligated the Ace3L (extein) under the permissive splicing temperature, confirming a functional Ace3L fitness protein is expressed in T. reesei strains.

[0139] Example 5 describes the large-scale fermentation of the modified LT351 (Ace3L-C80-iF19) and LT182 strains (Ace3L) described / constructed in Example 4, wherein Applicant has surprisingly observed that the modified LT351 (Ace3L-C80-iF19) has a higher growth rate and a higher yield when compared to the parental strain and to the LT182 strains (Ace3L). In particular, the parental and modified strains were grown in a defined medium with glucose as the carbon source at 34°C, followed by a protein production phase at 28 °C, with either a glucose / sophorose feed (inducing), or a glucose feed (noninducing) as carbon sources. For example, the growth rate of the parental strain during the growth phase at 34°C (a non-permissive temperature for intein iFl 9 splicing) was arbitrarily set at 100, and the relative growth rate by the modified LT182 strain (Ace3L) or the modified LT351 strain (Ace3L-C80-iF19) were plotted as shown in FIG 13, wherein the modified LT182 strain (Ace3L) showed an approximately 10%lower growth rate compared to the parental strain, and the modified LT351 strain (Ace3L-C80-iF19) demonstrated approximately 20% higher growth rate compared to the parental strain. For example, total protein yields obtained by the parental strain on glucose / sophorose (inducing conditions) at the end of fermentation (EOF) was arbitrarily set at 100, and the relative protein yields by modified LT182 strain (Ace3L) or modified LT35 Istrain (Ace3L-C80-iF 19) were plotted (FIG. 14). As shown in FIG. 14, when the strains were evaluated under inducing conditions (“Sop”, glucose / sophorose feed), the modified LT182 strain (Ace3L) demonstrated about a 1.15-fold (15%) improved total protein yield as compared to the parental strain, and the modified LT351 strain (Ace3L-C80-iF19) demonstrated approximately a 1.2- fold (-22%) improved yield as compared to the parental strain, and approximately 7% higher yield compared to the LT182 strain (Ace3L).

[0140] Additionally, as shown in FIG. 15, when the modified LT351 strain (Ace3L-C80-iF19) was evaluated under non-inducing conditions (“Glu”), the strain was able to produce protein well, at approximately 7% lower yield relative to itself under inducing conditions (“Sop”). Overall, the results presented herein demonstrate that the intein-modified LT351 strain (Ace3L-C80-iF19) has a higher growth rate as compared to the parental strain and the modified LT182 strain (Ace3L) at a non-permissive temperature for intein iF19 splicing, and produced the GA reporter effectively under both inducing and non-inducing conditions, and showed higher yields compared to the parental strain and to the modified LT182 strain (Ace3L).

[0141] Thus, certain one or more embodiments of the disclosure provide, inter alia, compositions and methods for designing, constructing, testing, screening, cultivating and the like recombinant filamentous fungal strains expressing intein-modified fitness proteins. Certain embodiments are therefore related to recombinant filamentous fungal strains expressing temperature-sensitive intein-modified fitness proteins. In particular, as generally exemplified with the / hrem-modified ade2 gene encoding the Adc2 protein (Example 1) and the iMtez'n-modified Ace3L genes (Examples 2-5) encoding the Ace3L protein, the function or activity of a fitness protein of interest can be modulated (controlled) according to methods and compositions of the disclosure.

[0142] For example, once a desired fitness protein has been identified, one of skill can analyze the DNA sequence (encoding the target protein) for a suitable of TGT, TGC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA or ACG codon to insert an intein DNA sequence. Alternatively, one of skill can analyze the amino acid sequence of a desired fitness protein for a suitable cysteine (Cys), serine (Ser), or threonine (Thr) amino acid position for intein modification. Alternatively, if a suitable TGT, TGC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA or ACG codon is not present in the DNA sequence encoding the desired fitness protein, one of skill may engineer the DNA sequence thereof to introduce a suitable codon for intein DNA insertion.

[0143] Moreover, in other embodiments Applicant has surprisingly observed that expression of a mutant histidine kinase allele (nz'WM743T; SEQ ID NO: 26) reduces the growth rate of Trichoderma during the growth phase, while it enhances production of the POI during the production phase. Moreover, a deletionof the nikl gene ( nikl) has no effect on the growth phase, nor on the production phase of Trichoderma, in comparison to the non-mutated parental strain containing the wild-type nikl allele. Another surprising observation is that inserting a temperature-regulated intein in niklM743T(e.g., FIG. 16 and FIG. 17) results in a strain that exhibits the nikl phenotype at the non-splicing temperatures (growth phase with significantly restored growth rate) and niklM743Tphenotype during the splicing temperatures (production phase with improved productivity).

[0144] Thus, in particular embodiments, filamentous fungal regulatory proteins (i.e., fitness proteins) are intein-modified. In certain embodiments, filamentous fungal regulatory proteins include, but are not limited to, transcriptional activator proteins, transcriptional repressor proteins, protein kinases, guanyltransferases, phosphatases, histone modification proteins and chromatin remodeling proteins. In other embodiments, a filamentous fungal fitness protein of interest (FPOI) is intein-modified, wherein the FPOI is selected from the group consisting of a xylanase regulator 1 (Xyrl) protein, an activator of cellulase 2 (Ace2) protein, an activator of cellulase 3 (Ace3) protein, a variant Ace3 protein, an activator of cellulase 4 (Ace4) protein, a carbon catabolite repressor 1 (Crel) protein, a histidine kinase (Nikl) protein, a GTP:a-D-mannose-l -phosphate guanyltransferase (Mpgl) protein, a beta-glucosidase regulator (BglR) protein, a Trire2_82512 protein and a Trire2_62386 protein.

[0145] Thus, as briefly set forth above and further described hereinafter, intein-modified fitness proteins of interest are constructed using standard molecular biological techniques and screened according to present disclosure. The intein, the target FPOI protein, or the intein-modified FPOI can be subjected to mutation and accordingly screened. Screening systems allow production of the protein and / or testing of its physical and / or functional characteristics. From an intein-modified FPOI, suitable candidates can be isolated and analyzed further. Analysis may include DNA sequencing, functional assays, structural assays, enzyme activity assays, and monitoring changes in activity, structure, or splicing in response to induction conditions. in. RECOMBINANT NUCLEIC ACIDS AND MOLECULAR BIOLOGY

[0146] As generally set forth above and further exemplified below, certain embodiments of the disclosure are related to the design and construction of intein-modified fitness proteins. More particularly, as set forth below in the Examples, recombinant nucleic acid constructs, recombinant fungal strains and the like may be constructed using routine methods well known in the art (e.g., Ausubel etal., 1987; Sambrook et al., 1989).

[0147] In certain aspects, modified (recombinant) filamentous fungal cells may be constructed via CRISPR-Cas9 editing. For example, a gene of interest can be modified, disrupted, deleted, or down- regulated by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g., Cas9 and Cpfl) or a guide DNA (e.g., NgAgo), 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 aprovided editing template to disrupt or delete or modify the gene. For example, the gene encoding the nucleic acid guided endonuclease (for this purpose Cas9 from .S'. pyogenes) or a codon optimized gene encoding the Cas9 nuclease is operably linked to a promoter active in the fungal cell and a terminator active in a fungal cell, thereby creating a fungal Cas9 expression cassette. Likewise, one or more target sites unique to the gene of interest are readily identified by a person skilled in the art. For example, to build a DNA construct encoding a gRNA-directed to a target site within the gene of interest, the variable targeting domain (VT) will comprise nucleotides of the target site which are 5' of the (PAM) protospacer adjacent motif (NGG), which nucleotides are fused to DNA encoding the 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 fungal cell expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in fungal cell and a terminator active in fungal cell.

[0148] 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 500 bp 5' of the targeted gene can be fused to about 500 bp 3' of the targeted gene to generate an editing template, which template is used by the fungal host’s machinery to repair the DNA break generated by the RNA-guided endonuclease (RGEN). Even shorter stretches of nucleotides in a form of double or single stranded DNA can be used as an editing template.

[0149] The Cas9 expression cassette, the gRNA expression cassette and the editing template can be codelivered to filamentous fungal cells using many different methods (e.g., protoplast fusion, electroporation, natural competence, or induced competence). The transformed cells arc screened by PCR amplifying the target gene with a forward and reverse primer. These primers can amplify the wildtype locus or the modified locus that has been edited by the RGEN.

[0150] 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 microprojectiles, gene gun or biolistic transformation, protoplast fusion and the like. General transformation techniques are known in the art (see, e.g., Ausubel et al., 1987, Sambrook et al., 2001 and 2012, and Campbell et al., 1989).

[0151] In certain other embodiments, the recombinant nucleic acid further comprises one or more selectable markers. Selectable markers for use in filamentous fungi include, but are not limited to, alsl, amdS, hygR, pyr2, pyr4, pyrG, sucA trpC, argB, a bleomycin resistance marker, a blasticidin resistance marker, a pyrithiamine resistance marker, a neomycin resistance marker, an adenine pathway gene, athymidine 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.

[0152] Generally, transformation of Trichoderma cells uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 105to 107 / mL, particularly 2xlO6 / mL. A volume of 100 L of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCh) 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.

[0153] In certain aspects, the disclosure relates to the expression / production of one or more proteins of interest which are endogenous to the filamentous fungal cell and / or the expression / production one or more proteins of interest which are heterologous to the filamentous fungal host cell.

[0154] In particular embodiments, a heterologous gene CDS (or ORF) encoding a protein is introduced into a filamentous fungal (host) cell. In certain embodiments, the heterologous gene CDS is cloned into an intermediate vector, before being transformed into a filamentous fungal (host) cells for expression. These intermediate vectors can be prokaryotic vectors, such as, e.g., plasmids, or shuttle vectors. The expression vector / construct 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 a 5' promoter operably linked to the heterologous nucleic acid sequence encoding the protein and may further comprise sequence signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. 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.

[0155] In addition to a promoter sequence, the expression cassette may also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence, or may be obtained from 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, the Aspergillus awamori or Aspergillus niger glucoamylase genes and / or the Mucor miehei carboxyl protease gene.

[0156] The particular expression vector used to transport the 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 A and M13, as well as plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ, as well as yeast 2p plasmids and centromeric yeast plasmids. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc.

[0157] 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 the 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 that they do not interfere with the replication or integration of the DNA in the fungal host.

[0158] The methods of transformation of the present disclosure may result in the stable integration of all or part of the transformation vector into the genome of the filamentous fungus. However, transformation resulting in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated. Any of the known procedures for introducing foreign (heterologous) nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, and any of the other known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g.. Sambrook et al., supra). Also of use is the Agrobacterium-mediated transfection method is suitable and well known.

[0159] After the expression vector(s) is / are introduced into the cells, the transfected cells are cultured under conditions favoring expression of genes under control of cellulase gene promoter sequences. Large batches of transformed cells can be cultured as described herein. Finally, product is recovered from the culture using standard techniques.IV. PROTEINS OF INTEREST

[0160] As stated above, certain embodiments of the disclosure provide genetically modified (recombinant) fungal cells expressing an intein-modified FPOI. In certain related embodiments, the disclosure provides modified fungal cells expressing an intein-modified FPOI and one or more endogenous or heterologous proteins of interest. In certain related embodiments, recombinant fungal cells of the disclosure produce increased amounts of a protein of interest (POI), wherein the POI includes, but is not limited to, enzymes, antibodies, receptor proteins, animal feed proteins, human food proteins, protein biologies and the like.

[0161] In certain embodiments, the POI is an enzyme selected from the group consisting of amylases, cellulases, hemicellulases, xylanases, peroxidases, proteases, lipases, phospholipases, esterases, cutinases, polyesterases, phytase, pectinases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, mannanases, a-glucanases, (1- glucanases, hyaluronidases, chondroitinases, laccases, amylases, glucoamylases, acetyl esterases, aminopeptidase, arabinascs, arabinosidascs, arabinofuranosidascs, carboxypcptidascs, catalases, nucleases, deoxyribonucleases, ribonucleases, epimerases, a-galactosidases, [3-galactosidases, glucan lysases, endo-P-glucanases, glucose oxidases, glucuronidases, invertases, and isomerases. In certain aspects, an enzyme is selected from an Enzyme Commission (EC) Number comprising EC 1, EC 2, EC 3, EC 4, EC 5, or EC 6.

[0162] Optimal conditions for the production of the POI 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 protein can be purified or isolated after expression. The protein 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 protein may be purified using a standard anti-protein 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.

[0163] In certain other embodiments, to confirm that a genetically modified fungal cell of the disclosure produces an increased level of a protein of interest, various methods of screening may be performed. The expression vector may encode a polypeptide fusion to the target protein which serves as a detectable label or the 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 the 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 protein of interest, such as, the detection of the increase of protein activity or amount per cell, protein activity or amount per milliliter of medium, allowing cultures or fermentations to continue efficiently for longer periods of time, or through a combination of these methods.

[0164] 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’hr wherein “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 the time of inoculation, which include the time of production as well as growth time.

[0165] The detection of protein yield on carbon is another method to evaluate protein production. The total protein yield is calculated as grams of protein produced divided by grams of substrate (glucose) consumed.VI. FERMENTATION

[0166] Certain embodiments are related to compositions and methods for producing proteins comprising growing, cultivating, or fermenting a modified filamentous fungal cell of the 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.

[0167] A suitable variation on the standard batch system is the “fed-batch fermentation” system. In this variation of a typical batch system, after the log phase is finished, the 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 that could lead to inhibition of cellular metabolism and protein production. Batch and fed-batch fermentations are common and well known in the art.

[0168] Continuous fermentation is a system where a defined fermentation medium is added continuously to a bioreactor, 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 arc primarily kept in log phase growth. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth 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.

[0169] 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 source material, molecular oxygen, and, of course, a starting inoculum of the filamentous fungal host to be employed.

[0170] 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 assureproper 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.

[0171] 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 also present preferably should be 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.

[0172] 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.

[0173] The fermentation temperature 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.

[0174] 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 other 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.

[0175] 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.

[0176] 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-containing substrate 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.

[0177] 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 femientation medium and whether or not the desired level of carbon source has been achieved.

[0178] 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.

[0179] If desired, part or all of the carbon and energy source material and / or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermenter.

[0180] 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 concentration 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 the like. The feed rates of the various materials can be varied so as to obtain maximal production rates and / or maximum yields.

[0181] 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 the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermenter employed is not critical.

[0182] The collection and purification of (e.g., cellulase) enzymes from the fermentation broth can also be done by procedures known to one skilled in the art. The fermentation broth will generally contain cellular debris, including cells, various suspended solids and other biomass contaminants, as well as the desired cellulase enzyme product, which are preferably removed from the fermentation broth by means known in the art.

[0183] Suitable processes for such removal include conventional solid-liquid separation techniques such as, e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes, to produce a cell-free filtrate. It may be preferable to further concentrate the fermentation broth or the cell-free filtrate prior to crystallization using techniques such as ultrafiltration, evaporation or precipitation.

[0184] Precipitating the proteinaceous components of the supernatant or filtrate may be accomplished by means of a salt, e.g., ammonium sulfate, followed by purification by a variety of chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography or similar art recognized procedures.VII. EXEMPLARY EMBODIMENTS

[0185] Non- limiting embodiments of the compositions and methods disclosed herein are as follows:

[0186] 1. A method for modulating the function of a fitness protein of interest (FPOI) in a filamentous fungal cell comprising (a) obtaining (or constructing) a filamentous fungal cell comprising gene encoding a FPOI, wherein the open reading frame (ORF) of the gene encoding the FPOI comprises at least one wild-type or engineered codon selected from the group consisting of TGT, TGC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA and ACG, modifying the ORF by inserting a polynucleotide encoding an intein immediately upstream of the at least one codon, (b) fermenting the modified cell for a period of time under a non-splicing intein temperature, wherein the FPOI is non-functional under the non-splicing intein temperature, and (c) fermenting the modified cell for a period of time under an intein splicing temperature, wherein the FPOI is functional under the intein splicing temperature.

[0187] 2. The method of embodiment 1, wherein the FPOI is selected from the group consisting of transcriptional activator proteins, transcriptional repressor proteins, kinases, phosphatases, guanyitransferases, glycosyltransferases, glycosidases, histone modification proteins, chromatin remodeling proteins and proteins involved in foaming.

[0188] 3. The method of embodiment 1, wherein the FPOI is selected from the group consisting of a xylanase regulator 1 (Xyrl) protein, an activator of cellulase 2 (Ace2) protein, an activator of cellulase 3 (Ace3) protein, a variant Ace3-L protein, an activator of cellulase 4 (Ace4) protein, a carbon catabolite repressor 1 (Crel) protein, a histidine kinase (Niki) protein, a M743T variant histidine kinase (NiklM743T) protein, a GTP:a-D-mannose-l -phosphate guanyltransferase (Mpgl) protein, a betaglucosidase regulator (BglR) protein, a Trire2.._82512 protein, a Trire2_.62386 protein, hydrophobin protein Hfbl and hydrophobin protein Hfb2.

[0189] 4. The method of embodiment 3, wherein the Xyrl 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% or 100% identity to SEQ ID NO: 15, the Acc2 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% or 100% identity to SEQ ID NO: 17, the Ace3 or Ace3L 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% or 100% identity to SEQ ID NO: 6, the Crel 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% or 100% identity to SEQ ID NO:21 and the Niki or variant NiklM7 l3Tprotein comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 27.

[0190] 5. The method of embodiment 1, wherein the intein comprises at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 25.

[0191] 6. The method of embodiment 5, wherein the intein comprises a tryptophan (W) to arginine (R) substitution at position 157 (W157R) and a glycine (G) to arginine (R) substitution at position 219 (G219R).

[0192] 7. The method of embodiment 1, wherein the non-splicing intein temperature is a temperature of 30.0°C and higher.

[0193] 8. The method of embodiment 1, wherein intein splicing temperature is a temperature of 29.0°C and lower.

[0194] 9. The method of embodiment 3, wherein the Ace3L protein comprises the intein inserted immediately upstream of a cysteine (Cys), a serine (Ser), or a threonine (Thr) residue present in SEQ ID NO: 6.

[0195] 10. The method of embodiment 1, wherein the modified cell expresses one or more lignocellulosic degrading enzymes.

[0196] 11. The method of embodiment 10, wherein the lignocellulosic degrading enzymes are selected from the group consisting of a cellobiohydrolase, a xylanase, an endoglucanase and a [5-gl ucosidase.

[0197] 12. The method of embodiment 1, wherein the modified cell comprises an introduced expression cassette encoding a heterologous protein of interest (POI).

[0198] 13. The method of embodiment 12, wherein the cassette encoding the heterologous POI comprises an upstream (5') cellulase gene promoter sequence operably linked to a downstream (3') polynucleotide (DNA) sequence encoding the heterologous POI, optionally comprising a terminator (DNA) sequence downstream and operably linked to the polynucleotide encoding the heterologous POI.

[0199] 14. The method of embodiment 13, wherein the heterologous POI is an enzyme is selected from the group consisting of acetyl esterases, amylases, aminopeptidase, arabinases, arabinosidases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, cutinases, chondroitinases, deoxyribonucleases, epimerases, esterases, glucoamylases, glucan lysases, glucose oxidases, glucuronidases, hemicellulases, hyaluronidases, invertases, isomerases, keratinases, laccases, lipases, lipoxygenases, ligninases, mannanascs, nucleases, oxidases, peroxidases, proteases, phospholipases, polyesterases, phytases, pectinase, phenol oxidases, pullulanases, pentosanases, reductases, ribonucleases, tannases, aglucanases, [1-glucanascs, a-galactosidases, Pgalactosidases, endo-P- glucanases and xylanases.

[0200] 15. A polynucleotide encoding an intein-modified fitness protein of interest (FPOI), wherein the intein-modified FPOI comprises at least one wild-type or genetically engineered cysteine (Cys), serine (Ser) or threonine (Thr) residue and an intein inserted immediately N-terminal to the at least one Cys, Ser or Thr residue.

[0201] 16. The polynucleotide of embodiment 15, wherein the intein comprises at least about 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 25, and comprises a tryptophan (W) to arginine (R) substitution at position 157 (W157R) and a glycine (G) to arginine (R) substitution at position 219 (G219R).

[0202] 17. The polynucleotide of embodiment 15, wherein the FPOI is selected from the group consisting of a xylanase regulator 1 (Xyrl) protein, an activator of cellulase 2 (Ace2) protein, an activator of cellulase 3 (Ace3) protein, a variant Ace3E protein, an activator of cellulase 4 (Ace4) protein, a carboncatabolite repressor 1 (Crel) protein, a histidine kinase (Niki) protein, a M743T variant histidine kinase (NiklM743T) protein, a GTP:a-D-mannose-l -phosphate guanyltransferase (Mpgl) protein, a betaglucosidase regulator (BglR) protein, a Trire2_82512 protein, a Trire2_62386 protein, hydrophobin protein Hfbl and hydrophobin protein Hfb2.

[0203] 18. The polynucleotide of embodiment 17, wherein the Ace3L protein comprises the intein inserted immediately upstream of the at least one Cys, Ser, or Thr residue present in SEQ ID NO: 6.

[0204] 19. The polynucleotide of embodiment 17, wherein the NikiM743Tprotein comprises the intein inserted immediately upstream of the at least one Cys, Ser, or Thr residue present in SEQ ID NO: 31.

[0205] 20. A polynucleotide expression construct (cassette) comprising an upstream (5') promoter sequence operably linked to a downstream (3') polynucleotide of embodiment 15, optionally comprising a terminator sequence downstream and operably linked to the polynucleotide of embodiment 15.

[0206] 21. The expression construct of embodiment 20, comprising at least about 95%, 96%, 97%, 98%, 99% or 100% identity to the Ace3L-C80-iF19 cassette of SEQ ID NO: 10.

[0207] 22. A recombinant filamentous fungal cell comprising an introduced expression of embodiment 20.

[0208] 23. The method of embodiment 1, wherein the filamentous fungal cell is selected from the group consisting of a Trichoderma sp. cell, an Aspergillus sp. cell, a Fusarium sp. cell, a Penicillium sp. cell, a Chrysosporium sp. cell, a Cephalosporium sp. cell, a Talaromyces sp. cell, a Geosmithia sp. cell, a Neurospora sp. cell and a Myceliophthora sp. cell.EXAMPLES

[0209] It should be understood that the following Examples, while indicating embodiments of the disclosure, are given by way of illustration only. From the above discussion and these Examples, one of skill in the 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 1CONSTRUCTING AND SCREENING INTEINS FOR CONDITIONAL SPLICING ACTIVITY IN FILAMENTOUS FUNGAL CELLS

[0210] As described in the instant example, all experiments were performed in a Trichoderma reesei host strain. More particularly, as set forth below, knockout (A) mutations of a gene of interest (ade2) were generated by RNP cutting and integration of a hph gene for hygromycin resistance. For instance, ade2 codes for a phosphoribosyl aminoimidazole carboxylase necessary for the synthesis of purines ('Jorgensen et al., 2014), wherein Aade2 phenotypes can be identified as red-colored colonies when supplementing with adenine, due to the accumulation and polymerization of the purine precursor 5- aminoimidazole ribonucleotide. The general methodology to test / screen for intein variants havingdesirable splicing activity in filamentous fungal cells consisted of ade2- complementation assays of the knockout strains (FIG. 4, T. reesei Aade2), wherein expression cassettes of genes of interest were cloned into the telomeric vector pRASl (carrying a pyr2 marker) in the following configurations:(1) Complementation: Standard ade2 complementation assay, serving as a positive (+) control;(2) iWT: Same as (1), including an insertion of the coding sequence of the wild-type VMA intein into the coding sequence of ade2 directly upstream of the codon encoding C183 (-1) or C242 (-2);(3) iF4: Same as (1), including an insertion of the coding sequence of the F4 variant of VMA intein (N278T, L319S) into the coding sequence of ade2 directly upstream of the codon encoding C183 (-1) or C242 (-2);(4) iF19: Same as (1), including an insertion of the coding sequence of the F19 variant of VMA intein (W157R, G219R) into the coding sequence of ade2 directly upstream of the codon encoding C183 (-1) or C242 (-2);(5) iDead: Same as (1), including an insertion of the coding sequence of the splicing-dead variant of VMA intein (N454tfe / ) into the coding sequence of ade2 directly upstream of the codon encoding C183 (-l) or C242 (-2); and(6) pRASl : Empty vector, serving as a negative (-) control.

[0211] For example, intein sequences obtained from DNA synthesis were codon-optimized for expression in T. reesei (strain QM6a) and inserted upstream (5') of codons encoding cysteine (Cys) residues of genes of interest. More particularly, in the case of ade2 (FIG. 2 and FIG. 3) intein sequences were inserted immediately upstream (5') of codons encoding cysteine positions Cl 83 and C242. Subcloning was carried out in E. coli, and assembled plasmids were transformed into their corresponding T. reesei knockout strain (i.e, Aade2 knockout strain).

[0212] Complementation Assays of ade2

[0213] The complementation of ade2 was analyzed in two (2) distinct manners: (i) using the ade2 gene as an auxotrophic marker (i.e., in the absence of adenine; adenine (-) negative), or (ii) as a reporter (i.e., when supplementing with adenine, adenine (+) positive). For example, in the absence of Ade2, the intermediate substrate 5'-phoshporibosyl-5-aminoimidazole (AIR) of the adenine synthesis pathway accumulates and forms polymers, giving rise to a red pigment. In assays using ade2 as an auxotrophic marker, the iWT colonies grew comparable to the Complementation colonies, indicating the generation of functional Ade2 protein via intein (iWT) splicing (FIG. 4A). As expected, none of the iDead colonies grew, confirming that the non-spliced (iDead) intein inserted in the Ade2 protein results in the disruption of the enzymatic activity (FIG. 4A). The colonies expressing the variant intein iF4 also showed no growth, suggesting the iF4 mutations likely caused slow, partial, or even complete inhibition of intein splicing (FIG. 4A).

[0214] In contrast, in iF19 assays, colony growth was dependent on the incubation temperature. For example, at 25.0°C the iF19 colonies grew comparable to iWT and Complementation colonies, while at32.5°C the iF19 colonies showed no growth (FIG. 4A). These results clearly demonstrate temperaturesensitive splicing of the iF19 variant at the permissive temperature of 25°C, whereas the iF4 variant showed no growth at 25°C, indicating the iF4 intein does not (auto) splice at the permissive temperature. Also noted herein, both of the tested cysteine positions within Ade2 protein (i.e., positions C183 and C242) gave rise to similar patterns. In assays using ade2 as a reporter in liquid culture (with adenine supplementation), only iF19 cultures showed condition-dependent changes in coloration (data not shown).

[0215] In an additional reporter assay with adenine supplementation, color switching of a parental population of iF19 cells was demonstrated by following the radial growth of mycelia discs on agar plates (FIG. 4B). For example, the plates were inoculated and incubated in quadruplicate, either at 25.0°C or at 32.5°C. Control plates stayed at the same temperature for five (5) days (either at 25.0°C or at 32.5°C). Assay plates were switched between the two (2) indicated temperatures after three (3) days, incubation there for one (1) day, and on the following day moved back to their initial temperature for one (1) more day (e.g., either 25.0°C — 32.5°C — 25.0°C, or 32.5°C 25.0 °C — 32.5°C). In iF19 assays, colonies with alternating white and red rings appeared (FIG. 4B), demonstrating the temperature dependency of the conditional intein splicing activity. Interestingly, mycelium rings that formed at previous incubation temperatures, did not change in color retroactively, suggesting that high metabolic activity is required for changes in coloration of the ade2 reporter system.EXAMPLE 2TEMPORAL CONTROL OF ACE3 TRANSCRIPTION FACTOR IN FILAMENTOUS FUNGAL CELLS

[0216] In the instant example, Applicant has designed, constructed, and tested exemplary fungal strains comprising an introduced Ace3 expression cassettes (e.g., see TABLE 1 below), wherein the Ace3 cassettes comprise a (5') intein insertion. For example, Ace3 is a T. reesei transcription factor (regulatory protein) known to regulate cellulase / hemi-cellulase production under inducing conditions (Hakkinen et al. 2014). More recently, a highly functional form of Ace3 (named “Ace3L”) was identified, as described in the PCT Publication No. WO2018 / 067599. In the case of the variant Ace3L transcription factor (TF), T. reesei strains expressing the Ace3L TF variant not only showed improved protein production in the presence of inducers (e.g., lactose, sophorose), but also showed significantly increased protein production in the absence of inducers. As set forth in the WO2018 / 067599 publication, the Ace3L variant comprises an intact (ZnzCySb) DNA binding domain at its N-terminus, and an eleven (11) amino acid truncation at its C-tcrminus, both of which arc essential features for its function in increasing protein production both in the absence and presence of inducers, as described above.

[0217] More particularly, in the instant example Applicant incorporated intein sequences into the variant Ace3L protein (SEQ ID NO: 6; TABLE 1) in an attempt to regulate (control) the Ace3L transcription factor, by using permissive and non-permissive intein splicing conditions described herein. For example,the six (6) Ace3L-intein expression vectors set forth in TABLE 1 were constructed using standard molecular biological procedures.TABLE 1ACE3L-INTEIN EXPRESSION CONSTRUCTS

[0218] As shown in TABLE 1, each Ace3L-intein expression vector comprises one (1) of three (3) different intein variants named iWT, iF 19. and iDead, which intein sequences were inserted upstream (5') of either one (1) of the two (2) cysteine (C) amino acid residues present in the Ace3L protein sequence (z.e., C residue positions C80 and C376; SEQ ID NO: 6 and FIG. 5). For example, the Ace3L C80 residue is one (1) of six (6) cysteine residues present in the Zn2Cys6 DNA binding domain, whereas the C376 residue of Ace3L is located outside of the (ZnzCyse) DNA binding domain (FIG. 5). As set forth in Example 1, the iWT intein is cable of splicing at growth temperatures between 18°C and 37°C; the iDead intein is a non-functional (dead) VMA intein which is not capable of splicing, and the iF19 intein is a temperature-sensitive VMA intein (variant) with a permissive splicing temperature below 28°C.

[0219] In particular, the expression vectors (TABLE 1) comprise a vector backbone having a bacterial ColEl origin of replication (on) and an AmpRgene for replication and selection in E. coli. In addition, T. reesei telomere sequences, a T. reesei pyr2 selection marker, a T. reesei did promoter sequence, and a native terminator sequence of the ace3 gene. For example, a representative vector map of vector pYL63 is shown in FIG. 6, depicting vector pYL63 comprising the Ace3L-C80-iF19 variant construct (SEQ ID NO: 12) set forth in TABLE 1. Likewise, the other vectors (<?.g., pYL62, pYL62, pYL68, pYL69 and pYL70) have the same sequences presented in FIG. 6, except for the specified Ace3L-intein variant sequences Ace3L-C80-iWT (SEQ ID NO: 9), Ace3L-C80-iF19 (SEQ ID NO: 10), Ace3L-C80-iDEAD (SEQ ID NO: 11), Ace3L-C376-iWT (SEQ ID NO: 12), Ace3L-C376-iF19 (SEQ ID NO: 13) and Ace3L- C376-IDEAD (SEQ ID NO: 14).

[0220] The expression vectors (TABLE 1) were transformed into a T. reesei parental strain using polyethylene glycol (PEG)-mediated protoplast transformation (Ouedraogo et al., 2015; Penttila et al., 1987). More particularly, the T. reesei parental strain was derived from T. reesei strain RL-P37 (NRRLDeposit No. 15709), wherein the T. reesei pyr2 gene has been deleted (Apyr2) as described by Sheir- Neiss and Montenecourt (1984). The transformants were grown on Vogel’s minimal medium agar plates to select for uridine prototrophy acquired by the pyr2 marker. Stable transformants were obtained by transfer on Vogel’s agar plate for two successive rounds, followed by two successive rounds of growth on non-selective PDA agar plates, and one round on Vogel’s agar plate, after which single colonies were obtained by plating dilution of spore suspension.[0221 J The parental (control) and modified strains described above were subsequently tested under inducing conditions (sophorose) and non-inducing conditions (glucose). In the non-inducing (glucose) conditions, cells were grown in 1.25 ml liquid broth of defined medium, supplemented with 2.5% glucose (weight / volume) in a regular twenty-four (24) deep well microtiter plate (MTP), and in the inducing (sophorose) conditions, cells were grown in 1.25 ml liquid broth of defined medium supplemented with 2.5% glucose / sophorose (weight / volume) in a twenty-four (24) deep well MTP, where sophorose serves as potent inducers for cellulase (enzyme) expression. For example, as described in PCT Publication No. WO2018 / 067599, protein production in the non-inducing (glucose) condition is dependent on a functional Ace3L TF. Thus, the parental and modified strains (TABLE 1) were tested under different temperature ranges to assess conditionally regulated (controlled) intein splicing of the Ace3L-intein constructs.

[0222] In particular, a temperature of 32.5°C (or higher) is a non-permissive temperature for intein iF19 splicing, whereas temperatures of either 27.5° C or 25°C were permissive temperatures for intein iF19 splicing. The MTP cultures were incubated at the indicated temperatures set forth in TABLE 2 below, at 250 rpm, 85% humidity for five (5) days. Following incubation at indicated temperatures, the supernatant from all cultures were harvested and total secreted protein in the supernatants measured by Bradford dyebinding assay (at 595 nm) using the Bio-Rad reagent (Thermo Scientific®; Catalogue No. 23236) and five ( 5 ) dilutions of bovine serum albumin (BSA) as a standard. The total protein production levels for the modified strains are presented in TABLE 2 as a ratio, which is relative to the total protein production levels produced by the parental (control) strain under sophorose (Glu / Sop) inducing conditions.TABLE 2 TOTAL SECRETED PROTEIN PRODUCTION OF TRICHODERMA STRAINS EXPRESSING ACE3L OR ACE3L- INTEINGlu / Sop1is an abbreviation of “Glucose / Sophorose”; an Inducing Condition; Glu2is an abbreviation of “Glucose”; a Non-Inducing Condition.

[0223] As presented above in TABLE 2, the modified LT167 strain (expressing Ace3L) showed the highest total protein production in all conditions tested (i.e., relative to Ace3 parent strain). For example, the modified (daughter) strains total protein production levels expressing the Ace3L-intein variants (i.e., LT325, LT329, LT335, LT318, LT320, LT322), including the LT325 strain (expressing Ace3L-C80- iWT), produced less secreted protein than the intein-free LT167 strain (expressing Ace3L), indicating that not all Ace3L-intein modified proteins constructed are capable of splicing in T. reesei under permissive splicing temperatures. In particular, the modified strains with an intein inserted at the C80 position of Ace3L (but not at the C376 position,' see FIG. 5) were cable of splicing at the permissive temperature.

[0224] More particularly, the modified LT325 strain (Ace3L-C80-iWT) secreted a similar amount of total protein on glucose / sophorose as the parental strain on glucose / sophorose (i.e., inducing conditions), and it was also able to secrete more total protein on glucose than the parental strain on glucose (i.e., non- inducing conditions) in all three (3) tested temperatures. The modified LT329 strain (Ace3L-C80-iF19) produced a similar amount of total protein as the LT325 strain (Ace3L-C80-iWT) at 27.5°C, or at 32.5°C, followed by 27.5°C, while it only produced basal levels of protein when continuously grown at 32.5°C (a non- permissive splicing temperature). In contrast, when an intein was inserted at position C376 of the Ace3L protein (e.g., modified LT318 and LT320 strains), the total protein level was either the same as, or lower than the parental strain under the same growth conditions, indicating that the intein inserted at position C376 was defective in splicing.

[0225] Together, these results demonstrate that the C80 position of the Ace3L protein (SEQ ID NO: 6) is a particularly suitable cysteine residue position for intein insertion, and that Ace3L-C80-iF19 constructenables temperature-controlled regulation of the Ace3L TF protein when expressed in a filamentous fungal cell.

[0226] Applicant further evaluated the temperature sensitivity of the modified LT329 (Ace3L-C80- IF19), which LT329 strain was cultivated under the same conditions as described above. The supernatant from all cultures were harvested and analyzed using Polyacrylamide Gel Electrophoresis (PAGE). Equal volumes of culture supernatants were subjected to a reducing environment for fifteen (15) minutes at 90°C, before addition of loading dye and resolution on a 4-12% NuPage™ (Invitrogen, Carlsbad CA) polyacrylamide gel with MOPS-SDS buffer. The gel was stained with SimplyBlue™ (Invitrogen) and imaged (FIG. 7). As shown in FIG. 7, at continuous permissive temperatures of 27.5°C and 25°C, the modified LT329 strain (Ace3L-C80-iF19) produced an increased amount of total protein on glucose / sophorose (inducing conditions) and on glucose (non-inducing conditions). More particularly, when the modified LT329 strain (Ace3L-C80-iF19) was grown at a non-permissive (splicing) temperature of 32.5°C, the total secreted protein was significantly reduced on glucose / sophorose (inducing conditions), and further reduced to the basal level on glucose (non-inducing conditions).

[0227] Likewise, when the temperature was switched from non-permissive (32.5°C) to permissive temperatures (27.5°C or 25°C) after two (2) days of incubation (FIG. 7), the modified LT329 strain (Ace3L-C80-iF19) was able to produce higher amounts of total protein relative to the cells which remained at the non-permissive temperature (32.5°C). These results demonstrate that the Ace3L TF protein, as expressed in the modified LT329 strain (Ace3L-C80-iF19) is active at the permissive temperature, showing improved protein production in the absence of inducers, which is a described benefit of the functional Ace3L variant TF protein (WO2018 / 067599).EXAMPLE 3LARGE SCALE FERMENTATION OF FILAMENTOUS FUNGAL CELLS EXPRESSING ACE3L TF WITH AN INTEIN INSERTION

[0228] The constitutive expression of Ace3L has been demonstrated to reduce cell growth rate during the growth phase, wherein the usage of a glucose-starvation induced did promoter mitigated the growth defect (PCT Publication No. WO2018 / 067599 and Luo et al., 2020). However, the growth rate of Ace3L remained slightly reduced as compared to the parental strain. The non-permissive and permissive temperature of intein iFl 9 auto-splicing surprisingly falls within the growth and production temperatures of T. reesei fermentation, respectively, allowing for tighter control of Ace3L fitness protein activity (function). In the present example, the wild-type parent, modified LT167 (Ace3L), and modified LT329 (Ace3L-C80-iF19) strains described in Example 2 were fermented in large scale biorcactors (-10-15 L) as generally described in U.S. Patent No. 7,713,725.

[0229] More specifically, cells were grown in a defined medium with glucose (as carbon source) at 30°C or 34°C, followed by protein production at 25°C on either glucose / sophorose (“Sop”, inducing condition) or glucose (“Glu”, non-inducing condition) as carbon sources. For example, the growth temperatures of30°C or 34°C are non-permissive temperatures for intein iF19 splicing, whereas the production temperature of 25°C is a permissive temperature for intein iF19 splicing (as demonstrated above in Example 2). More particularly, FIG. 8 shows the relative growth rates of the parental and modified LT167 (Ace3L) and LT329 (Ace3L-C80-iF19) strains at 34 °C, a non-permissive temperature for intein iF19 splicing. For instance, the growth rate of the parental stain was artificially set as 100, and the relative growth rates of the modified LT167 and LT329 strains were plotted (FIG. 8), wherein the modified LT167 (Ace3L) strain demonstrated an approximately 5% reduced growth rate, while the modified LT329 (Ace3L-C80-iF19) strain demonstrated an approximately 30% increased growth rate in comparison to the parental strain. FIG. 9 shows the total protein production yields from the large-scale fermentation of the parental (control) strain under inducing (sophorose) conditions, and the modified LT167 (Ace3L) and LT329 (Ace3L-C80-iF19) strains, under both inducing and non-inducing conditions. The total protein yield was calculated as grams of protein produced divided by grams of total glucose consumed during growth and protein production phase. For example, as presented in FIG. 9, total protein yields obtained by parental strain on glucose / sophorose (inducing condition) at the end of fermentation (EOF) was arbitrarily set at 100, and the relative protein yields by the modified LT167 and LT329 strains were plotted as shown in FIG. 9. Thus, under the sophorose (inducing) conditions, both modified strains (LT167 (Ace3L) and LT329 (Ace3L-C80-iF19)) demonstrated improved total protein yields (e.g., 1.3-fold higher) relative to parental (control) strain. In addition, as presented in FIG. 10, the LT329 (Ace3L-C80- iF / 9) strains was able to produce protein under the non-inducing conditions (glucose only, Glu), although the protein yield is approximately 45% lower yield compared to itself under inducing conditions (FIG. 10).

[0230] Thus, these results from large-scale fermentation are consistent with the results in small-scale microtitcr plates shown in Example 2, and further demonstrate the benefits in both growth rate and protein yield. More particularly, the results demonstrate that the modified LT329 strain (Ace3L-C80-iF19) has a higher growth rate compared to the parental strain at a non-permissive temperature for intein iF19 splicing during growth phase (FIG. 8). It also demonstrates that the Ace3L fitness protein expressed in the modified LT329 strain (Ace3L-C80-iF 19) is active (functional) at the permissive temperature for intein iF19 splicing during production phase. Under inducing conditions (sophorose), the modified LT329 strain (Ace3L-C80-iF19) showed improved protein production relative to the parent strain under the same conditions (FIG. 9), whereas under the non-inducing conditions, the modified LT329 strain (Ace3L-C80- iF19) was able to produce protein (FIG. 10), albeit to a lower level than it did under the inducing conditions, which is a described benefit of the functional Ace3L variant protein.EXAMPLE 4FILAMENTOUS FUNGAL CELLS EXPRESSING HETEROLOGOUS REPORTER PROTEIN AND TEMPERATURE REGULATED ACE3L-INTEIN

[0231] In the instant example, a parental 7. reesei strain expressing heterologous copies of a glucoamylase (GA) gene (US Patent No. 7,413,879) was modified as described herein. For example, the parental T. reesei GA expressing strain comprises a glucoamylase (GA) expression cassette, which cassette comprises an open reading frame (ORF) encoding the glucoamylase (GA) operably linked to an (5') upstream T. reesei cbhl promoter and operably linked to a (3') downstream 7. reesei cbhl terminator. The cassette further comprise a 1. reesei pyr2 gene as selectable marker. More particularly, an Ace3L intein expression vector named “pYL71” (FIG. 11) was constructed using standard molecular biological procedures, wherein the pYL71 vector comprises a heterologous T. reesei dicl promoter sequence (SEQ ID NO: 7) upstream (5') and operably linked to the Ace3L-C80-iF19 ORF (SEQ ID NO: 10) comprising the native ace3 gene terminator sequence (SEQ ID NO: 8). The pYL71 vector also contains a vector backbone with the bacterial ColEl ori and AmpRgene for replication and selection in E. coli.

[0232] Additionally, the vector was designed to enable targeted integration of the Ace3L-intein expression cassette at the sdil locus in T. reesei. The sdil gene encodes an iron-sulfur protein subunit of succinate dehydrogenase, wherein a single point mutation in the gene and its resulting amino acid substitution (z.e, H245L substitution) in the T. reesei Sdil protein confers resistance to the fungicide carboxin. For example, the same amino acid substitution (H245L) homolog was previously reported to impart resistance to carboxin in Ustilago maydis and other fungi (Broomfield and Hargreaves, 1992). This vector harbors a partial coding sequence of T. reesei sdil gene ( / '-upstream-fragment, 5' flank) and a downstream sequence of sdil gene (w / z-downstream-fragment, 3' flank). The point mutation at the sdi -upstream- fragment sequence confers carboxin resistance upon integration into the sdil gene locus. Selection of sdil in T. reesei may be performed as described in Kilaru et al. (2020).

[0233] Thus, a parental T. reesei glucoamylase (GA) expressing strain was transformed with the expression cassette of sdil-Ace3L-C%Q-iF19 amplified from vector pYL71 (FIG. 11). Transformation was performed using the PEG-mediated protoplast transformation protocol (Ouedraogo et al., 2015; Penttila et al., 1987). Transformants were selected for carboxin resistance, and further purified as previously described above in Example 2. The parental GA expressing reporter strain and the modified GA reporter strain (Ace3L-C80-iF19) were grown in microtiter plates (MTPs) at a permissive splicing condition (temperature of 27.5°C) and supernatant from the cell cultures were harvested and analyzed using PAGE as generally described above in Example 2.

[0234] As shown in FIG. 12, the parental cells produced a large amount of the GA reporter protein in defined medium with glucose / sophorose (Sop, inducing condition), and only a small amount of GA reporter in defined medium with glucose (Glu, non-inducing condition). In contrast, the modified LT351 strain (Ace31.-C80-IF 19) driven from the did promoter, produced about 1.6-fold higher GA reporter under the inducing (Sop) conditions (relative to the parental strain under inducing (Sop) conditions), andproduced similar amounts of GA reporter under non-inducing (Glu) conditions (relative to the parental strain under inducing conditions), or 3-fold higher GA reporter under non-inducing (Glu) conditions (relative to the parental strain under non-inducing conditions). These results demonstrate that the modified LT351 strain comprising the Ace3L-C80-iF19 cassette produces extracellular (secreted) proteins (GA reporter) in the absence of an inducer, and also produces more total protein than the parental strain under inducing (Sop) conditions. These results further confirm that the expressed Ace3L-C80-iF19 protein successfully spliced out the iF19 intein, and correctly ligated the Ace3L (extein) under the permissive splicing condition (e.g., temperature). For example, the functional Ace3L TF expressed in T. reesei strains has been shown to increase protein production in the absence of an inducing substrate, which is a described benefit of the functional Ace3L variant TF protein (PCT Publication No. WO2018 / 067599).EXAMPLE 5FILAMENTOUS FUNGAL CELLS EXPRESSING A HETEROLOGOUS REPORTER PROTEIN AND A TEMPERATURE REGULATED ACE3L- INTEIN

[0235] In the instant example, the parental GA expressing strain, modified LT351 strain (Ace3L-C80- iF19) and modified LT182 strain (Ace3L) described in Example 4, were fermented in large scale bioreactors (-10-15 L), as generally described in U.S. Patent No. 7,713,725. More specifically, cells were grown in a defined medium with glucose as the carbon source at 34°C, followed by a protein production phase at 28°C with either a glucose / sophorose feed (Sop; inducing) or a glucose feed (Glu, non-inducing) as carbon sources. The growth temperature of 34°C is a non-permissive temperature for intein iF19 splicing, and the production temperature of 28°C is a permissive temperature for intein iFl 9 splicing. In particular, FIG. 13 shows the relative growth rates of the parental and modified LT182 (Ace3L) and LT351 (Ace3L-C80-iF19) strains at 34°C, a non-permissive temperature for intein iF19 splicing. For instance, the growth rate of parental stain was artificially set as 100, and the relative growth rates of the modified LT182 and LT351 were plotted. As shown in FIG. 13, the modified LT182 (Ace3L) demonstrated an approximately 10% reduced growth rate, while the modified LT351 (Ace3L-C80-iF19) demonstrated an approximately 20% increased growth rate in comparison to the parental strain. The total protein yield was calculated as grams of protein produced divided by grams of total glucose consumed during growth and protein production phases. For example, the total protein yields obtained by the parental strain on glucose / sophorose (i.e.. under inducing conditions) at the end of fermentation (EOF) was arbitrarily set at 100, and the relative protein yields by modified LT 182 strain (Ace3L) or modified LT351 strain (Ace3L-C80-iF19) were plotted as shown in FIG. 14. For example, when the strains were evaluated under inducing conditions (“Sop”, glucose / sophorose feed), the modified LT182 strain (Ace3L) showed a 1.15-fold (15%) improvement in total protein yields as compared to the parental strain, and the modified LT351 strain (Ace3L-C80-iF19) demonstrated approximately a 1.2-fold (-22%) improved yields compared to the parental strain (FIG. 14). Additionally, when the modified LT351 strain (Ace3L- C80-iF19) was evaluated under non-inducing conditions (“Glu”, Glucose feed), the strain showed anapproximately 7% lower yield relative to the parental strain evaluated under inducing conditions (“Sop”, glucose / sophorose feed; FIG. 15).

[0236] Overall, the results presented herein demonstrate that the modified LT351 strain (Ace3L-C80- iF19 has a higher growth rate compared to the parental strain at a non-permissive temperature for intein iF19 splicing, and produced the GA reporter effectively under both inducing and non-inducing conditions, and showed higher yields compared to the parental strain at a permissive temperature for intein iF19 splicing and for protein production.EXAMPLE 6FILAMENTOUS FUNGAL CELLS EXPRESSING A HETEROLOGOUS PROTEIN AND A TEMPERATURE REGULATED INTEIN-MODIFIED HISTIDINE KINASE FITNESS PROTEIN

[0237] In the instant example, a parental strain expressing a heterologous alpha-amylase enzyme and carrying a variant allele (niklM7431SEQ ID NO: 26) of the native histidine-kinase (Niki) protein as a FPOI was compared to a derivative strain having the coding sequence of the iF19 intein inserted immediately upstream of a codon encoding a cysteine residue of variant histidine-kinase (NiklM743T; SEQ ID NO: 27) protein. More particularly, as presented in FIG. 16, in the instant example the iF19 intein coding sequence was inserted directly upstream of the codon encoding the cysteine residue at position 1,065 (C1065) of SEQ ID NO: 27 (FIG. 17). For example, strains were fermented in large scale bioreactors (-10-15 L), as generally described in U.S. Patent No. 7,713,725. More specifically, cells were grown in a defined medium with glucose as the carbon source at 34°C, followed by a protein production phase at 25°C with glucose / sophorose feed as carbon sources. Growth temperatures of 34°C are expected to be non-permissive for intein iF19 splicing, resulting in the knock-down of NiklM743T, whereas production temperatures of 25°C are expected to be permissive for intein iF 19 auto-splicing, allowing for NiklM743Tmaturation and activity.

[0238] Thus, at growth phase, when comparing growth rates relative to the parental strain (measured by dry-cell-weight accumulation), the growth rate of the m'WM7’r-intein-modified strain was found to be significantly increased, in this case by 25%, indicating effective NiklM743Tknock-down during non- permissive intein-splicing conditions at 34°C. At the end of fermentation (EOF), when comparing total protein yield relative to the parental strain (calculated as grams of protein produced divided by grams of total glucose consumed during growth and protein production phases), the total protein yield of the zzzHM74jr-intein-modified strain was found at the same level, indicating sufficient NiklM743Tmaturation during permissive intein-splicing conditions at 25°C.

[0239] Overall, the results presented herein demonstrate that the zzz7 / W74< / -intein-modil'icd derivative strain has a higher growth rate compared to the parental strain at non-permissive temperatures for intein iF19 splicing, and same -level total protein yield compared to the parental strain at permissive temperatures for intein iF19 splicing.REFERENCESU.S. Patent No. 7,713,725US Patent No. 7,413,879US Patent No. 8,420,387US Patent Publication No. US2004 / 0091966PCT Publication No. WO2011 / 153449Broomfield and Hargreaves, “A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis”, Curr. Genet., 22(2): 117-121, 1992.Hirata et al., “Molecular Structure of a Gene, VMA1, Encoding the Catalytic Subunit of H+-Translocating Adenosine Triphosphatase from Vacuolar Membranes of Saccharomyces cerevisiae”, J. Biol. Chem., Volume 265, Issue 12, pages 6726-6733, 1990.Jorgensen et al., “A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)” , Microbial Cell Factories, 13:33, 2014.Kilaru et al., “Optimised red- and green-fluorescent proteins for live cell imaging in the industrial enzymeproducing fungus Trichoderma reesei” Fungal Genet Biol., Vol. 138, May 2020.Le Crom et al., 2009Luo et al., “Modification of transcriptional factor ACE3 enhances protein production in Trichoderma reesei in the absence of cellulase gene inducer”, Biotechnol. Biofuels, 13:137, 2020.Mills et al., “Protein Splicing: How Inteins Escape from Precursor Proteins”, MINIREVIEW: Intein Mechanisms, J. Biol. Chem., Volume 289, Issue 21, pages 14498-14505, 2014.Needleman and Wunsch, “A general method applicable to the search for similarities in the amino acid sequence of two proteins”, J. Mol. Biol. 48: 443-453, 1970.Ouedraogo et al., 2015Penttila et al., 1987Perler et al., “Compilation and analysis of intein sequences”, Nucleic Acids Research, Volume 5, No. 6, pages 1087-1093, 1997.Shah and Muir, “Inteins: Nature’s Gift to Protein Chemists”, Chem. Sci., 5(1): 446-461, 2014.Sheir-Neiss and Montenecourt, 1984Tan et al., “Temperature-Sensitive Mutations Made Easy: Generating Conditional Mutations by Using Temperature-Sensitive Inteins That Function Within Different Temperature Ranges”, Genetics 18, pages 13-22, 2009.Xu and Perler, “The mechanism of protein splicing and its modulation by mutation”, The EMBO Journal, Volume 15, No. 19, pages 5146-5153, 1996.Zeidler et al., “Temperature-sensitive control of protein activity by conditionally splicing inteins”, Nature Biotechnology, Volume 22, No. 7, pages 871-876, 2004.

Claims

CLAIMS1. A method for modulating the function of a fitness protein of interest (FPOI) in a filamentous fungal cell comprising:(a) obtaining a filamentous fungal cell comprising gene encoding a FPOI, wherein the open reading frame (ORF) of the gene encoding the FPOI comprises at least one wild-type or engineered codon selected from the group consisting of TGT, TGC, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA and ACG, modifying the ORF by inserting a polynucleotide encoding an intein immediately upstream of the at least one codon,(b) fermenting the modified cell for a period of time under a non-splicing intein temperature, wherein the FPOI is non-functional under the non-splicing intein temperature, and(c) fermenting the modified cell for a period of time under an intein splicing temperature, wherein the FPOI is functional under the intein splicing temperature.

2. The method of claim 1, wherein the FPOI is selected from the group consisting of transcriptional activator proteins, transcriptional repressor proteins, kinases, phosphatases, guanyltransferases, glycosyltransferases, glycosidases, histone modification proteins, chromatin remodeling proteins and proteins involved in foaming.

3. The method of claim 1, wherein the FPOI is selected from the group consisting of a xylanase regulator 1 (Xyrl protein, an activator of cellulase 2 (Ace2) protein, an activator of cellulase 3 (Ace3) protein, a variant Ace3-L protein, an activator of cellulase 4 (Ace4) protein, a carbon catabolite repressor 1 (Crel) protein, a histidine kinase (Niki) protein, a GTP:a-D-mannose-l- phosphate guanyltransferase (Mpgl) protein, a beta-glucosidase regulator (BglR) protein, a Trire2_82512 protein, a Trire2_62386 protein, a hydrophobin Hfbl protein and a hydrophobin Hfb2 protein.

4. The method of claim 1, wherein the intein comprises at least 95% identity to SEQ ID NO: 25.

5. The method of claim 4, wherein the intein comprises a tryptophan (W) to arginine (R) substitution at position 157 (W157R) and a glycine (G) to arginine (R) substitution at position 219 (G219R).

6. The method of claim 1, wherein the non-splicing intein temperature is a temperature of 30.0°C and higher.

7. The method of claim 1, wherein intein splicing temperature is a temperature of 29.0°C and lower.

8. The method of claim 3, wherein the Ace3 protein comprises at least 80% identity to SEQ ID NO: 6.

9. The method of claim 8, wherein the Ace3 or Ace3L protein comprises the intein inserted immediately upstream of a cysteine (Cys), a serine (Ser), or a threonine (Thr) residue present in SEQ ID NO: 6.

10. The method of claim 1, wherein the modified cell expresses one or more lignocellulosic degrading enzymes.

11. The method of claim 10, wherein the lignocellulosic degrading enzymes are selected from the group consisting of a cellobiohydrolase, a xylanase, an endoglucanase and a -glucosidase.

12. The method of claim 1, wherein the modified cell comprises an introduced expression cassette encoding a heterologous protein of interest (POI).

13. The method of claim 12, wherein the cassette encoding the heterologous POI comprises an upstream cellulase gene promoter sequence operably linked to a downstream polynucleotide (DNA) sequence encoding the heterologous POI, optionally comprising a terminator (DNA) sequence downstream and operably linked to the polynucleotide encoding the heterologous POI.

14. The method of claim 13, wherein the heterologous POI is an enzyme is selected from the group consisting of acetyl esterases, amylases, aminopeptidase, arabinases, arabinosidases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, cutinases, chondroitinases, deoxyribonucleases, epimerases, esterases, glucoamylases, glucan lysases, glucose oxidases, glucuronidases, hemicellulases, hyaluronidases, invertases, isomerases, keratinases, laccases, lipases, lipoxygenases, ligninases, mannanases, nucleases, oxidases, peroxidases, proteases, phospholipases, polyesterases, phytases, pectinase, phenol oxidases, pullulanases, pentosanases, reductases, ribonucleases, tannases, aglucanases, P-glucanases, a-galactosidases, Pgalactosidases, endo-P-glucanases and xylanases.

15. The method of claim 1, wherein the filamentous fungal cell is selected from the group consisting of a Trichoderma sp. cell, an Aspergillus sp. cell, a Fusarium sp. cell, a Penicilliuni sp. cell, a Chrysosporium sp. cell, a Cephalosporium sp. cell, a Talaromyces sp. cell, a Geosmithia sp. cell, a Neurospora sp. cell and a Myceliophthora sp. cell.

16. A polynucleotide encoding an intein-modified fitness protein of interest (FPOI), wherein the intein-modified FPOI comprises at least one wild-type or genetically engineered cysteine (Cys), serine (Ser) or threonine (Thr) residue and an intcin inserted immediately N-tcrminal to the at least one Cys, Ser or Thr residue.

17. The polynucleotide of claim 16, wherein the FPOI is selected from the group consisting of transcriptional activator proteins, transcriptional repressor proteins, kinases, phosphatases, guanyltransferases, glycosyltransferases, glycosidases, histone modification proteins, chromatin remodeling proteins and proteins involved in foaming.

18. The polynucleotide of claim 16, wherein the FPOI is selected from the group consisting of a xylanase regulator 1 (Xyrl) protein, an activator of cellulase 2 (Ace2) protein, an activator of cellulase 3 (Ace3) protein, a variant Ace3-L protein, an activator of cellulase 4 (Ace4) protein, acarbon catabolite repressor 1 (Crel) protein, a histidine kinase (Niki) protein, a GTP:a-D- mannose-1 -phosphate guanyltransferase (Mpgl) protein, a beta-glucosidase regulator (BglR) protein, a Trire2_82512 protein, a Trire2_62386 protein, a hydrophobin Hfbl protein Hfbl and a hydrophobin Hfb2 protein.

19. The polynucleotide of claim 16, wherein the intein comprises at least 95% identity to SEQ ID NO: 25, and comprises a tryptophan (W) to arginine (R) substitution at position 157 (W157R) and a glycine (G) to arginine (R) substitution at position 219 (G219R).

20. The polynucleotide of claim 18, wherein the Ace3L protein comprises the intein inserted immediately upstream of the at least one Cys, Ser, or Thr residue present in SEQ ID NO: 6.

21. A polynucleotide expression construct comprising an upstream promoter sequence operably linked to a downstream (3') polynucleotide of claim 16, optionally comprising a terminator sequence downstream and operably linked to the polynucleotide of claim 16.

22. The expression construct of claim 21, comprising at least 90% identity to the Ace3L-C80-lF19 cassette of SEQ ID NO: 10.

23. A recombinant filamentous fungal cell comprising an introduced expression cassette of claim 21.

24. The recombinant cell of claim 23, selected from the group consisting of a Trichoderma sp. cell, an Aspergillus sp. cell, a Fusarium sp. cell, a Penicillium sp. cell, a Chrysosporium sp. cell, a Cephalosporium sp. cell, a Talaromyces sp. cell, a Geosmithia sp. cell, a Neurospora sp. cell and a Myceliophthora sp. cell.

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