Recombinant fungal cells and method for industrial-scale production of lectins

Abstract

This disclosure generally relates to methods and compositions for producing a desired heterologous lectin protein in recombinant filamentous fungal cells. Accordingly, specific embodiments relate to compositions and methods for lectin protein production, recombinant filamentous fungal strains including enhanced lectin protein productivity phenotypes, polynucleotides encoding one or more lectin proteins (e.g., expression constructs), industrial-scale fermentation, and lectin protein recovery processes.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of the International Application PCT / CN2023 / 104906 filed on 30 June 2023, which is incorporated herein by reference in its entirety.

[0002] This disclosure generally relates to the fields of microbial cells, molecular biology, fermentation, protein production, protein recovery, protein purification, and protein preparations. Specific aspects of this disclosure relate to the industrial-scale production and recovery of lectin proteins, recombinant filamentous fungal cells producing heterologous lectins, compositions and methods for recovering and / or purifying one or more lectins, and lectin preparations with enhanced purity.

[0003] Sequence listing reference The electronically submitted sequence listing text file named "NB42133-WO-PCT_SequenceListing.xml" was created on June 21, 2023, is 185KB in size, and is incorporated herein by reference in its entirety. [Background technology]

[0004] Lectins are typically defined as carbohydrate-binding proteins that can reversibly and highly specifically recognize and bind to simple or complex carbohydrates, while exhibiting no catalytic activity (Lagarda-Diaz et al., 2017). The lectin protein was originally hemagglutinin, named for its ability to aggregate red blood cells (and other cells). More recently, lectins such as the griffithsin (GRFT) protein from the red alga (Griffithsia genus), the KAA-2 protein from the red alga (Kappaphycus alvarezii), the concanavalin A (ConA) protein from Canavalia gladiola, the jacarin protein from A. heterophyllus, and the cyanobilin-N (CV-N) protein from N. ellipsosporum have been evaluated for their antiviral activity (Whitley et al., 2013).

[0005] For example, International Publication No. 2005 / 118627 and International Publication No. 2007 / 064844 describe a method for isolating natural griffithsin (GRFT) lectin from red algae (Griffithsia species), cloning its wild-type (grft) gene to produce recombinant polynucleotides, fermenting and producing these in E. coli host cells, and then isolating recombinant His-tagged GRFT protein from the E. coli host to characterize its antiviral activity. International Publication No. 2010 / 01424 generally describes a method for inhibiting hepatitis C virus infection in a host, which involves administering an effective amount of glycosylation-resistant GRFT (variant) protein (or its polypeptide conjugate) to the host in combination with another antiviral protein. For example, as described in this publication, the inventors focused on the fact that the antiviral protein combination of citobilin (SVN) and griffithcin (GRFT) has (nanomolecal) activity against hepatitis C virus (HCV).

[0006] U.S. Patent Application Publication No. 20110263485 describes a method for inhibiting human immunodeficiency virus (HIV) infection in a host, comprising administering to the host an effective amount of gp120 griffiscin and a peptide selected from gp41 binding protein, CCR5 binding protein, gp120 binding protein, or another griffiscin, the combination of which is a potent inhibitor of HIV infection. International Publication No. 2016 / 130628 discloses a variant griffiscin protein having mutations that alter the isoelectric point of the GRFT protein, which has been reported to modify its solubility within a range of pH values ​​that allow for improved product release. International Publication No. 2019 / 108656 generally describes microbiotic compositions comprising endosperm extracts and anti-HIV lectins, anti-HIV antibodies, or antigen-binding antibody fragments thereof.

[0007] Recombinant production of GRFT in tobacco plants (Nicotiana benthamiana) was described by O'Keefe et al. (2009), and GRFT, when expressed via an infectious tobacco mosaic virus (TMV)-based vector, accumulates to levels of approximately 1 gram of recombinant GRFT per kilogram of Nicotiana benthamiana leaf material. Despite the prospect of biologics such as griffiscin becoming HIV prophylactic agents, as intended in the publication of O'Keefe et al., practical use as topical fungicides is hindered by high production costs, and any manufacturing system that relies on growth under sterile conditions is unlikely to compete with the price of male condoms (which is essential if the product is available for use by people at risk of sexual transmission of HIV).

[0008] Hirayama et al. (2016) described the primary structure of KAA-2 lectin, elucidated using peptide mapping and complementary DNA (cDNA) cloning, and prepared its active recombinant form using an E. coli expression system. Gengenbach et al. (2019) described the transient expression of mistletoe lectin named "viscumin" (Viscum album) in intact tobacco (Nicotiana benthamiana) plants and the purification of recombinant viscumin from crude plant extract by affinity chromatography, comparing the performance and economics of the tobacco plant-based process with a corresponding E. coli expression-based process. As summarized by Gengenbach et al., the E. coli process had low recovery rates, required extensive dilution, and was complex, while the plant-based process involved only half the number of steps. According to a direct cost comparison between the two processes performed in the Gengenbach et al. study, the plant expression system was 50% cheaper than heterologous expression in natural host mistletoe (V. album) or Escherichia coli (E. coli) host cells.

[0009] Petrova et al. (2016) described a probiotic, Lactobacillus rhamnosus strain (GR-1), which was recorded to survive transplantation onto vaginal epithelium and interfere with urogenital pathogens. As described in this publication, a genomic region encoding a protein homologous to a lectin-like protein was identified (i.e., the llp1 gene encoding lectin-like protein 1 (Llp1)), and phenotypic analysis of a knockout mutant of the L. rhamnosus (GR-1) parent strain (GR-1_Δllp1) revealed a 2-fold decrease in adhesion to vaginal and uterovaginal epithelial cell lines compared to the wild type.

[0010] Recently, Petrova et al. (2018) described a probiotic L. rhamnosus strain expressing actinohibin (AH) or griffithsin (GRFT) (HIV inhibitory lectins) for in-situ delivery. As generally summarized in this publication, the L. rhamnosus strain is unable to produce intracellular or extracellular AH, and the observed results suggest that AH may be toxic during its transport from the L. rhamnosus cell wall by binding to essential glycosylated cell wall molecules such as peptidoglycans, extracellular polysaccharides, or glycosylated proteins in the Sec pathway. Petrova et al. (2018) further described L. rhamnosus strains constructed for extracellular expression of GRFT, and the results obtained suggest that L. rhamnosus strains can synthesize GRFT but do not secrete it from cells under the conditions tested. For example, as described in this publication, GRFT-corresponding bands were detected in the cell wall fraction of recombinant strains CMPG10731, CMPG10734, CMPG10767, and CMPG10768. This suggests the possibility of recombinant protein trapping in the cell wall. [Overview of the project] [Problems that the invention aims to solve]

[0011] Based on the above, it is clear that there remains an ongoing and unmet need in the art for improving host organisms capable of producing heterologous lectins for various antimicrobial applications, as well as an ongoing and unmet need for novel methods and compositions that enable cost-effective industrial-scale production, recovery, and / or purification of lectins. [Means for solving the problem]

[0012] As described and illustrated hereafter, the Disclosure provides, among other things, novel recombinant (modified) filamentous fungal cell lines expressing heterologous lectin proteins, polynucleotides (e.g., vectors, expression cassettes) containing nucleic acids encoding heterologous lectins, methods and compositions for causing heterologous lectins to be produced in recombinant filamentous fungal host cells, methods and compositions for recovering and optionally purifying lectins and their lectin protein preparations. As generally shown and described hereafter, one or more specific embodiments of the Disclosure provide recombinant filamentous fungal cells producing heterologous lectin proteins, the lectin proteins being secreted into a fermentation broth when grown / cultured / fermented under conditions suitable for lectin production. In certain other embodiments, the heterologous lectin proteins (and nucleic acids (DNA) encoding heterologous lectins) are derived from plant cells, cyanobacteria cells, algal cells, bacterial cells, insect cells, and animal cells, etc.

[0013] In one or more other specific embodiments, heterologous lectin proteins include: natural griffithsin (GRFT) lectin or functional GRFT variants derived therefrom; natural cytobilin (SVN) lectin or functional SVN variants derived therefrom; natural cyanobilin-N (CVN) lectin or functional CVN variants derived therefrom; natural K. alvarezii KAA-1 lectin or functional KAA-1 variants derived therefrom; natural K. alvarezii KAA-2 lectin or functional KAA-2 variants derived therefrom; natural Microcystis viridis (MVL) lectin or functional MVL variants derived therefrom; natural DCSIGN lectin or functional DCSIGN variants derived therefrom; natural Boodlea coacta agglutinin (BCA) lectin or functional BCA variants derived therefrom; natural Artocarpus Jacarina heterophyllus lectin or functional jacarina variants derived therefrom, Musa acuminata banana lectin or functional banana variants derived therefrom, Aaptos papilleta sponge lectin or functional sponge variants derived therefrom, Abrus precatorius lectin or functional apricot variants derived therefrom, Aegapodium podagraria lectin or functional apricot variants derived therefrom, Agaricus bisporus mushroom lectin or functional mushroom variants derived therefrom, Albizzia julibrissin lectin or functional albizzia julibrissin variants derived therefrom, Allomyrina dichotoma lectin or functional apricot variants derived therefrom, Aloe vera Aloe vera (Aloe plant) lectin or functional aloe plant variant derived therefrom, natural wild soybean (Amphicarpaea)(bracteata) lectin or functional wild bean variant derived therefrom, wild eel (Anguilla) lectin or functional eel variant derived therefrom, wild sea hare (Aplysia depilans) lectin or functional mollusca variant derived therefrom, wild peanut (Arachis hypogaea) lectin or functional peanut variant derived therefrom, wild purple porcelain (Bauhinia purpurea) lectin or functional purple porcelain variant derived therefrom, wild Bryonia diocia (white bryony) lectin or functional white bryony variant derived therefrom, wild giant hawk moth (Caragana Arborescens) lectin or functional giant hawk moth variant derived therefrom, wild horseshoe crab (Carcinoscorpius) Horseshoe crab lectin or functional horseshoe crab variant derived therefrom, natural Microcystis aeruginosa (cyanobacteria) microbilin (MVN) lectin or functional MVN variant derived therefrom, natural Eucheuma serra (red algae) ESA-2 lectin or functional ESA-2 variant derived therefrom, natural Musa acuminate (banana) BanLec lectin or functional BanLec variant derived therefrom, natural Aspidistra elatior AEL lectin or functional AEL variant derived therefrom, natural Chaetopterus variopedatus (marine worm) CVL lectin or functional CVL variant derived therefrom, broad bean (Vicia faba) lectin or functional broad bean variant derived therefrom, natural lentil (Lens culinaris) or functional lentil variants derived therefrom, natural pea (Pisum)Examples include, but are not limited to, sativum lectin or functional pea variants derived therefrom, jacarin-like lectin or functional jacarin-like variants derived therefrom, CVN-like lectin or functional CVN-like variants derived therefrom, OAA-like lectin or functional OAA-like variants derived therefrom, galectin-1-like lectin or functional galectin-1-like variants derived therefrom, and lysine-like lectin or functional lysine-like variants derived therefrom.

[0014] In certain other embodiments, one or more polynucleotides (e.g., an expression cassette) encoding a heterologous lectin protein are introduced into the cell. In certain relevant embodiments, such a polynucleotide (DNA) expression cassette encoding a heterologous lectin protein comprises at least an upstream (5') promoter region sequence operably ligated to a downstream nucleic acid encoding a secretory (signal peptide) sequence operably ligated to a downstream (3') nucleic acid encoding the heterologous lectin protein, and optionally comprises a terminator sequence located downstream and operably ligated to a nucleic acid encoding the heterologous lectin protein. In certain embodiments, the preferred upstream promoter region (DNA) sequence is a functionally strong promoter region sequence in recombinant cells. Examples of strongly promoter functional Trichoderma species cells include, but are not limited to, cellobiohydrolase promoters (e.g., chb1 promoter, chb2 promoter), endoglucanase promoter (e.g., eg2 promoter), β-glucosidase promoter (bgl1 promoter), xylanase promoter (e.g., xyn3 promoter), rev3 promoter, bxl promoter, tkl1 promoter, dld1 promoter, axe1 promoter, hxk1 promoter, dic1 promoter, opt promoter, gut1 promoter, and pki1 promoter. In other embodiments, nucleic acid (DNA) sequences encoding secretory (signal peptide) sequences encode functional signal peptide sequences in recombinant filamentous fungal cells. Functional secretory (signal peptide) sequences are protein signal sequences that can be utilized by filamentous fungal (host) cells for extracellular secretion of heterologous lectin proteins into fermentation broth (supernatant). For example, functional secretory (signaling) peptide sequences in Trichoderma species cells include, but are not limited to, the cellobiohydrolase-1 secretory sequence (cbh1ss) of SEQ ID NO: 15 and the aspartate endopeptidase secretory sequence (pep1ss) of SEQ ID NO: 16.

[0015] In certain other embodiments, heterologous lectin proteins are expressed as monomers, dimers, fusion proteins, or combinations thereof. In other embodiments, the recombinant cells of the Disclosure include gene modifications that cause the cells to lose the production of one or more endogenous proteins (e.g., one or more endogenous secretory enzymes).

[0016] Therefore, other embodiments relate to expression cassettes encoding heterologous lectin proteins. In one or more embodiments, the expression cassette encoding heterologous lectin proteins comprises at least an upstream promoter region sequence (functional in ascomycetes cells) operably ligated to a downstream nucleic acid (functional in ascomycetes cells) encoding a secretion sequence operably ligated to a downstream nucleic acid (functional in ascomycetes cells) encoding a heterologous lectin.

[0017] Therefore, certain other embodiments of this disclosure provide, among other things, a method for producing heterologous lectin proteins in filamentous fungal cells. In certain embodiments, such a method includes, but is not limited to, obtaining filamentous fungal (host) cells, introducing one or more expression cassettes encoding heterologous lectin proteins into the cells for secretory expression, and growing / culturing / fermenting the modified cells under conditions suitable for lectin protein production. In certain one or more embodiments of the method, the polynucleotide (DNA, vector, expression cassette) encoding the heterologous lectin protein includes at least an upstream promoter region sequence operably ligated to a downstream nucleic acid encoding a secretory (signal peptide) sequence operably ligated to a downstream nucleic acid encoding the heterologous lectin protein. In certain relevant embodiments or aspects, the lectin protein is secreted into a fermentation broth (i.e., grown / cultivated / fermented under conditions suitable for lectin production). In other embodiments of the method, one or more heterologous lectin proteins are derived from plant cells, cyanobacteria cells, algal cells, bacterial cells, fungal cells, insect cells, or animal cells. In yet another embodiment, one or more expression cassettes encoding heterologous lectin proteins are incorporated into the cell's genome. In certain other embodiments of the method, heterologous lectin proteins may be expressed as lectin monomers, lectin dimers, and lectin trimers, etc. In other relevant embodiments, heterologous lectin proteins may be expressed as lectin fusion proteins. In certain other relevant embodiments, lectin multimers (i.e., lectin dimers, lectin trimers) may be constructed using an amino acid linker sequence inserted between the N-terminus and C-terminus of a lectin protein (e.g., first and second lectin protein sequences having a linker sequence containing about 4-6 residues). Similarly, lectin fusion proteins may be constructed to have an N-terminal (protein) fusion and / or a C-terminal (protein) fusion.

[0018] In other embodiments of the method, the recombinant (modified) filamentous fungal (host) cells comprise one or more genetic modifications that result in the absence of production of one or more endogenous proteins (e.g., secreted endogenous enzymes) in the cells. Thus, other embodiments of the method include, but are not limited to, harvesting the final fermentation broth containing the secreted lectin, performing a clarification process on the harvested broth, performing a concentration process on the clarified broth, and recovering the lectin from the clarified and concentrated broth and its protein preparations, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] [Figure 1]The mature amino acid sequences of exemplary lectins, such as wild-type red algae (Griffithsia species) griffithsin protein (GRFT, SEQ ID NO: 1), variant X31A griffithsin protein (A-GRFT, SEQ ID NO: 2), variant M78Q griffithsin protein (Q-GRFT, SEQ ID NO: 4), synthetic griffithsin dimer protein (GRFT.op1-dimer, SEQ ID NO: 6), wild-type Kappaphycus alvarezil KAA-2 protein (SEQ ID NO: 4), and wild-type Kappaphycus alvarezil KAA-1 protein (SEQ ID NO: 10), are shown. As shown in Figure 1, the wild-type griffithsin (WT GRFT) protein contains 121 amino acid residues, and the amino acid "X" at position 31 of WT GRFT is an unknown, naturally occurring amino acid residue (Figure 1A, SEQ ID NO: 1). As shown in Figure 1B, variant X31A griffithsin (A-GRFT, SEQ ID NO: 2) contains 121 amino acid residues, and the naturally absent residue X at position 31 of WT GRFT (Figure 1A, SEQ ID NO: 1) is replaced by alanine (A) at position 31 of A-GRFT (SEQ ID NO: 2). As shown in Figure 1C, variant M78Q griffithsin (Q-GRFT, SEQ ID NO: 4) contains 121 amino acid residues, and the naturally absent residue X at position 31 of WT GRFT (Figure 1A, SEQ ID NO: 1) is replaced by alanine (A) at position 31 of Q-GRFT (SEQ ID NO: 4), and the methionine (M) residue at position 78 of WT GRFT (Figure 1A, SEQ ID NO: 1) is replaced by glutamine (Q) at position 78 of Q-GRFT (SEQ ID NO: 4). As shown in Figure 1D, a synthetic GRFT-dimer (i.e., fusion protein) was constructed (GRFT.op1-dimer, SEQ ID NO: 6), which contains two copies of GRFT fused with the linker peptide YGPGS. The wild-type KAA-2 lectin protein contains 269 amino acid residues (SEQ ID NO: 8), as shown in Figure 1E, and the wild-type KAA-1 lectin protein contains 134 amino acid residues (SEQ ID NO: 10), as shown in Figure 1F. [Figure 2]Exemplary lectins, such as the wild-type Musa acuminate (Banlec) protein (Figure 2A) containing 144 amino acid residues shown in SEQ ID NO: 23, the wild-type Renouxia sp. (AYR06195) protein (Figure 2B) containing 121 amino acid residues shown in SEQ ID NO: 25, the wild-type Artocarpus integrifolia (jacalin) protein (Figure 2C) containing 157 amino acid residues shown in SEQ ID NO: 27, the wild-type Nostoc ellipsosporum (CV-N) protein (Figure 2D) containing 101 amino acid residues shown in SEQ ID NO: 29, and the wild-type Microcystis viridis (MVL) protein (Figure 2E) containing 114 amino acid residues shown in SEQ ID NO: 31, are shown with their mature amino acid sequences. [Figure 3] A schematic diagram of an exemplary lectin polynucleotide expression cassette of the present disclosure is shown. More specifically, as shown in Figure 3, the expression cassette can be constructed for extracellular expression / secretion of the lectin protein in gram-positive (host) cells. As shown in Figures 3A and 3B, the promoter sequence (abbreviated as "pro"), signal sequence (abbreviated as "sig-seq"), and optional terminator sequence (abbreviated as "term") of the cassette are usually selected to be functional in the desired host. As shown in Figure 3A, the DNA sequence (lectin) encoding the mature lectin protein is operably linked by being placed downstream (3') of the (5') nucleic acid sequence (sig-seq) encoding the (secretion) signal peptide sequence. Similarly, Figure 3B shows a lectin cassette in which the DNA sequence contains an optional transcription terminator (term) placed downstream (3') of the DNA sequence encoding the mature lectin and is operably linked. For enhancing the expression of the cassette in specific filamentous (host) cells, the DNA sequence (lectin) encoding the mature lectin protein can be codon-optimized using techniques and methods known to those skilled in the art. [Figure 4]SDS-PAGE analysis of small-scale expression screening of griffiscin, griffiscin dimer, and KAA-2 lectin-expressing strains without pH treatment is shown, with red arrows indicating the presence of lectins with expected molecular weights. As shown in Figure 4, wild-type griffiscin protein was expressed in cassettes containing two different codon-optimized sequences: lanes 1-5 are clones derived from codon-optimized griffiscin (GRFT-1-GRFT-5, op2), lanes 6-8 are clones derived from codon-optimized griffiscin (GRFT-6-GRFT-8, op1), lane 9 contains the codon-optimized M78Q variant of griffiscin, and lane 10 contains an empty host strain (T. reesei). [Figure 5] Figure 5 shows SDS-PAGE analysis of small-scale expression screening of griffithsin, griffithsin dimer, and KAA-2 lectin-expressing strains treated with pH 2.3. Red arrows indicate the presence of lectins with expected molecular weights. As shown in Figure 5, small-scale cultures of GRFT (codon-optimized sequence: op1), GRFT (codon-optimized sequence: op2), and the variant GRFT-M78Q (codon-optimized sequence: op1) were incubated at pH 2.3. The cultures were centrifuged, and the supernatant was analyzed by SDS-PAGE. Lanes 1, 2, 4, 5, and 6 contain codon-optimized GRFT (op2), lanes 7, 8, and 9 contain codon-optimized GRFT (op1), and lanes 10 and 11 contain codon-optimized GRFT-M78Q variant (op1). A protein band of approximately 14 kDa (similar to the molecular weight of Griffithsin protein, approximately 12.7 kDa) is detected in most lanes (except for lane 6, which showed no expression). Lane 3 contains an empty host T. reesei strain. [Figure 6]Figure 6 shows SDS-PAGE analysis for small-scale expression screening of selected sets of lectins, as shown in the table and listed. As shown in Figure 6, red arrows indicate the presence of lectins with expected molecular weights, with estimated expression levels as low (+), intermediate (++), and high (+++). [Figure 7] SDS-PAGE analysis for a small-scale expression screening of a selected set of lectins fused to the CBM21 protein is shown, and the list of screened lectins is shown in the table in Figure 7. As shown in Figure 7, red arrows indicate the presence of lectins with expected molecular weights, and their expression levels were estimated as low (+), intermediate (++), high (+++), and NA. [Figure 8] This shows the total soluble protein produced by three lectin-expressing strains in a 2-liter fermentation run, including strain BFZ25 (Griffithsin), strain BFZ72 (Griffithsin dimer as a fusion to the CBH1 core protein), and strain BFZ27 (KAA-2 as a fusion to the CBH1 core protein). [Figure 9] This shows the SDS-PAGE analysis of the soluble fraction in a 2-liter fermented orchid sample harvested after 180 hours of fermentation. As shown in Figure 9, lanes 1, 3, and 5 contain the soluble sample after treatment at pH 2.3 and clarification by centrifugation. Lanes 2, 4, and 6 contain pH-neutralized samples from lanes 1, 3, and 5 after treatment with the EndoH (endoglycosylase) enzyme. [Figure 10] This is a schematic diagram of plasmid pGX256. As shown in Figure 10, the abbreviation "TrTEL" is the telomere of Trichoderma reesei, "KanR" is the kanamycin resistance gene, "AmpR" is the ampicillin resistance gene, "pyr2" is the oroto acid phosphoribosyltransferase coding gene, "cbh1 promoter" is the promoter of cellobiohydrolase 1, "cbh1 terminator" is the terminator of cellobiohydrolase 1, and "amdS_Ani_AAA33295" is the acetamidase coding gene. [Figure 11]This is a schematic diagram of plasmid pGX256 containing lectin CDS. As shown in Figure 11, the abbreviation "TrTEL" is the telomere of Trichoderma reesei, "KanR" is the kanamycin resistance gene, "AmpR" is the ampicillin resistance gene, "pyr2" is the oroto acid phosphoribosyltransferase coding gene, "cbh1 promoter" is the promoter of cellobiohydrolase 1, "cbh1 terminator" is the terminator of cellobiohydrolase 1, and "amdS_Ani_AAA33295" is the acetamidase coding gene. [Figure 12]SDS-PAGE analysis of AFP-fusion lectins is shown. More specifically, as shown in Figure 12, the following lectins were expressed: Griffithsia species lectin (GRFT, SEQ ID NO: 1), wild-type Meristotheca papulosa lectin (MPA-2, SEQ ID NO: 58), variant griffithcin M78Q (Q-GRFSEQ, SEQ ID NO: 4), wild-type Canavalia ensiformis lectin (concanavalin A, SEQ ID NO: 60), wild-type Nostoc ellipsosporum lectin (CV-N, SEQ ID NO: 29), wild-type Artocarpus integrifolia lectin (jacarin, SEQ ID NO: 27), wild-type Microcystis viridis lectin (MVL, SEQ ID NO: 31), and wild-type Musa acuminate lectin (Banlec, SEQ ID NO: 23), wild-type Cytonema varium lectin (Citbilin, SEQ ID NO: 54), wild-type Oscillatoria agardhii lectin (OAA-like lectin, SEQ ID NO: 62), wild-type Homo sapiens lectin (DCSIGN, SEQ ID NO: 35), wild-type Eucheuma serra lectin (ESA-2, SEQ ID NO: 64), wild-type Kappaphycus alvarezil lectin (KAA-1, SEQ ID NO: 10), wild-type Microcystis aeruginosa lectin (MVN, SEQ ID NO: 33), wild-type Eucheuma denticulatum lectin (EDA2, SEQ ID NO: 56), and wild-type Mus (Mus) lectin Musculus lectin (galectin-1, SEQ ID NO: 66). [Figure 13]The SDS-PAGE analysis of CBM21 fusion lectins is shown. More specifically, as shown in Figure 13, the following lectins were expressed: variant griffithsin M78Q (SEQ ID NO: 4), wild-type CV-N lectin (SEQ ID NO: 29), wild-type MVL lectin (SEQ ID NO: 31), wild-type cytobilin lectin (SEQ ID NO: 54), wild-type DCSIGN lectin (SEQ ID NO: 35), wild-type KAA-1 lectin (SEQ ID NO: 10), wild-type EDA2 lectin (SEQ ID NO: 56), wild-type MPA-2 lectin (SEQ ID NO: 58), wild-type concanavalin A lectin (SEQ ID NO: 60), wild-type Banlec lectin (SEQ ID NO: 23), wild-type OAA-like lectin (SEQ ID NO: 62), wild-type ESA-2 lectin (SEQ ID NO: 64), wild-type MVN lectin (SEQ ID NO: 33), and wild-type galectin-1 lectin (SEQ ID NO: 66). [Figure 14] This shows a hemagglutination assay for wild-type jacarin protein (SEQ ID NO: 27). [Figure 15] This shows hemagglutination assays for GRFT protein (SEQ ID NO: 1), Q-GRFT protein (SEQ ID NO: 4), KAA-2 protein (SEQ ID NO: 8), KAA-1 protein (SEQ ID NO: 10), jacarin protein (SEQ ID NO: 27), CV-N protein (SEQ ID NO: 29), and DCSIGN protein (SEQ ID NO: 35). [Modes for carrying out the invention]

[0020] A brief explanation of biological sequences Sequence ID 1 is the amino acid sequence of the wild-type mature griffithsin (WT-GRFT) protein isolated from a species of the genus Griffithsia. Sequence ID 2 is the amino acid sequence of the (X31A) variant GRFT (A-GRFT) protein, which has an alanine (A) substitution at position 31 of the unknown "X" amino acid residue of Sequence ID 1. Sequence ID 3 is the DNA sequence encoding the A-GRFT protein, a variant of Sequence ID 2, and its DNA sequence has been codon-optimized for expression in Trichoderma cells. Sequence ID 4 is the amino acid sequence of the (M78Q) variant griffithsin (Q-GRFT) protein, which has a substitution of methionine (M) to glutamine (Q) at amino acid position 78. Sequence ID 5 is the DNA sequence encoding the Q-GRFT protein, a variant of Sequence ID 4, and its DNA sequence has been codon-optimized for expression in Trichoderma cells. Sequence ID 6 is the amino acid sequence of a synthetic Griffith sin dimer protein named "GRFT.op1-dimer". Sequence ID 7 is a synthetic DNA sequence that encodes the Griffith cin dimer protein (GRFT.op1-dimer) of Sequence ID 6. Sequence ID 8 is the amino acid sequence of wild-type tropical sedge (Kappaphycus alvarezil) KAA-2 lectin. Sequence ID 9 is a synthetic DNA sequence encoding the KAA-2 lectin of Sequence ID 8, and its DNA sequence has been codon-optimized for expression in Trichoderma cells. Sequence ID 10 is the amino acid sequence of wild-type Kappaphycus alvarezil KAA-1 lectin. Sequence ID 11 is a synthetic DNA sequence encoding the KAA-1 lectin of Sequence ID 10, and its DNA sequence has been codon-optimized for expression in Trichoderma cells. Sequence ID 12 is the amino acid sequence of the T. reesei Cbh1 core domain. Sequence ID 13 is the DNA sequence that encodes the T. reesei Cbh1 core domain of Sequence ID 12. Sequence ID 14 is the DNA sequence of the T. reesei promoter (Pcbh1). Sequence ID 15 is a DNA sequence that encodes the T. reesei Cbh1 signal sequence (Cbh1ss). Sequence ID 16 is a DNA sequence that encodes the T. reesei Pep1 signal sequence (Pep1ss). Sequence ID 17 is the DNA sequence of the T. reesei cbh1 terminator (Tcbh1). Sequence ID 18 is the DNA sequence of the T. reesei pyr2 gene. Sequence ID 19 is a DNA sequence that encodes the KEX2 cleavage site sequence in budding yeast (S. cerevisiae). Sequence ID 20 is the amino acid sequence of the KEX2 site in budding yeast (S. cerevisiae) with improved cleavage efficiency. Sequence ID 21 is a synthetic DNA sequence that encodes the amino acid linker sequence (YGPGS, Sequence ID 22) of the GRFT dimer protein. Sequence ID 22 is the amino acid linker sequence of the GRFT dimer (fusion) protein (GRFT.op1-dimer, Sequence ID 6). Sequence ID 23 is the amino acid sequence of Banlec lectin from wild-type Malayan pine tree (Musa acuminate). Sequence ID 24 is a synthetic DNA sequence that encodes the Banlec lectin of Sequence ID 23. Sequence ID 25 is the amino acid sequence of AYR06195, a lectin from the wild-type red alga *Renouxia* species. Sequence ID 26 is a synthetic DNA sequence that encodes the red algae lectin of Sequence ID 25. Sequence ID 27 is the amino acid sequence of wild-type copra honey (Artocarpus integrifolia) jacarin lectin. Sequence ID 28 is a synthetic DNA sequence that encodes the jacarin lectin of Sequence ID 27. Sequence ID 29 is the amino acid sequence of wild-type Nostoc ellipsosporum CV-N lectin. Sequence ID 30 is a synthetic DNA sequence that encodes the CV-N lectin of Sequence ID 29. Sequence ID 31 is the amino acid sequence of wild-type Microcystis viridis MVL lectin. Sequence ID 32 is a synthetic DNA sequence that encodes the Microcystis viridis MVL lectin of Sequence ID 31. Sequence ID 33 is the amino acid sequence of wild-type Microcystis aeruginosa MVN lectin. Sequence ID 34 is a synthetic DNA sequence that encodes the Microcystis aeruginosa MVN lectin, as shown in Sequence ID 33. Sequence ID 35 is the amino acid sequence of wild-type human (Homo sapiens) DCSIGN lectin. Sequence ID 36 is a synthetic DNA sequence that encodes the human (Homo sapiens) DCSIGN lectin of Sequence ID 35. Sequence ID 37 is a synthetic guide RNA sequence (TrC114F) that targets a single gene locus in T. reesei. Sequence ID 38 is a synthetic (DNA) plasmid named "pLH1061" [pI1-Pcbh1-GRFT.op1]. Sequence ID 39 is a synthetic (DNA) plasmid named "pLH1065" [pI1-Pcbh1-GRFT.M78Q-op1]. Sequence ID 40 is a synthetic (DNA) plasmid named "pLH1098" [pI1-Pcbh1-CBH1 core-KEX2-GRFT.op1-linker-GRFT.op1]. Sequence ID 41 is a synthetic (DNA) plasmid named "pLH1080" [pI1-Pcbh1-CBH1 core-KEX2-KAA2]. Sequence ID 42 is a 5' DNA primer named "OT4268" [a 5' primer for a pI1 integration cassette at a selected target locus (TrC114F)]. Sequence ID 43 is a 3' DNA primer named "OT4269" [a 3' primer for a pI1 integration cassette at the target locus (TrC114F)]. Sequence ID 44 is a 5' DNA primer named "OT4333" [a 5' diagnostic primer for integration cassettes at the TrC114F locus]. Sequence ID No. 45 is a 3' DNA primer named "OT4334" [a 3' diagnostic primer for integration cassettes at the TrC114F locus]. Sequence ID 46 is a synthetic DNA integration cassette named "HRD1" [a chromosomal integration cassette for GRFT.op1]. Sequence ID 47 is a synthetic DNA integration cassette named "HRD2" [chromosome integration cassette for GRFT.M78Q.op1]. Sequence ID 48 is a synthetic DNA integration cassette named "HRD3" [a chromosomal integration cassette for CBH1 core-KEX2-GRFT.op1-linker-GRFT.op1]. Sequence ID 49 is a synthetic DNA integration cassette named "HRD4" [chromosome integration cassette for CBH1 core-KEX2-KAA2]. Sequence ID 50 is a synthetic DNA integration cassette named "HRD5" [a chromosomal integration cassette for GRFT.op2]. Sequence ID 51 is the codon-optimized DNA sequence [GRFT.op2] encoding the variant griffith cin (A-GRFT) protein. Sequence ID 52 is the amino acid sequence of the starch-binding domain of Rhizopus arrhizus CBM21. Sequence ID 53 is a synthetic DNA sequence encoding the Rhizopus arrhizus CBM21 starch-binding domain, and its DNA sequence has been codon-optimized for expression in Trichoderma cells. Sequence ID 54 is the amino acid sequence of wild-type Cytonema varium Hg-24-1 cytobilin lectin. Sequence ID 55 is a synthetic DNA sequence that encodes the cytonema varium Hg-24-1 cytobilin lectin, which is also sequence ID 54. Sequence ID 56 is the amino acid sequence of wild-type Eucheuma denticulatum EDA2 lectin. Sequence ID 57 is a synthetic DNA sequence that encodes the Eucheuma denticulatum EDA2 lectin, which is also sequence ID 56. Sequence ID 58 is the amino acid sequence of wild-type Meristotheca papulosa MPA-2 lectin. Sequence ID 59 is a synthetic DNA sequence that encodes the Meristotheca papulosa MPA-2 lectin, which is the same sequence as Sequence ID 58. Sequence ID 60 is the amino acid sequence of concanavalin A lectin from wild-type Canavalia ensiformis. Sequence ID 61 is a synthetic DNA sequence encoding the concanavalin A lectin from Canavalia ensiformis, which is also sequence ID 60. Sequence ID 62 is the amino acid sequence of wild-type Oscillatoria agardhii NIES-204 OAA-like lectin. Sequence ID 63 is a synthetic DNA sequence that encodes the Oscillatoria agardhii OAA-like lectin of Sequence ID 62. Sequence ID 64 is the amino acid sequence of wild-type Eucheuma serra ESA-2 lectin. Sequence ID 65 is a synthetic DNA sequence that encodes the Eucheuma serra ESA-2 lectin, which is also sequence ID 64. Sequence ID 66 is the amino acid sequence of wild-type mouse (Mus musculus) galectin-1 lectin. Sequence ID 67 is a synthetic DNA sequence that encodes the mouse (Mus musculus) galectin-1 lectin, which is sequence ID 66. Sequence ID 68 is a DNA sequence that encodes the T. reesei APF signal sequence (APFss). Sequence ID 69 is DNA T. reesei (TrTEL). Sequence ID 70 is a DNA sequence containing the Escherichia coli (E. coli) KanR gene coding sequence (KanR CDS). Sequence ID 71 is a DNA sequence containing the Escherichia coli (E. coli) AmpR gene coding sequence (AmpR CDS). Sequence ID 72 is a DNA sequence containing the Aspergillus nidulans amdS promoter (PamdS) sequence [FGSC A4]. Sequence ID 73 is a DNA sequence containing the A. nidulans amdS terminator [FGSC A4]. Sequence ID 74 is a DNA sequence containing the A. nidulans amdS gene (amdS CDS) which codes for [FGSC A4]. Sequence ID 75 is the amino acid sequence of the A. nidulans AmdS protein [FGSC A4] encoded by Sequence ID 74. Sequence ID 76 is the synthetic (DNA) sequence of plasmid pGX256 [telomere-based cloning vector]. Sequence ID 77 is the synthetic (DNA) sequence of plasmid pLH1053[pI1-Pcbh1-GRF.op2]. Sequence ID 78 is the amino acid sequence of wild-type sunflower (H. annuus) lectin. Sequence ID 79 is a synthetic DNA sequence that encodes wild-type sunflower (H. annuus) lectin.

[0021] As briefly shown above and described hereafter, certain embodiments of the present disclosure provide, among other things, novel recombinant filamentous fungal cells expressing heterologous lectin proteins, where the lectin proteins may be the same lectin, combinations of different lectin proteins, recombinant polynucleotides encoding heterologous lectins for introduction into filamentous fungal host cells for expression of heterologous lectins (e.g., vectors, expression cassettes), fermentation broths containing lectin proteins (and lectin preparations obtained therefrom), lectin proteins recovered from fermentation broths (and lectin preparations obtained therefrom), and purified lectin preparations, etc. Thus, certain embodiments of the present disclosure provide novel methods for the recovery and / or purification of lectins obtained from recombinant filamentous fungal cells expressing one or more lectins.

[0022] I. Definition Before describing the strain and method in detail, the following terms are defined for clarity. Terms not defined should be in accordance with their common meaning as used in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the composition and method apply.

[0023] All publications and patents cited herein are incorporated herein by reference.

[0024] Where a range of values ​​is indicated, unless otherwise explicitly indicated in the context, it is understood that each intermediate value up to one-tenth of the lower limit between the upper and lower limits of that range, and all other specified values ​​or intermediate values ​​within that specified range, are included within the scope of the Composition and Method. These smaller upper and lower limits may independently be included within the smaller range and are also included within the scope of the Composition and Method of the Invention, subject to any limits specifically excluded within the specified range. Where a specified range includes one or both limits, the range excluding one or both of those limits is also included within the Composition and Method.

[0025] In this specification, a particular range is indicated by a number preceded by the term “approximately”. In this specification, the term “approximately” is used to provide literal support for the exact number preceded by it and for numbers that are close to or approximate the number preceded by the term. When determining whether a number is close to or approximates a specifically named number, any close or approximate number that is not named may be a number that, in the context in which it is presented, provides a substantially equal number to the specifically named number. For example, in relation to a number, the term “approximately” means that, unless the term is specifically defined in the context, the range of that number - 10%~ + This refers to a range of 10%. In another example, the phrase "a pH of approximately 6" refers to a pH value between 5.4 and 6.6 unless a specific pH value is defined elsewhere.

[0026] The headings given herein are not intended to limit the various aspects or embodiments of the compositions and methods that can be obtained by referring to the entire specification. Accordingly, the terms defined immediately below are defined in more detail by referring to the entire specification.

[0027] In accordance with the modes for carrying out the invention, the following abbreviations and definitions apply. Note that unless otherwise explicitly indicated in the context, the singular forms "a," "an," and "the" include multiple referents. For example, a reference to "enzyme" includes multiple such enzymes, and a reference to "dosage" includes one or more doses and their equivalents known to those skilled in the art.

[0028] It should also be noted that the claims may be written in a manner that excludes any optional element. Therefore, this statement is intended to serve as a precedent for the use of exclusive technical terms such as “only,” “only,” “except,” and “not include,” or for the use of “negative” limitations in connection with the enumeration of elements of the claims.

[0029] It should be further noted that the term "contains" as used herein means "contains, but is not limited to, the component following the term 'contains'." While the component following the term 'contains' is required or essential, a composition containing that component may further contain other non-essential or optional components.

[0030] It should also be noted that the term "consisting of" as used herein means "including, but not limited to," the component following the term "consisting of." Therefore, the component following the term "consisting of" is required or essential, while other components are not present in the composition.

[0031] As will be apparent to those skilled in the art by reading this disclosure, each of the individual embodiments described and explained herein has individual components and features that can be readily separated from or combined with any features of some other embodiments without departing from the scope or spirit of the compositions and methods described herein. Any of the described methods may be carried out in the order of events described or in any other logically possible order.

[0032] As used herein, the terms “wild-type” and “natural” are used interchangeably and refer to genes, proteins, fungal cells, or strains found in nature.

[0033] 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 manipulated genetic modification or is modified by the introduction of a heterologous nucleic acid molecule, or a cell (e.g., a microbial cell) that has been modified so that the expression of a heterologous or endogenous nucleic acid molecule or gene can be controlled. Recombinants also refer to cells derived from or that are progeny of non-natural cells having one or more such modifications. Genetic modifications include, for example, modifications that introduce an expressible nucleic acid molecule encoding a protein, or the addition, deletion, substitution of other nucleic acid molecules or other functional modifications of the cell’s genetic material. For example, recombinant cells may express genes or other nucleic acid molecules not found in the same or homologous form in natural (wild-type) cells, or they may achieve alterations in the expression patterns of endogenous genes, such as overexpression, underexpression, minimal expression, or no expression at all.

[0034] The creation of "recombinant," "recombined," or "recombined" nucleic acids generally involves assembling two or more nucleic acid fragments, which result in the creation of a chimeric gene.

[0035] As used herein, the term "gene" is synonymous with the term "alleus," which refers to a nucleic acid that codes for and directs the expression of a protein or RNA. Since filamentous fungi generally reproduce vegetatively as haploid organisms, a single copy of a particular gene (i.e., a single allele) is sufficient to confer a specific phenotype.

[0036] As used herein, the term “gene” means a segment of DNA involved in the production of a polypeptide (protein) chain, which may or may not include the regions before and after the coding region (e.g., the 5' untranslated (5'UTR) or “leader” sequence, the 3'UTR or “trailer” sequence, the promoter sequence, and the terminator sequence, etc.) and intervening sequences (introns) between individual coding segments (exons). For example, a gene (DNA) sequence (GOI) of interest may encode a lectin protein of interest, a structural protein, a commercially important industrial protein or peptide, such as an enzyme (e.g., protease, mannanase, xylanase, amylase, glucoamylase, cellulase, oxidase, phytase, lipase, etc.). The gene of interest may be a naturally occurring gene, a mutant (modified) gene, or a synthetic gene.

[0037] As used herein, the term “promoter” refers to a nucleic acid sequence that functions to direct the transcription of a downstream gene or its open reading frame (ORF). Promoters are generally appropriate for host cells in which the target gene is expressed (e.g., filamentous fungal cells). Promoters, along with other transcriptional and translational regulatory nucleic acid sequences (also referred to as “regulatory sequences”), are essential for the expression of a given gene. Generally, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences and terminator sequences, including core promoters and enhancer or activator or repressor sequences, and transcriptional and translational initiation and termination 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.

[0038] As used herein, the term “promoter activity” refers to the ability of a nucleic acid to direct the transcription of a downstream (3') polynucleotide in a host cell. To test promoter activity, recombinant nucleic acids can be generated by operably ligating a (promoter) nucleic acid to a downstream polynucleotide. Recombinant nucleic acids can be introduced into cells to evaluate the transcription of polynucleotides. In certain cases, polynucleotides may encode proteins, and the transcription of polynucleotides can be evaluated by assessing protein production in cells.

[0039] As used herein, the term “operatably linked” refers to a functional linkage between two or more nucleic acid sequences. Therefore, a nucleic acid sequence is operatably linked if it functionally relates to another nucleic acid sequence. For example, a promoter or terminator sequence is operatably linked to a coding sequence if it influences the transcription of that sequence; a ribosome binding site is operatably linked to a coding sequence if it is positioned to facilitate translation; and a nucleic acid sequence encoding a secretion leader (i.e., a signal peptide) is operatably linked to a nucleic acid sequence encoding a polypeptide (e.g., an ORF) if it is expressed as a preprotein involved in the secretion of the polypeptide. Generally, “operatably linked” means that the linked DNA (nucleic acid) sequences are adjacent, and in the case of a secretion leader, they are adjacent and in the leading phase. Enhancers, however, do not need to be adjacent. Linking (i.e., operatably linking) two or more nucleic acid sequences can be achieved using any method to those skilled in the art.

[0040] As used herein, “functional genes” are genes that can be used by cellular components to produce active gene products, typically proteins. In contrast, “non-functional genes” are those that cannot be used by cellular components to produce active gene products (i.e., functional proteins), or have a reduced ability to be used by cellular components to produce active gene products (i.e., functional proteins).

[0041] As used herein, "functional protein" refers to a protein that has a function or activity such as enzyme function / activity, binding function / activity (e.g., DNA binding), and surface activity properties, and has not been modified by mutagenesis, cleavage, or other means to eliminate or reduce its function / activity.

[0042] As used herein, terms such as "modified filamentous fungal cells," "mutant or variant filamentous fungal cells," "recombinant fungal cells," and "modified filamentous fungal strains" may be used interchangeably and refer to filamentous fungal cells derived from (or obtained from) control or parent filamentous fungal cells belonging to the subphylum Pezizomycotina. For example, a "modified" filamentous fungal cell may be derived from (or obtained from) a control or parent filamentous fungal cell, and the modified cell contains at least one gene modification not found in the control or parent cell.

[0043] As used herein, the term "ascomycete fungal cell" refers to all organisms within the phylum Ascomycota in the Kingdom of Fungi. Examples of ascomycete fungal cells include, but are not limited to, filamentous fungi within the subphylum Pezizomycotina, such as species of Trichoderma, Aspergillus, Myceliophthora, and Penicillium.

[0044] As used herein, the term "filamentous fungus" refers to all filamentous forms of the subphylum Eumycota and Oomycota. Examples of filamentous fungi include, but are not limited to, species of the genera Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma. In some embodiments, the filamentous fungus may be Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae.

[0045] In some embodiments, the filamentous fungi are Fusarium species, such as Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, and Fusarium roseum. These include Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, and Fusarium venenatum. In other embodiments, filamentous fungi include Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Scytalidium thermophilum, and Thielavia terrestris.In certain other embodiments, the filamentous fungi include Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0046] Examples of parent Trichoderma reesei strains used herein include, but are not limited to, T. reesei strain QM6a (ATCC deposit number 13631), T. reesei strain RL-P37 (NRRL deposit number 15709), and T. reesei strain RUT-C30 (ATCC deposit number 56765). Examples of parent Aspergillus niger strains include, but are not limited to, A. niger strain ATCC deposit number 1015. Examples of parent Aspergillus oryzae strains include, but are not limited to, A. oryzae strain RIB40 (ATCC deposit number 42149). An exemplary parent Myceliophthora thermophila strain is M. thermophila strain ATCC deposit number 42464, but is not limited to this. For example, Trichoderma strains Rut-RUT-C30 and RL-P37 are mutagenic (cellulase overproduction) derivatives of the Trichoderma native isolate QM6a (Sheir-Neiss and Montenecourt, 1984), and strain NG14 is the immediate common ancestor. In certain embodiments, suitable Trichoderma strains may be derived from and / or obtained from T. reesei strains containing a deletion of the T. reesei pyr2 gene (Δpyr2), as generally described by Sheir-Neiss and Montenecourt (1984) and International Publication No. 2011 / 153449 (the entirety of which is specifically incorporated herein by reference).

[0047] As used herein, the terms “lignocellulose-degrading enzyme,” “cellulase enzyme,” and / or “cellulase” are interchangeable and include glycoside hydrolase (GH) enzymes such as cellobiohydrolase, xylanase, endoglucanase, and β-glucosidase, which hydrolyze the β-(1,4)-linked glycosidic bonds of cellulose (hemicellulose) to produce glucose.

[0048] In certain embodiments, the cellobiohydrolase may be an enzyme classified under Enzyme Committee Number (EC3.2.1.91), the endoglucanase may be an enzyme classified under EC3.2.1.4, the endo-β-1,4-xylanase may be an enzyme classified under EC3.2.1.8, the β-xylosidase may be an enzyme classified under EC3.2.1.37, and the β-glucosidase may be an enzyme classified under EC3.2.1.21.

[0049] As used herein, the "endoglucanase" protein may be abbreviated as "EG," the "cellobiohydrolase" protein as "CBH," the "β-glucosidase" protein as "BG," and the "xylanase" protein as "XYL." Therefore, as used herein, the gene encoding the EG protein (gene CDS or ORF) may be abbreviated as "eg," the gene encoding the CBH protein (or ORF) may be abbreviated as "cbh," the gene encoding the BG protein (or ORF) may be abbreviated as "bg," and the gene encoding the XYL protein (or ORF) may be abbreviated as "xyl."

[0050] As used herein, the terms “polypeptide” and “protein” (and / or their respective plural forms) are used interchangeably to refer to polymers of any length containing amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers modified naturally or by any other operation or modification, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeling components. This definition also includes polypeptides containing one or more analogues of amino acids (e.g., non-natural amino acids) and other modifications known in the art.

[0051] In certain embodiments, one or more lectin proteins of interest may be designed and constructed as fusion proteins. For example, in certain non-limiting embodiments, a lectin fusion protein may comprise an N-terminal fusion of one or more amino acid residues ("N-fusion") and / or a C-terminal fusion of one or more amino acid residues ("C-fusion").

[0052] As used herein, the term "inducible polypeptide / protein" refers to a protein derived from or obtainable from a protein by the addition of one or more amino acids to either or both of the N-terminus and / or C-terminus, the substitution of one or more amino acids at one or more different sites in the amino acid sequence, the deletion of one or more amino acids at one or more ends of the protein or at one or more sites in the amino acid sequence, and / or the insertion of one or more amino acids at one or more sites in the amino acid sequence. The preparation of protein derivatives can be achieved by modifying the DNA sequence encoding a native protein, transforming that DNA sequence into a suitable host, and expressing the modified DNA sequence to form an induceable protein.

[0053] Related (and induced) proteins include "variant proteins." Variant proteins differ from the reference / parent protein (e.g., wild-type protein) by substitutions, deletions, and / or insertions of a small number of amino acid residues. The number of different amino acid residues between a variant protein and the parent 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 may share amino acid sequence identity with the reference protein by at least about 50-60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more. Variant proteins may also differ from the reference protein in selected motifs, domains, epitopes, and conserved regions.

[0054] As used herein, the term “similar sequence” refers to a sequence within a protein that provides similar function, tertiary structure, and / or conserved residues to the protein of interest (i.e., typically the original protein of interest). For example, in an epitope region containing an α-helix or β-sheet structure, the substituted amino acids in the similar sequence preferably maintain the same specific structure. The term also refers to nucleotide sequences and amino acid sequences. In some embodiments, similar sequences are developed such that amino acid substitutions result in a variant enzyme exhibiting similar or improved function. In some embodiments, the tertiary structure and / or conserved residues of amino acids within the protein of interest are located in or near the segment or fragment of interest. Therefore, if the segment or fragment of interest contains, for example, an α-helix or β-sheet structure, the substituted amino acids preferably maintain their specific structure.

[0055] As used herein, the term “homologous protein” refers to a protein that has similar activity and / or structure to a reference protein. Homologs are not necessarily intended to be evolutionarily related. Therefore, the term is intended to encompass the same, similar, or corresponding proteins (i.e., in terms of structure and function) obtained from various organisms. In some embodiments, it is desirable to identify homologs that have a quaternary, tertiary, and / or primary structure similar to that of a reference protein.

[0056] 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 in the Wisconsin Genetics software package (Genetics Computer Group, Madison, WI)). Other lectin gene / protein homologs can be identified by reference to one or more exemplary lectin proteins, as shown and described in Section II below, which are well suitable for production in one or more modified filamentous fungal strains of this disclosure.

[0057] For example, PILEUP is a useful program for determining sequence homology levels. PILEUP creates a multisequence alignment from a group of related sequences using progressive pairwise alignment. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng and Doolittle (1987). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap. Another example of a useful algorithm is the BLAST algorithm. One particularly useful BLAST program is the WU-BLAST-2 program. The parameters "W", "T", and "X" determine the sensitivity and speed of the alignment. The BLAST program uses, by default, word length (W) 11, BLOSUM62 scored matrix alignment (B) 50, expected value (E) 10, M' 5, N' 4, and comparison of both strands.

[0058] With respect to at least two nucleic acids or polypeptides, the terms “substantially similar” and “substantially identical” as used herein typically mean that the polynucleotide or polypeptide contains sequences having at least about 40% to 100% sequence identity. Therefore, in one or more embodiments, a substantially similar or substantially identical nucleic acid or polypeptide of this disclosure contains at least about 40%, 50%, 60%, 70%, 80%, 90%, or 100% identity with one or more sequences described herein. In certain relevant embodiments, one or more nucleic acid sequences and / or one or more protein sequences of this disclosure contain at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 50%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, or 62% identity with one or more sequences described herein. This includes 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. Sequence identity can be determined using standard parameters with known programs such as BLAST, ALIGN, and CLUSTAL. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. Databases can also be searched using FASTA. One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conserved amino acid substitutions are immunologically cross-reactive. Therefore, polypeptides are substantially identical to a second polypeptide if, for example, two peptides differ only by conserved substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions (e.g., within a range of moderate to high stringency).

[0059] As used herein, “nucleic acid” refers to DNA, cDNA, and RNA of genomic or synthetic origin, which may be double-stranded or single-stranded, whether representing nucleotides or polynucleotide sequences and fragments or parts thereof, as well as sense strands or antisense strands.

[0060] As used herein, the term “expression” refers to the transcription and stable accumulation of sense RNA (mRNA) or antisense RNA derived from the nucleic acid molecules of this disclosure. Expression may also refer to the translation of mRNA into polypeptides. Therefore, the term “expression” includes, but is not limited to, all processes involved in polypeptide production, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0061] As used herein, the terms “modification” and “gene modification” are interchangeable and not limited to: (a) introduction, substitution or removal of one or more nucleotides within a gene or introduction, substitution or removal of one or more nucleotides within a regulatory element required for the transcription or translation of a gene; (b) gene disruption; (c) gene transformation; (d) gene deletion; (e) downregulation of a gene (e.g., antisense RNA, siRNA, and miRNA); (f) specific mutagenesis (including, but not limited to, CRISPR / Cas9-based mutagenesis); and / or (g) random mutagenesis of one or more genes disclosed herein.

[0062] As used herein, gene modifications such as "the introduction, substitution, or removal of one or more nucleotides in a protein-coding gene" include the coding sequence (i.e., exons) and non-coding intervening (intron) sequences of a gene.

[0063] As used herein, “gene disruption,” “gene disruption,” “gene inactivation,” and “gene inactivation” are interchangeable and broadly refer to any gene modification that substantially disrupts / inactivates a target gene. Exemplary gene disruption methods include, but are not limited to, the complete or partial loss of any part of a gene, including a polypeptide coding sequence (CDS), promoter, enhancer, or other regulatory element, or the mutagenesis of such gene. Mutagenesis includes substitutions, insertions, deletions, inversions, and all combinations and variations thereof that disrupt / inactivate a target gene and substantially reduce or prevent the expression / production of a functional gene product. In certain embodiments of this disclosure, such gene disruption prevents a host cell from expressing / producing the encoded lov gene product.

[0064] In other embodiments, the protein of interest expressed / produced by the fungal cells of this disclosure (e.g., lectin POI) may be detected, measured, and assayed by protein quantification methods, gene transcription methods, mRNA translation methods, etc., including but not limited to protein transport / mobility (SDS-PAGE), mass spectrometry, HPLC, size exclusion, ultracentrifugation sedimentation velocity analysis, transcriptomics, proteomics, fluorescent tags, epitope tags, and fluorescent protein (GFP, RFP, etc.) chimeric / hybrid methods.

[0065] As used herein, functionally and / or structurally similar proteins are considered “related proteins.” Such related proteins may originate from organisms of different genera and / or species, or even different classes (e.g., bacteria and fungi). Related proteins also include homologs and / or orthologues determined by primary sequence analysis, secondary or tertiary structure analysis, or immunological cross-reactivity.

[0066] As used herein, the term “promoter” refers to a nucleic acid sequence capable of controlling the expression of a coding sequence (CDS) or functional RNA. Generally, the coding sequence (CDS) is located downstream (3') of the promoter (pro) sequence. A promoter may be derived entirely from a native gene, or it may consist of various elements derived from various naturally occurring promoters, or it may further include synthetic nucleic acid segments. It is understood by those skilled in the art that various promoters may direct gene expression in various cell types or developmental stages, or in response to various environmental or physiological conditions. Promoters that cause the most gene expression in most cell types are generally referred to as “constitutive promoters.” Furthermore, since the precise boundaries of regulatory sequences are not fully understood in most cases, it is recognized that DNA fragments of various lengths may possess identical promoter activity.

[0067] The term “introduce,” as defined herein, as used in phrases such as “introduce at least one polynucleotide open reading frame (ORF) or its gene or vector into fungal cells,” includes, but is not limited to, methods known in the art for introducing polynucleotides into cells, such methods including, but are not limited to, protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, and transfection.

[0068] As used herein, “transformed” or “transformed” means that cells have been transformed by the use of recombinant DNA technology. Transformation typically occurs by inserting one or more nucleotide sequences (e.g., polynucleotides, ORFs, or genes) into the cell. The nucleotide sequence to be inserted may be a heterologous nucleotide sequence (i.e., a sequence not naturally present in the transformed cell).

[0069] As used herein, “transformation” refers to the introduction of foreign DNA into a host cell such that the DNA is maintained as a chromosomal integration or a self-replicating extrachromosomal vector. As used herein, “transformed DNA,” “transformed sequence,” and “DNA construct” refer to the DNA used to introduce the sequence into the host cell. The DNA may be produced in vitro by PCR or any other suitable technique. In some embodiments, the transformed DNA includes an incoming sequence, while in other embodiments, the transformed DNA further includes an incoming sequence whose homology box is flanked. In yet another embodiment, the transformed DNA includes other non-homologous sequences (i.e., stuffer sequences or flanks) appended to the ends. The ends may be closed so that the transformed DNA forms a ring, for example, to be inserted into a vector.

[0070] As used herein, “introduced sequence” refers to a DNA sequence introduced into a fungal cell chromosome. In some embodiments, the introduced sequence is part of a DNA construct. In other embodiments, the introduced sequence encodes one or more proteins of interest. In some embodiments, the introduced sequence includes a sequence (i.e., homologous or heterologous) that may or may not already be present in the genome of the transformed cell. In some embodiments, the introduced sequence encodes one or more proteins of interest, genes, and / or mutant or modified genes. In alternative embodiments, the introduced sequence encodes a functional wild-type gene or operon, a functional mutant gene or operon, or a non-functional gene or operon. In some embodiments, the introduced sequence is a non-functional sequence inserted into a gene to disrupt its function. In another embodiment, the introduced sequence includes a selection marker. In further embodiments, the introduced sequence includes two homology boxes.

[0071] As used herein, “homology box” refers to a nucleic acid sequence homologous to a sequence within a fungal cell chromosome. More specifically, a homology box is an upstream or downstream region having approximately 80–100% sequence identity, approximately 90–100% sequence identity, or approximately 95–100% sequence identity to the direct flanking coding region of a gene or part of a gene that is deleted, disrupted, inactivated, and downregulated according to the present invention. These sequences indicate where the DNA construct is incorporated into the fungal cell chromosome and which portion of the fungal cell chromosome is replaced by the incoming sequence. Without intending to limit the present disclosure, homology boxes may range from approximately 1 base pair (bp) to 200 kilobases (kb). Preferably, homology boxes include approximately 1 bp–10.0 kb, 1 bp–5.0 kb, 1 bp–2.5 kb, 1 bp–1.0 kb, and 0.25 kb–2.5 kb. Homology boxes may also include approximately 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb, and 0.1 kb. In some embodiments, homology boxes are flanked at the 5' and 3' ends of the selection marker, and the homology boxes contain nucleic acid sequences that directly flank the coding region of the gene.

[0072] As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence that can be expressed in a host cell, wherein the expression of the selectable marker confers to a cell containing the gene to the ability to grow in the presence of the corresponding selector or in the absence of essential nutrients.

[0073] As used herein, the terms “selectable marker” and “selection marker” refer to nucleic acids (e.g., genes) that can be expressed in host cells and facilitate the selection of a host containing a vector. Examples of such selectable markers include, but are not limited to, antimicrobial agents. Therefore, the term “selectable marker” refers to a gene that indicates that a host cell has taken up the incoming DNA of interest or that some other reaction has occurred. Typically, a selectable marker is a gene that confers antimicrobial agent resistance or metabolic advantage to a host cell, making it possible to distinguish cells containing foreign DNA from cells that have not accepted any foreign sequence during transformation.

[0074] In this specification, the host cell "genome," fungal cell "genome," or filamentous fungal cell "genome" includes chromosomal genes and extrachromosomal genes.

[0075] As used herein, the terms “plasmid,” “vector,” and “cassette” often refer to extrachromosomal elements that carry genes typically not part of the cell’s central metabolism and are usually in the form of a circular double-stranded DNA molecule. Such elements may be linear or circular single-stranded or double-stranded DNA or RNA self-replicating sequences, genomic integration sequences, phages, or nucleotide sequences of any source, in which a number of nucleotide sequences are joined or rejoined into a unique structure that can introduce a promoter fragment and DNA sequence for a selected gene product into the cell along with a suitable 3' untranslated sequence.

[0076] As used herein, the term “vector” refers to any nucleic acid that can replicate (proliferate) within a cell and carry a novel gene or DNA segment (e.g., “introduced sequence”) into the cell. Therefore, the term refers to a nucleic acid construct designed for transport between various host cells. Examples of vectors include viruses, bacteriophages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes such as YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), and PLAC (plant artificial chromosome), which are either “episomes” (i.e., autonomously replicate) or can be incorporated into the chromosomes of host cells.

[0077] As used herein, "transformation cassette" refers to a specific vector that contains a gene and, in addition to that gene, has an element that promotes the transformation of a particular host cell.

[0078] As used herein, "expression vector" refers to a vector capable of incorporating and expressing heterologous DNA within a cell. Many prokaryotic and eukaryotic expression vectors are commercially available and known to those skilled in the art. The selection of an appropriate expression vector is within the scope of the knowledge of those skilled in the art.

[0079] As used herein, the term “expression cassette” refers to a nucleic acid construct (e.g., the vector or vector element described above) that is recombinantly or synthetically constructed and has a set of designated nucleic acid elements that enable the transcription of a specific nucleic acid within a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the DNA construct also includes a set of specific nucleic acid elements that enable the transcription of a specific nucleic acid within a target cell. In certain embodiments, the DNA construct of this disclosure includes a selection marker and an inactivated chromosome segment or gene segment or DNA segment as defined herein.

[0080] As used herein, a “targeted vector” is a vector that contains a polynucleotide sequence homologous to a region in the chromosome of a host cell being transformed, and that can drive homologous recombination in that region. For example, a targeted vector is used to introduce gene modifications into the chromosome of a host cell via homologous recombination. In some embodiments, a targeted vector includes other non-homologous sequences (i.e., stuffer sequences or flanking sequences) that are appended, for example, to the ends. The ends can be closed so that the targeted vector forms a ring, for example, by being inserted into the vector.

[0081] As used herein, the terms “purified,” “isolated,” and “enriched” mean that a biomolecule (e.g., polypeptide or polynucleotide) has been modified from its native state by separating some or all of its naturally occurring components from which it originally associates. Such isolation or purification may be achieved by separation techniques recognized in the art, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, heat treatment, ammonium sulfate precipitation or other protein salting-out, crystallization, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation, to remove unwanted whole cells, cell fragments, impurities, foreign proteins, or enzymes from the final composition. Subsequently, components that impart additional benefits, such as activators, anti-inhibitors, desired ions, pH-adjusting compounds, or other enzymes or chemicals, may be added to the purified or isolated biomolecular composition.

[0082] As used herein, "protein preparation" refers to any material containing one or more proteins, typically a substantially aqueous solution.

[0083] As used herein, the terms “broth,” “culture broth,” “fermentation broth,” and / or “total fermentation broth” may be used interchangeably and refer to preparations produced by cellular fermentation without any post-processing steps after fermentation is complete. For example, total fermentation broth is typically produced when a microbial culture is grown to saturation and incubated under carbon-restricted conditions that allow for protein synthesis (e.g., protein expression by host cells and, by choice, secretion of proteins into the cell culture medium). Typically, total fermentation broth is unfractionated and contains used cell culture medium, metabolites, extracellular polypeptides, and microbial cells.

[0084] As used herein, the term “treated broth” refers to broth that has been conditioned by altering its chemical composition and / or physical properties. “Conditioning” of broth may include one or more treatments such as cell lysis, pH modification, heating, cooling, addition of chemicals (e.g., calcium, salts, flocculants, reducing agents, enzyme activators, enzyme inhibitors, and / or surfactants), mixing, and / or holding the broth for a set period of time (e.g., 0.5 to 200 hours) without further treatment.

[0085] As used herein, the “cell lysis” process includes any cell lysis technique known in the art, including, but not limited to, enzymatic treatment (e.g., lysozyme, proteinase K treatment), chemical means (e.g., ionic liquids), physical means (e.g., French press, ultrasound), and simply holding the culture without feed.

[0086] As used herein, the terms “recovered,” “recovered,” and “recovering” refer to the at least partial separation of proteins from one or more components of a microbial broth and / or the at least partial separation of proteins from one or more solvents (e.g., water or ethanol) in the broth.

[0087] In certain embodiments, a broth from which host cells have been fermented for the production of lectin proteins is clarified, with or without broth treatment. As used herein, “clarified” broth means broth that has been subjected to at least one clarification process to remove cell debris and / or other insoluble components. Clarification processes as understood in the art include, but are not limited to, centrifugation, cross-flow membrane filtration, and solid / liquid filtration.

[0088] "Cell fragments" refer to the cell wall and other insoluble components that are released or formed after the disruption of the cell membrane (for example, after a cell lysis process).

[0089] In certain embodiments, solvent separation as understood in the art includes, but is not limited to, ultrafiltration, evaporation, spray drying, and freeze-drying. The resulting solution is referred to as a "clarified broth concentrate," "UF concentrate," or "ultrafiltration concentrate."

[0090] As used herein, the term "cell aggregate" refers to the cellular components (including intact cells and lysed cells) present in liquid (deep) culture. Cell aggregates can be expressed in terms of dry cell weight (DCW) or wet cell weight (WCW).

[0091] As used herein, the terms "lectin" and "lectin protein" are interchangeable and refer to carbohydrate-binding proteins (or glycoproteins) that can reversibly and highly specifically recognize and bind to simple or complex carbohydrates, while exhibiting no catalytic activity. Therefore, lectin proteins as described herein have the same meaning as lectins as described in the Art (see, for example, Lagarda-Diaz et al., 2017).

[0092] As used herein, the terms "functional lectin variant," and as used in phrases such as "natural lectin and its functional (lectin) variant" and "natural griffithsin (GRFT) protein and its functional GRFT variant," refer to variant (mutant) lectins derived from natural (parent) lectin proteins, and functional (lectin) variants include carbohydrate-binding activity. In certain embodiments, functional lectin variants may include reduced carbohydrate-binding activity compared to the parent (natural) lectin, the same carbohydrate-binding activity compared to the parent (natural) lectin, or increased carbohydrate-binding activity compared to the parent (natural) lectin.

[0093] II. Lectin Proteins As briefly shown above, lectins are proteins (or glycoproteins) that have a non-catalyzed carbohydrate-binding site. As is generally understood in this art, lectins are not the same as antibodies because, unlike enzymes, their carbohydrate-binding properties never change, and they are not induced as an immune response. For example, some of the most well-known lectins are found in leguminous seeds and are thought to play a role in innate immunity and defense mechanisms in plants (Peumans and Van Damme, 1998). More recently, the use of lectins in mitigating viral infections (e.g., HIV, MERS-CoV, SARS-CoV-2, HCV, and Ebola) has attracted considerable attention (see International Publication No. 2005 / 118627, International Publication No. 2007 / 064844, International Publication No. 2010 / 01424, International Publication No. 2016 / 130628, International Publication No. 2019 / 108656, and U.S. Patent Application Publication No. 20110263485). However, as described in the background art above, the economics of recombinant lectin production (e.g., using currently available host expression systems and their downstream recovery processes) has considerably limited the acceptance and / or use of lectins as antimicrobial compositions.

[0094] For example, International Publication No. 2005 / 118627 and International Publication No. 2007 / 064844 describe a method for isolating natural griffithsin (GRFT) lectin from red algae (Griffithsia species), cloning its wild-type (grft) gene to produce recombinant polynucleotides, fermenting and producing these in E. coli host cells, and then isolating the recombinant His-tagged GRFT protein from the E. coli host to characterize its antiviral activity. However, as described in International Publication No. 2005 / 118627 and International Publication No. 2007 / 064844, the recombinant GRFT protein encoded by the nucleic acid of Example 2 (and its C-terminal His-tagged GRFT protein) did not efficiently translocate to the peripheral fraction of Escherichia coli (E. coli) after GRFT protein expression, and most of the produced GRFT protein accumulated in the inclusion cells of Escherichia coli (E. coli) without cleavage of the pelB signal sequence located at the N-terminus of the griffisin protein. Therefore, as previously described, a step was taken to express griffisin in the cytosolic fraction of Escherichia coli (E. coli) using N-terminal (His)-tagged GRFT or a His-tagged variant of GRFT.

[0095] Similarly, International Publication No. 2010 / 01424 describes a method for inhibiting hepatitis C virus infection in a host, comprising administering to the host an effective amount of glycosylation-resistant GRFT protein (or its polypeptide conjugate) in combination with another antiviral protein. For example, as described in this publication, the inventors noted that the antiviral protein combination of citobilin (SVN) and griffiscin (GRFT) has (nanomolecal) activity against hepatitis C virus (HCV). U.S. Patent Application Publication No. 20110263485 further describes a method for inhibiting human immunodeficiency virus (HIV) infection in a host, comprising administering to the host an effective amount of gp120 griffiscin and a peptide selected from gp41 binding protein, CCR5 binding protein, gp120 binding protein, or another griffiscin, the combination of which is a potent inhibitor of HIV infection.

[0096] International Publication No. 2016 / 130628 discloses a variant Griffiths sin protein with mutations that alter the isoelectric point of the GRFT protein, which has been reported to modify its solubility within various pH ranges, enabling improved product release.

[0097] International Publication No. 2019 / 108656 describes, in general, microbiotic compositions comprising endosperm extracts and anti-HIV lectins, anti-HIV antibodies, or antigen-binding antibody fragments thereof. More specifically, the inventors have utilized transgenic plants expressing two or more cyanobilin-N (CVN) proteins, griffiscin (GRFT) proteins, cytobilin (SVN) proteins, and other anti-HIV lectin proteins. However, as stated in International Publication No. 2019 / 108656, the production of such microbiotic components is expensive because it requires a fermenter-based expression platform, downstream processing facilities must adhere to Good Manufacturing Practices (GMP) to ensure the removal of viruses or endotoxins, and the ability, scalability, and cost issues affecting the fermenter are exacerbated when two or three distinct products are required for each fungicide, with individual manufacturing processes.

[0098] Recombinant production of GRFT in tobacco plants (Nicotiana benthamiana) was described by O'Keefe et al. (2009), and when GRFT is expressed via an infectious tobacco mosaic virus (TMV)-based vector, it accumulates to levels of approximately 1 gram of recombinant GRFT per kilogram of Nicotiana benthamiana leaf material. Hirayama et al. (2016) described the primary structure of KAA-2 lectin, elucidated using peptide mapping and complementary DNA (cDNA) cloning, and prepared its active recombinant using an E. coli expression system.

[0099] Gengenbach et al. (2019) described the transient expression of a mistletoe lectin named "viscumin" (Viscum album) in intact Nicotiana benthamiana plants and the purification of recombinant viscumin from crude plant extracts by affinity chromatography, comparing the performance and economics of the tobacco plant-based process with a corresponding process based on Escherichia coli (E. coli) expression. For example, as described by Gengenbach et al., full-length viscumin was produced in Nicotiana benthamiana leaves at levels of up to 7 mg / kg of purified product, and the yield of full-length viscumin was comparable to that of plant lectins expressed in yeast, but approximately six times lower than that of refolded viscumin A and B chains expressed in Escherichia coli (E. coli). As summarized by Gengenbach et al., the E. coli (E. coli) process had low recovery rates, required extensive dilution, and was complex, while the plant-based process involved only half the number of steps. According to a direct cost comparison between the two processes performed in the Gengenbach et al. study, the plant-based expression system was 50% cheaper than heterologous expression in natural host mistletoe (V. album) or E. coli (E. coli) host cells.

[0100] Based on the foregoing, this disclosure addresses a number of ongoing and unmet needs in the art relating to lectin proteins. More specifically, as described herein and shown in the Examples section, the applicant has surprisingly found that recombinant (modified) filamentous fungal cells / strains are particularly well-suited for use in the industrial-scale production of heterologous (foreign) lectin proteins.

[0101] For example, as generally described in Example 1, filamentous fungal cells were designed, constructed, and evaluated for their ability to express exemplary lectin proteins, such as the Griffith cymvariant M78Q (Q-GRFT, SEQ ID NO: 4), GRFT dimer (fusion) protein (GRFT.op1-dimer, SEQ ID NO: 6), and K. alvarezii KAA-2 lectin (SEQ ID NO: 8), as shown in Figure 1. Similarly, Example 2 generally involves other heterologous lectin proteins (see, for example, Figure 2 and Table 1), such as wild-type Malayan tree lectin (Musa acuminate) (Banlec, SEQ ID NO: 23), wild-type sunflower (H. annuus) lectin (SEQ ID NO: 78), wild-type Renouxia species lectin (AYR06195, SEQ ID NO: 25), wild-type copra honey (Artocarpus integrifolia) lectin (Jacarin, SEQ ID NO: 27), wild-type Nostoc ellipsosporum lectin (CV-N, SEQ ID NO: 29), wild-type Microcystis viridis lectin (MVL, SEQ ID NO: 31), and wild-type Microcystis erginosa lectin (Microcystis This document describes the design and construction of vectors for expressing *Aeruginosa* PCC7806 lectin (MVN, SEQ ID NO: 33) and wild-type human DCSIGN lectin (SEQ ID NO: 35). Example 3 further describes a method for incorporating a lectin expression cassette into the genome of a suitable filamentous fungal host cell (e.g., via Cas9-induced targeted integration).

[0102] As shown in Example 4, screening of lectin-expressing clones (Examples 1-3) was first performed in small-scale cultures (24-well microtiter plates) at 28°C for 5 days and evaluated via SDS-PAGE analysis (see, for example, Figures 4-7). For example, as shown in Figure 4 (without pH2 treatment) and Figure 5 (pH2 treatment), the expression analysis of Trichoderma host strains transformed with the embedded cassette HRD1 (see Table 2) was evaluated, and 5 μL of culture supernatant from each clone was loaded onto an SDS-PAGE gel (Figures 4 and 5). As shown in Figure 4, lanes 1-5 contain clones derived from codon-optimized griffithsin sequences-op2 (GRFT-1-GRFT-5), lanes 6-8 contain clones derived from codon-optimized griffithsin sequences-op1 (GRFT-6-GRFT-8), lane 9 contains the codon-optimized M78Q(op1) variant, and lane 10 contains an empty host strain (T. reesei). For example, as indicated by the red arrows in the gel image (Figure 4), lanes 1-9 each contain a protein band of approximately 14 kDa, which is similar to the molecular weight of the griffithsin (GRFT) protein (approximately 12.7 kDa).

[0103] Similarly, as shown in Figure 5, several small cell cultures were treated at pH 2 to access lectin purification based on stability in a lower pH range. In particular, small cultures of GRFT codon-optimized sequences "op1" and "op2" and the codon-optimized GRFT variant (M78Q, op1) were incubated in 100 mM NaH2PO4 buffer, pH 2, at 28°C for 2 hours, the cultures were centrifuged, and the supernatant was analyzed by SDS-PAGE (Figure 5). For example, as indicated by the red arrows in the gel image (Figure 5), lanes 1-11 contain a protein band of approximately 14 kDa (i.e., analogous to the molecular weight of GRFT (approximately 12.7 kDa)), and the intensity of the (14 kDa) band is generally higher than the (14 kDa) band in Figure 4 (without pH 2 treatment), indicating that GRFT proteins can be partially purified by low pH treatment (e.g., approximately pH 2). In addition, a GRFT-op1 strain with the "op1" codon optimized (i.e., based on a small-scale screening expression profile enhanced against "op2"), such as the one named "BFZ25," was selected for further study in a 2-liter bioreactor.

[0104] Using a similar small-scale culture screening process, we evaluated Trichoderma transformants containing various embedded cassettes (see, for example, Table 2), such as strain BFZ72 encoding a GRFT fusion protein (e.g., CBH1 core-KEX2-GRFT.op1-linker-GRFT.op1) and strain BFZ27 encoding a KAA-2 fusion protein (CBH1 core-KEX2-KAA2). More specifically, as generally shown in Figures 6 and 7, the results of additional lectin small-scale expression screening in T. reesei included GRFT, Q-GRFT, CV-N, MVL, SVN, DCSIGN, KAA-1, EDA2, MPA-2, ConA, Jacarin, BanLec, OAA, ESA-2, MVN, and Galectin-1, all expressed under the control of the chb1 promoter (Pcbh1) and containing starch-binding domain (CBM21) fusions.

[0105] As described in Example 5, the production of secretory lectins was further evaluated via fed-batch fermentation carried out in a 2-liter bioreactor. More specifically, as shown in Example 5, fed-batch fermentation was performed using T. reesei strain BFZ25 (Pcbh1-GRFT.op1) for the expression of griffisin (GRFT) monomers under the control of the cbh1 promoter (Pchb1), and T for the expression of griffisin (GRFT) dimers as fusion proteins to the Cbh1 core protein (CBH1 core) and Kex2 linker (KEX2) under the control of the cbh1 promoter (Pcbh1). The T. reesei strain BFZ72 (Pcbh-cbh1 core-Kex2-GRFT.op1-linker-GRFT.op1) and the T. reesei strain BFZ27 (Pcbh1-cbh1 core-KEX2-KAA) were used for the expression of KAA-2 lectin (KAA) as a fusion protein to the Cbh1 core protein (CBH1 core) and Kex2 linker (KEX2) under the control of the cbh1 promoter (Pcbh1). For example, as described in Example 5 and shown in Figure 8, the entire broth sample from the fed-batch fermentation was collected every 24 hours, and the total fermentation time was 188 hours. As shown in Figure 8, the total soluble protein secreted in the fermentation run was plotted against the effective fermentation time (EFT, hours) for three fermentation runs (strains BFZ25, BFZ27, and BFZ72). Similarly, the fermentation supernatant was analyzed by SDS-PAGE as shown in Figure 9, and major protein bands were detected at approximately 12.7 kDa (GRFT monomer), approximately 25.8 kDa (GRFT dimer), and approximately 27.9 kDa (KAA-2). The presence of GRFT monomers, GRFT dimers, and KAA-2 was further confirmed by protein mass spectrometry of total secreted proteins in the fermentation culture supernatant. The total protein secretion titer after the completion of the 188-hour fermentation run is shown in Table 4 (Example 5).

[0106] Example 6 of this disclosure further describes the design, construction, and evaluation of exemplary filamentous fungal strains expressing / producing other heterologous lectins. More specifically, Example 6 describes polynucleotides (expression cassettes) encoding heterologous lectins, such as jacarin-like lectin, CVN-like lectin, OAA-like lectin, lysine-like lectin, galectin-1-like lectin, SVN-like lectin, DC-SIGN-like lectin, and ConA-like lectin, and these polynucleotide cassettes (see, for example, Figures 10 and 11) were introduced into exemplary filamentous fungal cells and evaluated in 96-well plates and 1-liter bioreactors. For example, as shown in Figures 12 and 13, the expression of griffisin, jacarin-like, CVN-like, OAA-like, lysine-like, galectin-like, SVN-like, DC-SIGN, and ConA-like lectins in broth supernatant was evaluated via SDS-PAGE. As shown in Figures 12 and 13, griffiscin (monomer) protein, jacarin-like lectin, CVN-like lectin, and DC-SIGN-like lectin appear as a single band with a molecular weight of approximately 10 kDa to 16 kDa, while OAA-like lectin appears as a single band with a molecular weight of approximately 10 kDa to 40 kDa.

[0107] Therefore, as briefly shown above and further described in the following sections, this disclosure demonstrates that recombinant filamentous fungal cells are particularly well-suited host expression systems for the production of heterologous lectins. This was surprising and unexpected given the current state of the art of lectins, namely recombinant lectins produced and recovered from plant expression systems (e.g., tobacco) and / or Gram-negative expression systems (e.g., Escherichia coli). More specifically, this disclosure addresses various ongoing and unmet needs in the art for improving host organisms capable of producing heterologous lectins for various antimicrobial applications, as well as ongoing and unmet needs for novel methods and compositions enabling cost-effective industrial-scale production, recovery, and / or purification of lectins. Accordingly, certain embodiments of this disclosure relate, among other things, to recombinant filamentous fungal (host) cells producing heterologous lectins, fermentation of host cells expressing heterologous lectins, industrial-scale production of lectins, and recovery and purification of one or more lectins produced. For example, in certain embodiments, lectin recovery and purification may be carried out as generally described later in Example 7. In certain other embodiments, lectin fermentation and recovery may be carried out as generally described later in Section IV (Lectin Fermentation and Recovery).

[0108] Therefore, in certain other embodiments, the present disclosure provides a method for assaying the hemagglutination activity of lectins. In particular, lectin hemagglutination ability (activity) was evaluated using erythrocytes from 15 different animal sources (i.e., dogs, rabbits, guinea pigs, mice, rats, humans, chickens, turkeys, ducks, geese, pigs, cattle, horses, sheep, and goats), as described in Example 8. For example, as shown in Figures 14 and 15, the hemagglutination activity of lectin samples could be easily determined by visually identifying a precipitate button at the bottom of the well for hemagglutination-negative (-) samples, even if the suspension showed no signs of agglutination in the well for hemagglutination-positive (+) samples. More specifically, as shown in Figure 15, the most important positive results were recorded in Figure 15, and the corresponding effective animal erythrocytes are summarized in Table 6 (Example 8).

[0109] Therefore, as intended and described herein, specific embodiments of this disclosure relate, among other things, to nucleic acids encoding lectin proteins, recombinant cells expressing / producing one or more lectin proteins, lectin protein recovery, lectin protein purification, and lectin (protein) preparation. More specifically, in specific embodiments, native lectin proteins and / or variant lectin proteins and / or DNA (nucleic acid) sequences encoding them may be derived from / obtained from known lectin proteins. In specific embodiments, the lectin proteins are derived from host organisms that naturally produce lectin proteins. Therefore, in specific embodiments, the lectin proteins of this disclosure are derived from eukaryotic cells or cyanobacterial cells. In specific embodiments, the eukaryotic cells are photosynthetic plant cells, insect cells, or animal cells.

[0110] In certain embodiments, the lectin protein may be derived from one or more antiviral lectins described in U.S. Patent Publication No. 20040204365, U.S. Patent Publication No. 20020127675, U.S. Patent Publication No. 20110189105 and U.S. Patent Publication No. 20110263485 and / or International Publication No. 2005 / 118627, International Publication No. 2008 / 022303, International Publication No. 2010 / 014248, International Publication No. 2014 / 197650, International Publication No. 2016 / 130628 and International Publication No. 2019 / 108656 (each of which is incorporated herein by reference in its entirety). Therefore, in certain embodiments, the lectin protein is cytobilin (SVN), griffiscin (GRFT), cyanobilin-N (CVN), their functional fragments, and / or their functional variants (mutants).

[0111] In other embodiments, the lectin protein is an antiviral lectin as described in International Publication No. 2019 / 108656, such as jacarpin lectin, Musa acuminata lectin, Boodlea coacta lectin, Microcystis viridis lectin, and / or one or more of their functional fragments and / or functional variants that retain the ability to bind to carbohydrates on the viral envelope as described in International Publication No. 2019 / 108656.

[0112] In other embodiments, the lectin protein is derived from, but is not limited to, eukaryotic lectin sources described by Singh and Sarathi (2012), including, Aaptos papilleta (sponge) lectin, Abrus precatorius lectin, Aegapodium podagraria lectin, Agaricus bisporus lectin, Albizzia julibrissin (mimosa tree seed) lectin, Allomyrina dichotoma lectin, Aloe arborescens (aloe plant) lectin, Amphicarpaea bracteata lectin, Anguilla anguilla (eel) lectin, and Aplysia Examples include lectins from *Depilans* (Mollusca, Mediterranean), peanut lectins from *Arachis hypogaea*, jacarin lectins from *Artocarpus heterophullus*, purpurea lectins from *Bauhinia purpurea*, white bryony lectins from *Bryonia diocia*, arborescens lectins from *Caragana arborescens*, horseshoe crab lectins from *Carcinoscorpius rotundacauda*, and / or functional fragments and / or functional variants thereof that retain the ability to bind to specified carbohydrates (parts) as described in reference to Singh and Sarathi (2012).

[0113] In certain embodiments, the lectin proteins of this disclosure may be classified into groups including, but are not limited to, galactose (Gal)-specific lectins, glucose (Glu)-specific lectins, fucose (Fuc)-specific lectins, mannose (Man)-specific lectins, N-acetylgalactosamine (GalNAc)-specific lectins, N-acetylglucosamine (GluNAc)-specific lectins, and sialic acid-specific lectins.

[0114] Therefore, in certain embodiments, lectins suitable for use according to this disclosure may be derived from and / or isolated from eukaryotic lectin-source organisms. For example, in certain embodiments, lectins (proteins) may be isolated from eukaryotic (source) organisms using affinity chromatography processes known to those skilled in the art (i.e., one of the aforementioned carbohydrate moieties (Gal, Man, GalNAc, etc.) is attached to an inactive (chromatographic) matrix so as to retain a lectin protein having binding specificity to the carbohydrate moiety).

[0115] In certain other embodiments, the lectin protein is microbilin (MVN) lectin derived from the cyanobacterium Microcystis aeruginosa (PCC7806), the MVN lectin possessing mannose-specific affinity. For example, as described in Breitenbach Barroso Coelho et al. 2018 (incorporated herein by reference), MVN lectin can inhibit HIV-1 infection, multinucleated cell formation between infected and uninfected CD4 T cells, and HIV-1 infection. In other embodiments, the lectin protein is cytobilin (SVN) derived from the cyanobacterium Cytonema varium, which inhibits viral replication by binding with high affinity to mannose residues on the viral envelope glycoprotein, as observed in the Zaire Ebola virus (see, e.g., Breitenbach Barroso Coelho et al., 2018). In yet another embodiment, the lectin protein is ESA-2 lectin derived from the red alga Eucheuma serra, which exhibits anti-HIV activity and potent inhibition against influenza A virus (H1N1) infection (see, e.g., Breitenbach Barroso Coelho et al., 2018). In yet another embodiment, the lectin protein is BanLec (jacarine-related) lectin derived from the fruit of the banana (Musa acuminate), which recognizes high-mannose glycans found on the viral envelope of HIV-1 and other viruses (see, e.g., Breitenbach Barroso Coelho et al., 2018).

[0116] In certain other embodiments, the lectin protein is a mannose-binding plant lectin derived from the rhizome of Aspidistra elatior (AEL), which has been demonstrated to have considerable in vitro inhibitory activity against vesicular stomatitis virus, coxsackievirus B4, and respiratory syncytial virus (see, for example, Breitenbach Barroso Coelho et al., 2018).

[0117] In certain other embodiments, the lectin protein is CVL lectin (β-galactose-specific) derived from the marine worm Chaetopterus variopedatus, which has anti-HIV-1 activity (see, for example, Breitenbach Barroso Coelho et al., 2018).

[0118] In certain other embodiments, the lectin proteins of this disclosure may be derived from the seeds of broad bean (Vicia faba), lentil (Lens culinaris), and pea (Pisum sativum), as commonly described in El-Araby et al., 2020 (incorporated herein by reference). Crude extracts of the three legumes were purified by affinity chromatography using mannose agarose, as commonly shown in reference to El-Araby et al. (2020), and the purified broad bean, lentil, and pea lectins had molecular weights of 18 kDa, 14 kDa, and 17 kDa, respectively, as determined by amino acid sequence analysis. For example, when these purified lectins were tested against bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia) and fungi (Candida albicans), the minimum inhibitory concentration (MIC) values ​​ranged from 1.95 μg / ml to 250 μg / ml.

[0119] In another embodiment, the lectin proteins of this disclosure are antitumor (anticancer) lectins derived from mistletoe (Viscum album) as described in Pevzner et al., 2004 and Gengenbach et al., 2019 (incorporated herein by reference), such as mistletoe lectin I (MLI) and mistletoe lectin III (MLIII) B-subunits.

[0120] As intended herein, one or more natural lectins and / or valiantrectins derived therefrom may be evaluated for function or activity by means such as, but not limited to, hemagglutination activity assays (Example 8), carbohydrate / glycan binding affinity assays, antimicrobial inhibitor assays, and combinations thereof, as shown and described in El-Araby et al. (2020). Thus, in certain embodiments, the natural lectins and / or valiantrectins of this disclosure include antimicrobial activity (e.g., antiviral activity, antifungal activity, antimicrobial activity).

[0121] Therefore, certain embodiments of the present disclosure provide recombinant filamentous fungal cells expressing one or more heterologous nucleic acids (polynucleotides) encoding lectin proteins. In certain embodiments, the recombinant filamentous fungal cells express heterologous polynucleotides encoding native lectin proteins or functional variants derived from native lectin proteins. In certain embodiments, the heterologous polynucleotides encoding lectin proteins are expression cassettes introduced into the recombinant cells. In certain embodiments, at least one expression cassette is introduced into the filamentous fungal cells. In other embodiments, at least two expression cassettes are introduced into the filamentous fungal cells. For example, Figure 3 shows a schematic diagram of an exemplary lectin polynucleotide cassette suitable for extracellular expression / secretion of lectin proteins (e.g., Figures 3A-3B). Therefore, in certain embodiments, the filamentous fungal host cells of the present disclosure include one or more lectin expression cassettes introduced therein, and the host cells express and secrete lectins when cultured under appropriate conditions.

[0122] In certain embodiments, the filamentous fungal cells / strains of this disclosure are selected from, derived from, or obtained from ascomycete fungal cells. Examples of ascomycete (filamentous) fungal cells include, but are not limited to, species of the subphylum Pezizomycotina, such as species of the genera Trichoderma, Aspergillus, Myceliophthora, and Penicillium. In other embodiments, filamentous fungi include, but are not limited to, species of the genera Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma.

[0123] In some embodiments, the filamentous fungi are species of the genus Aspergillus, such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae.

[0124] In some embodiments, the filamentous fungi are Fusarium species, such as Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, and Fusarium roseum. These include Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, and Fusarium venenatum.

[0125] In other embodiments, filamentous fungi include Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Scytalidium thermophilum, and Thielavia terrestris.

[0126] In further embodiments, the filamentous fungi include species of the genus Trichoderma, such as Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0127] III. Recombinant Polynucleotides and Molecular Biology As previously stated, specific embodiments of this disclosure relate to modified filamentous fungal strains that express / produce / secrete heterologous lectin proteins. Therefore, specific embodiments of this disclosure relate, in particular, to molecular biology, gene modification, polynucleotides, genes, gene-coding sequences (CDS), ORFs, vectors, expression cassettes, fusion proteins, protein linker sequences, and cleavable protein linker sequences.

[0128] In certain embodiments, the expression cassette includes or comprises nucleic acid (DNA) sequences encoding a fusion protein, such as an upstream (5') DNA fusion sequence (N-terminal fusion) and / or a downstream (3') DNA fusion sequence (C-terminal fusion).

[0129] In other embodiments, one or more expression cassettes are incorporated into the genome of a filamentous fungal cell. In certain embodiments, the filamentous fungal cell contains at least two introduced expression cassettes encoding the same or different lectin proteins. In certain other embodiments, the recombinant filamentous fungal cell has the production of one or more endogenous genes encoding one or more proteases deleted. In certain embodiments, one or more proteases include, but are not limited to, subtilisin-like serine proteases, aspartate proteases, trypsin-like serine proteases, glutamate proteases, and aminopeptidases. Various molecular biology methodologies are available to delete the expression / production of such secreted proteases in filamentous fungal strains as described herein (e.g., gene disruption, gene deletion, downregulation genes, etc.). For example, in certain embodiments, it may be advantageous to eliminate (or reduce) the production of highly expressed and secreted proteases in order to mitigate the protease degradation of lectin proteins secreted into fermentation broth, as intended herein.

[0130] In other embodiments, recombinant filamentous fungal cells include one or more introduced expression cassettes encoding protease inhibitors. A non-limiting example of a protease inhibitor protein is the natural "oomugiamylase subtilisin inhibitor" (BASI) protein. In certain embodiments, a wild-type gene encoding a protease inhibitor (e.g., BASI) is codon-optimized for expression in filamentous fungal cells, and the protease inhibitor is secreted into the fermentation broth. Other suitable protease inhibitors and the genes encoding them are known in the art and may be used to mitigate the unwanted / undesirable protease degradation of lectin proteins secreted into the fermentation broth.

[0131] In certain other embodiments, recombinant filamentous fungal cells are deficient in the production of one or more endogenous genes encoding one or more lignocellulosidases. In certain embodiments, one or more secreted lignocellulosidases include, but are not limited to, cellobiohydrolases (e.g., CBH1, CBH2), endoglucanases (e.g., EG1, EG2), β-glucosidase (BG), and xylanase (e.g., XYL). Various molecular biological methodologies are available to deficient in the expression / production of such secreted lignocellulosidases in filamentous fungal strains, as described herein (e.g., gene disruption, gene deletion, downregulation genes, etc.). For example, in certain embodiments, it may be advantageous to eliminate (or reduce) the production of highly expressed and secreted lignocellulosidases in order to reduce the burden on the protein expression / secretion pathways of filamentous fungal cells, as intended herein.

[0132] In certain embodiments, the disclosure provides recombinant nucleic acids (polynucleotides) comprising a gene or gene CDS encoding a lectin protein. In particular, certain embodiments provide polynucleotide constructs encoding lectin proteins (e.g., expression cassettes) for the expression and secretion of lectin proteins into culture media / fermentation broth. In certain embodiments, one or more expression cassettes for the secretion of lectin proteins may generally be represented by one or more schematic diagrams.

[0133] For example, an expression cassette encoding a secreted lectin protein may be schematically represented as 5'-[pro]-[sig-seq]-[lectin CDS]-3', where the expression cassette comprises a promoter (pro) region sequence (5' to 3' direction), a nucleic acid (sig-seq) encoding a protein (signal) secretion sequence operably linked to it, and a nucleic acid (lectin CDS) encoding a lectin protein operably linked to it. In certain embodiments, the promoter (pro) region sequence is a strong, functional promoter in the host filamentous fungal strain. Examples of strong, functional promoter (pro) region sequences in Trichoderma species fungal cells include, but are not limited to, T. reesei cellobiohydrolase promoters (e.g., Pcbh1, Pcbh2), xylanase promoters (e.g., xyn3), glucoamylase promoters (e.g., gla1), and endoglucanase promoters (e.g., egl2). While specific promoter (pro) sequences are illustrated herein, those skilled in the art can screen, identify, and select other suitable promoter sequences that are active / functional in host filamentous fungal cells. Similarly, while specific protein (signal) secretion sequences (sig-seq) are illustrated herein (e.g., Cbh1 secretion sequence (SEQ ID NO: 15), Pep1 secretion sequence (SEQ ID NO: 16)), those skilled in the art can screen, identify, and select other suitable protein signal / secretion sequences that are functional in filamentous fungal cells. For example, in certain embodiments, lectin protein secretion in one or more filamentous fungal cells of this disclosure can be identified using one or more signal (secretion) peptide sequences derived from highly secreted filamentous fungal proteins known in the art.In certain other embodiments, lectin protein secretions in one or more filamentous fungal cells include: Talaromyces β-mannanase secretory sequence, Talaromyces glucoamylase secretory sequence, Trichoderma Cbh2 secretory sequence, Trichoderma glucoamylase secretory sequence, Humicola Cel45 secretory sequence, Neurospora chitin synthase secretory sequence, Aspergillus α-galactosidase (GlaA) secretory sequence, Aspergillus PepN secretory sequence, Trichoderma harzianum aspartylprotease (PapA) secretory sequence, and Myceliophthora IMI Examples include, but are not limited to, xylanase secretion sequences, and can be screened and identified by reference to one or more intrinsic protein secretion / signal sequences and their functional variants.

[0134] In certain other embodiments, one or more cassettes comprise one or more upstream (N-linker) and / or downstream (C-linker) nucleic acids encoding one or more (protein / peptide / amino acid) linker amino acid sequences. In certain embodiments, the protein / peptide / amino acid linker sequence is a cleavable sequence (e.g., a KEX2 cleavable site). For example, during protein secretion in fungal cells, a particular protein is cleaved by KEX2 of serine peptidases (EC3.4.21.61) or KEX2, a member of the “kexin” family. As described in U.S. Patent Application Publication No. 2014 / 0024067 and U.S. Patent No. 8,936,917 (each incorporated herein by reference in its entirety), KEX2 is a highly specific calcium-dependent endopeptidase that cleaves a peptide bond immediately C-terminal to a pair of basic amino acids ("KEX2 site") in a protein substrate (e.g., a lectin) during the secretion of that (lectin) protein. For example, the KEX2 (cleavable) site may be included in the construction of an expression cassette encoding one or more lectin proteins, as commonly described in U.S. Patent Publication No. 2014 / 0024067. Similarly, U.S. Patent No. 8,936,917 describes a modified KEX2 cleavage site having a pre-sequence (VAVE), which improves the cleavage efficiency at the KEX2 site after pre-sequencing. While the KEX2 cleavage site is exemplified, other protease cleavage sites functional in filamentous fungal cells can be used to cleave peptide linkers between protein fusion partners and lectin proteins. Examples of additional protease cleavable linkers include, but are not limited to, STE13 described by La Maquer et al. (2019) and the self-cleaving 2A peptide described by Subramanian et al. (2017).

[0135] In other embodiments, one or more cassettes encoding lectin proteins include operablely linked and terminator region sequences (terms) located at their 3' ends.

[0136] In certain embodiments, the polynucleotides of this disclosure may comprise one or more selectable markers. Selectable markers used for filamentous fungi include, but are not limited to, alll, amdS, hygR, pyr2, pyr4, pyrG, sucA, bleomycin resistance markers, blastosidine resistance markers, pyrithiamine resistance markers, chlorimulone ethyl resistance markers, neomycin resistance markers, adenine pathway genes, tryptophan pathway genes, and thymidine kinase markers. In certain embodiments, the selectable marker is pyr2, whose composition and use are generally described in International Publication No. 2011 / 153449.

[0137] Standard techniques for filamentous fungal transformation and fungal culture (well known to those skilled in the art) are used to transform the fungal host cells of this disclosure. Therefore, techniques for introducing DNA constructs or vectors into fungal host cells include transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection-mediated and DEAE-dextrin-mediated transfection), incubation with calcium phosphate DNA precipitate, high-speed gun, gene gun, or bioristic transformation and protoplast fusion using DNA-coated microprojectiles. General transformation techniques are well known in the art.

[0138] In many cases, the transformation of Trichoderma species fungal cells is typically 10 5 ~10 7 / mL, especially about 2x10 6Protoplasts or cells subjected to permeabilization treatment at a density of / mL are used. 100 μL of these protoplasts or cells in a suitable solution (e.g., 1.2 M sorbitol and 50 mM CaCl2) are mixed with the desired DNA. Generally, high concentrations of polyethylene glycol (PEG) are added to the uptake solution. Additives such as dimethyl sulfoxide, heparin, spermidine, and potassium chloride may also be added to the uptake solution to promote transformation. Similar procedures are available for other fungal host cells (see, for example, U.S. Patent Nos. 6,022,725 and 6,268,328, both of which are incorporated by reference).

[0139] Therefore, the methods and compositions of this disclosure generally rely on conventional techniques in the field of recombinant genetics. For example, in certain embodiments, a gene encoding a heterologous lectin protein of interest is introduced into filamentous fungal (host) cells. In certain embodiments, the gene (or gene CDS) is cloned into an intermediate vector before being transformed into filamentous fungal (host) cells for replication and / or expression. These intermediate vectors may be prokaryotic vectors such as plasmids or shuttle vectors. In certain embodiments, the expression of the gene encoding the lectin protein or gene CDS is under the control of a heterologous promoter, which may be a heterologous constitutive promoter or a heterologous inducible promoter, in particular a strong promoter capable of overexpressing the lectin protein.

[0140] Expression vectors typically contain a transcription unit or "expression cassette" that includes all the additional elements necessary for the expression of heterologous sequences. For example, a typical expression cassette contains an upstream (5') promoter operably linked to the nucleic acid sequence encoding the protein of interest, and may further include a nucleic acid sequence encoding the protein (signal) secretion sequence, nucleic acid sequences necessary for the efficient polyadenylation of the transcript, a ribosome binding site, and a translation termination sequence. Additional elements of the cassette may include enhancers and, if genomic DNA is used as a structural gene, introns having functional splice donor sites and acceptor sites.

[0141] In addition to the promoter sequence, the expression cassette may also contain a transcription termination region downstream of the structural gene to achieve efficient termination. The termination region may be obtained from the same gene as the promoter sequence or from a different gene. In this invention, any fungal terminator is likely to be functional, but preferred terminators include: a terminator derived from the Trichoderma cbh1 gene, the Aspergillus nidulans trpC gene, and a terminator derived from the Aspergillus awamori or Aspergillus niger glucoamylase gene.

[0142] The specific expression vector used to transport genetic information into cells is not particularly important. Any conventional vector used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include bacteriophages λ and M13, as well as plasmids, such as pBR322-based plasmids, pSKF, pET23D, and fusion expression systems, such as MBP, GST, and LacZ. To provide a convenient isolation method, an epitope tag, such as c-myc, may also be added to the recombinant protein.

[0143] Elements that can be included in the expression vector may be replicons, genes encoding antibiotic resistance that allow selection of bacteria possessing the recombinant plasmid, or specific restriction sites in the non-essential region of the plasmid to allow insertion of heterologous sequences. The specific antibiotic resistance gene to be selected is not definitive, because any of the many resistance genes known in the art may be suitable. The prokaryotic sequence is preferably selected so as not to interfere with DNA replication or integration in the filamentous fungal host.

[0144] The transformation method of the present invention may result in the stable integration of all or part of the transformation vector into the genome of a filamentous fungus. However, transformation that results in the maintenance of a self-replicating extrachromosomal transformation vector is also intended. Since many standard transfection methods can be used to generate filamentous fungal cell lines expressing large amounts of heterologous proteins, any known procedure for introducing foreign nucleotide sequences into fungal host cells may be used. These include the use of calcium phosphate transfection, polyblens, protoplast fusion, electroporation, bioristics, liposomes, microinjection, plasma vectors, viral vectors, and any other known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells. Agrobacterium-mediated transfection methods, such as those described in U.S. Patent No. 6,255,115, are also useful.

[0145] After the expression vector is introduced into the cells, the transformed cells are cultured under conditions favorable to gene expression. Large batches of transformed cells can be cultured as described herein. Finally, the protein product is recovered from the culture using standard techniques. Thus, this disclosure provides enhancement of the expression and production of the desired protein of interest, as described herein.

[0146] In certain embodiments or aspects of the present disclosure, a filamentous fungal cell line may include one or more gene modifications, including but not limited to (a) the introduction, substitution, or removal of one or more nucleotides within a gene (gene CDS or its ORF) or within a regulatory element required for the transcription or translation of a gene (gene CDS or ORF), (b) gene disruption, (c) gene transformation, (d) gene deletion, (e) gene downregulation, (f) specific mutagenesis, and / or (g) random mutagenesis of a gene (gene CDS or its ORF).

[0147] As generally shown above and described below, those skilled in the art can easily construct recombinant / modified / variant filamentous fungal strains by performing one or more gene modifications by reference to one or more nucleic acid sequences and / or protein sequences disclosed herein. For example, gene deletion techniques eliminate or reduce protein expression / production and / or eliminate or reduce the expression / production of encoded proteins by allowing partial or complete removal of a gene. In such a method, gene deletion can be achieved by homologous recombination using an embedded plasmid / vector constructed to contain adjacent 5' and 3' regions that flank the gene. The adjacent 5' and 3' regions can be introduced into filamentous fungal cells, for example, on an embedded plasmid / vector associated with a selectable marker that allows the plasmid to be incorporated into the cell.

[0148] In other embodiments, modified strains of filamentous fungi include gene modifications that disrupt or inactivate a gene of interest. Exemplary methods of gene disruption / inactivation include disrupting any portion of a gene containing a polypeptide coding sequence (CDS), promoter, enhancer, or other regulatory element, and such disruption includes substitution, insertion, deletion, inversion, and combinations thereof and variations thereof. A non-limiting example of gene disruption techniques is inserting (integrating) an integration plasmid containing a nucleic acid fragment homologous to the gene of interest into one or more genes of the Disclosure, which creates overlap between homologous regions and the integration (insertion) vector DNA between the overlapping regions. In certain other non-limiting examples, gene disruption techniques include inserting an embedded plasmid containing a nucleic acid fragment homologous to the gene of interest into the gene of interest, which creates overlap between homologous regions and the embedded (inserted) vector DNA between overlapping regions. The inserted vector DNA then, for example, separates the gene's promoter from the protein-coding region or disrupts (disrupts) the gene's coding or non-coding sequence, resulting in enhanced protein-producing phenotype. The disruption construct may be a selectable marker gene (e.g., pyr2) accompanied by 5' and 3' regions homologous to the gene of interest. The selectable marker allows for the identification of transformants containing the disrupted gene. Therefore, in certain embodiments, gene disruption may involve modification of regulatory elements of the gene, such as the promoter, ribosome-binding sites (RBS), untranslated regions (UTR), and codon changes.

[0149] In other embodiments, modified strains of filamentous fungi are constructed (i.e., genetically modified) by introducing, substituting, or removing one or more nucleotides in a gene or regulatory element required for its transcription or translation. For example, nucleotides may be inserted or removed to introduce an immature stop codon, remove a start codon, or cause a frameshift of an open reading frame (ORF). Such modifications may be achieved by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art.

[0150] In other embodiments, modified strains of filamentous fungi are constructed by a gene transformation process. For example, in a gene transformation method, a nucleic acid sequence corresponding to a target gene is mutagenesized in vitro to generate a defective nucleic acid sequence, which is then transformed into parental cells to generate variant cells containing the defective gene. Homologous recombination replaces the endogenous gene with the defective nucleic acid sequence. It may be desirable that the defective gene or gene fragment also encodes a marker that can be used to select transformants containing the defective gene. For example, the defective gene may be introduced onto a non-replicating plasmid or a thermosensitive plasmid associated with a selectable marker. Selection for plasmid integration is influenced by selection for the marker under conditions that do not allow plasmid replication. Selection for a second recombination event resulting in gene substitution is influenced by colony investigation for the disappearance of the selectable marker and the acquisition of the mutant gene.

[0151] In other embodiments, modified strains of filamentous fungi are constructed using established antisense (gene silencing) techniques with a nucleotide sequence complementary to the nucleic acid sequence of the gene of interest. More specifically, gene expression by a filamentous fungal strain can be reduced (downregulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which can be transcribed intracellularly and hybridized into intracellularly produced mRNA. Thus, under conditions where the complementary antisense nucleotide sequence can hybridize into mRNA, the amount of translated protein is reduced or eliminated. Examples of such antisense methods, but not limited to, include RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), and antisense oligonucleotides, all of which are well known to those skilled in the art.

[0152] In other embodiments, modified strains of filamentous bacteria are constructed by random or specific mutagenesis using methods well known in the art, including but not limited to chemical mutagenesis and transposition. Gene modification can be performed by subjecting parental cells to mutagenesis and screening for mutant cells in which gene expression is reduced or eliminated. Mutagenesis can be specific or random and can be performed, for example, by using a suitable physical or chemical mutagenerator, using a suitable oligonucleotide, or by subjecting a DNA sequence to PCR-generated mutagenesis. Furthermore, mutagenesis can be performed by using any combination of these mutagenesis methods. Examples of physical or chemical mutagenerators suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrite, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When using such agents, mutagenesis is typically performed by incubating the parent cells to be mutageneised in the presence of the optimal mutagenesis agent under appropriate conditions, and then selecting mutant cells that show reduced gene expression or no expression at all.

[0153] In certain other embodiments, modified strains of filamentous fungi are constructed by site-directed gene editing techniques. For example, in certain embodiments, variant strains of filamentous fungi are constructed (i.e., genetically modified) using transcription activator-like endonucleases (TALENs), zinc finger endonucleases (ZFNs), and homing (mega) endonucleases, etc. More specifically, the portion of the gene to be modified (e.g., coding regions, non-coding regions, leader sequences, propeptide sequences, signal sequences, transcriptional terminators, transcriptional activators, or other regulatory elements required to express the coding region) is subjected to gene modification by ZFN gene editing, TALEN gene editing, and homing (mega) endonucleases, etc., methods of these modifications are well known and available to those skilled in the art.

[0154] In certain other embodiments, modified strains of filamentous fungi are constructed by CRISPR / Cas9 editing. More specifically, compositions and methods for fungal genome modification using CRISPR / Cas9 systems have been described and are well known in the art (see, for example, International Publication Nos. 2016 / 100571, 2016 / 100568, 2016 / 100272, and 2016 / 100562). Thus, the gene of interest can be disrupted, deleted, mutated, or otherwise genetically modified by a nucleic acid-inducible endonuclease, which finds the target DNA by binding either a guide RNA (e.g., Cas9) or guide DNA (e.g., NgAgo) to a target sequence on the DNA, and the endonuclease can cause single-strand or double-strand breaks in the DNA. This targeted DNA break serves as a substrate for DNA repair and, in combination with a provided editing template, can disrupt or delete a gene. For example, a gene encoding a nucleic acid-inducible endonuclease (e.g., a codon-optimized gene encoding Cas9 or Cas9 nuclease from S. pyogenes) can be operably ligated to a promoter and a terminator active in filamentous fungal cells to create a filamentous fungal Cas9 expression cassette. Similarly, one or more target sites specific to a gene of interest can be readily identified by those skilled in the art. For example, to construct a DNA construct encoding a gRNA directed to a target site in a gene of interest, a variable targeting domain (VT) contains a nucleotide of the target site, which is a 5' (PAM) protospacer flanking motif (TGG), and this nucleotide is fused to DNA encoding a Cas9 endonuclease recognition domain (CER) for S. pyogenes Cas9. DNA encoding gRNA is created by combining DNA encoding the VT domain and DNA encoding the CER domain.Therefore, a filamentous fungal expression cassette for gRNA is created by operably ligating the DNA encoding the gRNA to a promoter and a terminator that are active within the filamentous fungal cell.

[0155] In certain embodiments, DNA breaks induced by endonucleases are repaired / replaced by incoming sequences. For example, nucleotide editing templates are provided so that the cell's DNA repair mechanism can utilize them to accurately repair DNA breaks caused by the Cas9 expression cassette and gRNA expression cassette described above. For example, an editing template can be created by fusing a 5' target gene of approximately 500 bp to a 3' target gene of approximately 500 bp, and this template is used by the mechanisms of a filamentous fungus host to repair DNA breaks caused by RGEN (RNA-induced endonuclease).

[0156] Cas9 expression cassettes, gRNA expression cassettes, and editing templates can be co-delivered to filamentous fungal cells using a variety of methods (e.g., protoplast fusion, electroporation, innate competence, or induced competence). Transformed cells are screened by PCR by amplifying target loci with forward and reverse primers. These primers can amplify wild-type loci or modified loci edited by RGEN. These fragments are then sequenced using sequencing primers to identify edited colonies.

[0157] Another method for genetically modifying a target gene is to alter its expression level. For example, nuclease-deficient variants of such nucleotide-induced endonucleases (e.g., Cas9 D10A, N863A or Cas9 D10A, H840A) can be used to modulate gene expression levels by enhancing or antagonistizing the transcription of the target gene. These Cas9 variants are inactive for all nuclease domains present in the protein sequence but retain RNA-induced DNA-binding activity (i.e., these Cas9 variants cannot cleave any strand of DNA once they bind to a homogeneous target site). Therefore, nuclease-deficient proteins (i.e., Cas9 variants) can be expressed as filamentous fungal expression cassettes, and when combined with filamentous fungal gRNA expression cassettes, the resulting Cas9 variant proteins are directed to specific target sequences within the cell. The binding of Cas9 (variant) proteins to specific gene target sites can reduce the amount of gene products produced by blocking the binding or movement of transcription mechanisms on cellular DNA. Therefore, any of the genes disclosed herein can be targeted for gene expression reduction using this method. Gene silencing can be monitored in cells containing nuclease-deficient Cas9 expression cassettes and gRNA expression cassettes using methods such as RNA-seq.

[0158] IV. Fermentation and recovery of lectins As will be further detailed hereafter and illustrated in the examples, this disclosure describes and illustrates processes (methods) particularly suitable for harvesting, clarifying, recovering, and purifying a fermented broth from which one or more lectin proteins have been secreted. Therefore, certain embodiments relate, in particular, to collecting the broth after fermentation has finished, harvesting the collected broth, and recovering one or more lectins from the harvested broth (e.g., clarifying the harvested broth, concentrating the clarified broth, purifying the clarified broth concentrate, etc.). In certain embodiments, the purified lectin (protein) preparation is derived from the fermented broth collected and harvested as described herein.

[0159] In certain embodiments, the disclosure provides recombinant filamentous cells that produce a lectin protein of interest. Thus, one or more embodiments relate to culturing (fermenting) filamentous fungal cells for the production of lectin proteins. Generally, fermentation methods well known in the art are used to ferment filamentous fungal cells. In some embodiments, the fungal cells are grown under batch or continuous fermentation conditions.

[0160] Classical batch fermentation is a closed system where the composition of the culture medium is set at the start of fermentation and not altered during fermentation. At the start of fermentation, the desired organism is inoculated into the medium. This method allows fermentation to proceed without adding any components to the system. Typically, batch fermentation is considered "batch" in terms of the addition of a carbon source, and attempts are often made to control factors such as pH and oxygen concentration. The composition of metabolites and biomass in the batch system changes constantly until fermentation is stopped. In batch culture, cells progress through a static delay phase to a high-proliferation logarithmic phase, and finally to a quiescent phase where the growth rate slows down or stops. If left untreated, cells in the quiescent phase will eventually die. Generally, cells in the logarithmic phase are responsible for producing the majority of the product.

[0161] A suitable variation of the standard batch system is the "fed-batch fermentation" system. In this variation of the typical batch system, the substrate is gradually added as fermentation progresses. The fed-batch system is useful when catabolite inhibition is likely to inhibit cellular metabolism and when a limited amount of substrate in the culture medium is desired. In the fed-batch system, it is difficult to measure the actual substrate concentration, so it is estimated based on changes in measurable factors such as pH, dissolved oxygen, and the partial pressure of exhaust gases such as CO2. Batch fermentation and fed-batch fermentation are common and well-known in the art.

[0162] Continuous fermentation is an open system in which a specified fermentation medium is continuously added to a bioreactor, and an equal amount of acclimatized medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant density, with the cells primarily in the logarithmic growth phase. Continuous fermentation allows for the regulation of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, limiting nutrients such as a carbon or nitrogen source can be maintained at a fixed ratio while all other parameters are regulated. In other systems, numerous factors affecting growth can be continuously modified while maintaining a constant cell concentration, as measured by the turbidity of the medium. Continuous systems attempt to maintain steady-state growth conditions. Therefore, cell loss resulting from the removal of medium must be balanced with respect to the cell growth rate during fermentation. Methods for regulating nutrients and growth factors in the continuous fermentation process, as well as techniques for maximizing the rate of product formation, are well known in industrial microbiology.

[0163] To ensure proper microbial growth, maximize the assimilation of carbon and energy sources by cells during the microbial transformation process, and achieve maximum cell yield at maximum cell density in the fermentation medium, it is essential to supply appropriate amounts of inorganic nutrients in appropriate proportions, in addition to carbon and energy sources, oxygen, assimilated nitrogen, and microbial inoculation material.

[0164] The composition of aqueous inorganic media can vary widely, in part, depending on the microorganisms and substrates used, as is known in the art. In addition to nitrogen, the inorganic media should contain appropriate amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium in appropriate soluble and assimilated ionic composite forms, and preferably, certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine should also be present, herein too, in appropriate soluble and assimilated forms, all of which are known in the art.

[0165] Fermentation is an aerobic process in which the necessary molecular oxygen is supplied by a molecular oxygen-containing gas, such as air, oxygen-enriched air, or even substantially pure molecular oxygen, which is provided to maintain the contents of the fermenter at an appropriate partial pressure of oxygen that is effective in promoting the vigorous growth of microbial species.

[0166] Microorganisms also require an assimilated nitrogen source. This assimilated nitrogen source can be any nitrogen-containing compound or a compound capable of releasing nitrogen in a form suitable for metabolic utilization by microorganisms. Various organic nitrogen source compounds, such as protein hydrolysates, can be used, but typically, inexpensive nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts, such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds, can be utilized. Ammonia gas itself is convenient for large-scale operations and can be used by bubbling it through an aqueous ferment (fermentation medium) in appropriate amounts. Simultaneously, such ammonia can also be used to assist in pH control.

[0167] The pH range of aqueous microbial fermentation products (fermentation mixtures) should be within the exemplary range of approximately 2.0 to 8.0. For filamentous fungi, the pH is typically within the range of approximately 2.5 to 8.0, and for T. reesei, the pH is typically within the range of approximately 3.0 to 7.0. The preferred pH range for microorganisms depends to some extent on the culture medium used and the specific microorganism, and therefore changes somewhat with changes in the medium, as can be easily determined by those skilled in the art. In certain embodiments, by carrying out fermentation in a manner that allows control of carbon-containing substrates as limiting factors, good conversion of carbon-containing substrates to cells is achieved, while contamination by a considerable amount of unconverted substrates in the cells is avoided. The latter is not a problem with respect to water-soluble substrates, as any trace residues can be easily washed away. However, this can be a problem with non-water-soluble substrates, requiring additional product treatment steps such as appropriate washing steps.

[0168] As mentioned above, the time it takes to reach this level is not significant and can vary depending on the specific microorganisms and the fermentation process performed. However, methods for determining the carbon source concentration in the fermentation medium and for determining whether the desired carbon source level has been achieved are well known in the art.

[0169] Fermentation can be carried out in batches or as a continuous operation, and fed-batch operation may be preferred in terms of ease of control, production of uniform amounts of product, and the most economical use of all equipment.

[0170] If necessary, some or all of the carbon and energy source materials, and / or some of the assimilated nitrogen sources such as ammonia, may be added to the aqueous inorganic medium before supplying it to the fermenter.

[0171] Preferably, each flow introduced into the reactor is controlled at a predetermined speed, or controlled according to the need determined by monitoring, such as the concentration of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the fermenter exhaust gas, and cell density or transmittance, which can be measured by dry cell weight. The supply rates of various materials can be varied to obtain the fastest possible cell growth rate and the highest possible yield of microbial cells relative to the substrate charge, in line with the efficient utilization of carbon and energy sources.

[0172] In batch or preferred fed-batch operations, all equipment, reactors or fermentation means, tanks or vessels, piping systems and associated circulation or cooling devices, etc., are first sterilized, for example, at about 121°C for at least about 15 minutes, usually using steam. Next, the culture of selected microorganisms is inoculated into the sterilized reactor in the presence of all necessary nutrients, including oxygen, and a carbon-containing substrate. The type of fermenter used is not important.

[0173] The collection and purification of lectin proteins from fermentation broth can be carried out by procedures known to those skilled in the art. For example, the cost of lectin protein production can be reduced by constructing the recombinant fungal strains of the present disclosure, as described above, to secrete one or more lectin proteins into the fermentation broth, thereby simplifying the lectin protein recovery process (e.g., eliminating the need for cell lysis). Fermentation broth generally contains cells, various suspended solids and other biomass contaminants, as well as cell fragments containing the desired lectin protein, which are removed from the fermentation broth by means known in the art.

[0174] Therefore, in certain embodiments, lectin protein preparations are recovered according to the compositions and methods of this disclosure. In other embodiments, lectin preparations are recovered and purified according to the methods of this disclosure. As used herein, “purified,” “isolated,” or “enriched” with respect to “lectin” (protein) means that the lectin is converted from a less pure state by separating the lectin from some or all of the associated contaminants. Contaminants include, but are not limited to, microbial cells, metabolites, solvents, chemicals, colors, inactive forms of the target lectin, aggregates, processing aids, inhibitors, fermentation media, cell fragments, nucleic acids, proteins other than the target lectin, host cell proteins, and cross-contaminations from the production apparatus.

[0175] Therefore, in relation to the term "purified lectin" as used herein, purification can be achieved by any separation technique recognized in the Art, including, but not limited to, ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, heat treatment, ammonium sulfate precipitation or other protein salting-out, crystallization, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis or gradient separation, to remove unwanted whole cells, cell fragments, impurities, foreign proteins or enzymes from the final composition.

[0176] Subsequently, the purified or isolated lectin composition may be further enriched with components that provide additional benefits, such as activators, inhibitors, desired ions, pH-adjusting compounds, or other enzymes or chemicals.

[0177] As used herein, the "purity" of a lectin is a relative term and does not mean limited when used in phrases such as "the recovered lectin has a higher purity, the same purity, or a lower purity than before the recovery process". For example, the relative "purity" of a lectin (protein) before and after the recovery process can be determined using methods known in the art, including but not limited to general quantification methods (e.g., Bradford, UV-Vis, activity assay), electrophoretic analysis (SDS-PAGE), analytical HPLC, mass spectrometry, and hydrophobic interaction chromatography.

[0178] Non-limiting examples for accessing the relative purity of a lectin include, but are not limited to, SDS-PAGE analysis and / or the A 280 ratio of non-lectin (impurities) to the lectin.

[0179] For example, the relative lectin purity via SDS-PAGE can be determined by the visual abundance of the lectin (protein) band compared to the non-lectin protein (unwanted contaminants, impurities) bands present in the preparation. Additionally, the relative purity of a lectin can be determined by the non-lectin to lectin (A 280 ) ratio. For example, the A 280 ratio is a measure of the amount of absorbance at 280 nm contributed by non-lectin impurities to the absorbance at 280 nm contributed by 1 unit of lectin in the protein preparation (e.g., non-lectin A 280 / lectin A 280 ), and a smaller number means higher purity. More specifically, the method for determining the lectin (A 280 ) concentration can be measured by HPLC using the purified lectin as a standard substance, and the concentration by HPLC is converted to lectin A 280 using 1 mg / mL lectin = 0.936 at 280 nm absorbance. The method for determining the non-lectin (A 280 ) concentration in the preparation is to zero with MilliQ water and, if necessary, A 280Non-lectin A can be measured using a 1 cm channel glass cuvette diluted to <1. 280 The concentration was determined from the measured values ​​of the prepared sample, specifically lectin A 280 It is calculated by subtracting [a certain value].

[0180] Therefore, according to certain embodiments, lectin (protein) preparations are recovered from fermentation broth, and the recovered lectin preparations are purified to a higher degree after undergoing one or more recovery processes described herein. For example, fermentation broth (e.g., the total broth at the end of fermentation) may be subjected to one or more protein recovery processes, including but not limited to broth conditioning, broth clarification, protein enrichment and / or protein purification processes (e.g., protein concentration, filtration, precipitation, crystallization, crystal separation and crystal sludge dissolution processes), buffer exchange processes and sterile filtration processes. In certain embodiments, fermentation broth is subjected to a broth treatment (broth conditioning) process to improve its subsequent broth handling properties. In certain other embodiments or aspects, purified lectin (protein) preparations may be derived from or recovered from collected and harvested fermentation broth.

[0181] Therefore, as described herein, a person skilled in the art may readily adapt or modify one or more of the compositions and / or methods disclosed herein for the recovery of specific lectin proteins and / or combinations thereof, so the methods / processes of this disclosure are not limited. Generally, fermentation broths obtained by fermenting filamentous fungal cells that express and secrete lectin proteins may be treated by harvesting, clarifying and concentrating the broth, as generally described herein.

[0182] In certain other embodiments, modified filamentous fungal cells containing introduced cassettes encoding lectins may be described by volumetric productivity, defined as the amount of protein (g) produced during fermentation per nominal volume (L) of the bioreactor per total fermentation time (h). For example, volumetric productivity may be measured by methods known in the art (e.g., ELISA, HPLC, Bradford assay, and LC / MS).

[0183] In certain other embodiments, modified filamentous fungal cells containing introduced cassettes encoding lectins may be described according to total protein yield, where total protein yield is defined as the amount of protein produced per gram of supplied carbohydrates (g) compared to the (unmodified) parent strain. Thus, the total protein yield (g / g) used herein may be calculated using the following formula: Yf = Tp / Tc In the formula, "Yf" is the total protein yield (g / g), "Tp" is the total protein produced during fermentation (g), and "Tc" is the total carbohydrates supplied during the fermentation (bioreactor) run (g).

[0184] Total protein yield may also be described as carbon conversion efficiency / carbon yield, for example, as the percentage (%) of supplied carbon incorporated into the total protein. Therefore, in certain embodiments, modified filamentous fungal cells containing the introduced cassette may be described according to carbon conversion efficiency (e.g., the percentage (%) of increased supplied carbon incorporated into the total protein).

[0185] In certain other embodiments, modified filamentous fungal cells containing the introduced cassette may be described according to their specific productivity (Qp) of lectin protein. For example, detection of specific productivity (Qp) is a suitable method for evaluating the rate of lectin protein production, and Qp can be determined using the following formula: "Qp = gP / gDCW·hr" In the formula, "gP" is the amount of protein produced in grams in the tank, "gDCW" is the amount of dry cell weight (DCW) in grams in the tank, and "hr" is the fermentation time (hours) from the time of planting, which includes the production time and the growth time.

[0186] V. Exemplary Embodiments Non-limiting embodiments of this disclosure include, but are not limited to, the following: 1. Recombinant filamentous fungal cells expressing polynucleotides encoding heterologous lectins. 2. Recombinant cells of Embodiment 1, which secrete lectins into broth when cultured / fermented under conditions suitable for lectin production. 3. Recombinant cells of Embodiment 1, wherein the lectin is derived from plant cells, cyanobacteria cells, algal cells, bacterial cells, fungal cells, insect cells, or animal cells. 4. Lectins include natural griffithsin (GRFT) lectin or its derivative variant GRFT lectin, natural citobilin (SVN) lectin or its derivative variant SVN lectin, natural cyanobilin-N (CVN) lectin or its derivative variant CVN lectin, natural K. alvarezii KAA-1 lectin or its derivative variant KAA-1 lectin, natural K. alvarezii KAA-2 lectin or its derivative variant KAA-2 lectin, natural Microcystis viridis (MVL) lectin or its derivative variant MVL lectin, natural DCSIGN lectin or its derivative variant DCSIGN lectin, natural Boodlea coacta agglutinin (BCA) lectin or its derivative variant BCA lectin, and natural parahodo (Artocarpus) Jacarina heterophyllus lectin or its derivative variant, jacarina lectin; Musa acuminata (banana) lectin or its derivative variant, banana lectin; Aaptos papilleta (sponge) lectin or its derivative variant, sponge lectin; Abrus precatorius lectin or its derivative variant, azuki bean lectin; Aegapodium podagraria lectin or its derivative variant, aegapodium podagraria lectin; Agaricus bisporus lectin or its derivative variant, mushroom lectin; Albizzia julibrissin lectin or its derivative variant, albizzia julibrissin lectin; Allomyrina dichotoma lectin or its derivative variant, aloe vera Aloe lectin (Aloe plant) arborescens or variant aloe plant lectin derived therefrom, natural AmphicarpaeaBracteata lectin or its derivative variant, wild eel (Anguilla) lectin or its derivative variant, wild sea hare (Aplysia depilans) lectin or its derivative variant, wild peanut (Arachis hypogaea) lectin or its derivative variant, wild purple sedge (Bauhinia purpurea) lectin or its derivative variant, wild Bryonia diocia (white bryony) lectin or its derivative variant, wild hawk moth (Caragana Arborescens) lectin or its derivative variant, wild horseshoe crab (Carcinoscorpius) Horseshoe crab lectin or its derivative variant, horseshoe crab lectin; natural Microcystis aeruginosa (cyanobacteria) microbilin (MVN) lectin or its derivative variant, MVN lectin; natural Eucheuma serra (red algae) ESA-2 lectin or its derivative variant, ESA-2 lectin; natural Musa acuminate (banana) BanLec lectin or its derivative variant, BanLec lectin; natural Aspidistra elatior AEL lectin or its derivative variant, AEL lectin; natural Chaetopterus variopedatus (marine worm) CVL lectin or its derivative variant, CVL lectin; broad bean (Vicia faba) lectin or its derivative variant, broad bean lectin; natural lentil (Lens Lentil lectins derived from culinaris or the wild pea (Pisum pulcherrimum),Recombinant cells of Embodiment 1, selected from the group consisting of sativum lectin or a variant pea lectin derived therefrom, jacarin-like lectin or a variant jacarin-like lectin derived therefrom, CVN-like lectin or a variant CVN-like lectin derived therefrom, OAA-like lectin or a variant OAA-like lectin derived therefrom, galectin-1-like lectin or a variant galectin-1-like lectin derived therefrom, and lysine-like lectin or a variant trichine-like lectin derived therefrom. 5. Recombinant cells of Embodiment 1, wherein the polynucleotide encoding the lectin is an transexpression cassette comprising at least an upstream (5') promoter sequence operably ligated to a downstream nucleic acid encoding a secretory (signal peptide) sequence operably ligated to a downstream (3') nucleic acid encoding the lectin, and optionally comprising a downstream terminator sequence located downstream and operably ligated to a nucleic acid encoding the lectin. 6. Recombinant cells of Embodiment 1, in which polynucleotides encoding lectins are incorporated into the cell's genome. 7. Recombinant cells of Embodiment 1, comprising at least two introduced polynucleotides encoding lectins. 8. Recombinant cells of Embodiment 1, wherein the cells are ascomycete filamentous fungal cells. 9. Recombinant cells of Embodiment 1, selected from the group consisting of Acremonium, Aspergillus, Emericella, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma species cells. 10. The promoter is further defined as a functionally strong promoter in the recombinant cell of Embodiment 5. 11. Recombinant cells of Embodiment 10, wherein the strong promoter is selected from the group consisting of cellobiohydrolase promoter, endoglucanase promoter, β-glucosidase promoter, xylanase promoter, rev3 promoter, bxl promoter, tkl1 promoter, dld1 promoter, axe1 promoter, hxk1 promoter, dic1 promoter, opt promoter, gut1 promoter, and pki1 promoter. 12. Recombinant cells of Embodiment 5, wherein the promoter is a functional cellobiohydrolase-1 promoter (Pcbh1) containing at least 95% sequence identity to SEQ ID NO: 14. 13. Recombinant cells of Embodiment 5, wherein the nucleic acid encoding the secretory (signal peptide) sequence encodes a cellobiohydrolase-1 secretory sequence (cbh1ss) having at least 95% sequence identity with respect to SEQ ID NO: 15. 14. Recombinant cells of Embodiment 5, wherein the nucleic acid encoding the secretory (signal peptide) sequence encodes a functional aspartate endopeptidase secretory sequence (pep1ss) having at least 95% sequence identity with respect to SEQ ID NO: 16. 15. Recombinant cells of Embodiment 1 or Embodiment 2, wherein the lectin is a monomer. 16. Recombinant cells of Embodiment 1 or Embodiment 2, wherein the lectin is a dimer. 17. Recombinant cells of Embodiment 1 or Embodiment 2, wherein the lectin is a fusion protein. 18. Lectin dimers are the first (1 st ) Lectin protein sequence and second (2 nd Recombinant cells of Embodiment 16, comprising an amino acid linker sequence between the lectin protein sequence and the lectin protein sequence, the linker sequence comprising approximately 4 to 6 amino acid residues. 19. Recombinant cells of Embodiment 18, wherein the linker amino acid sequence includes SEQ ID NO: 22. 20. Recombinant cells of Embodiment 17, wherein the lectin fusion protein comprises an N-terminal protein fusion and / or a C-terminal protein fusion. 21. Recombinant cells of Embodiment 20 comprising an N-terminal starch-binding domain (SBD) protein operably linked to a C-terminal lectin protein, and / or a C-terminal SBD protein operably linked to an N-terminal lectin protein. 22. Recombinant cells of Embodiment 21, in which the SBD protein has approximately 95% identity to the Rhizopus arrhizus SBD protein of Sequence ID No. 52. 23. Recombinant cells of Embodiment 20 comprising an N-terminal cellobiohydrolase-1 core (Cbh1-core) protein operably linked to a C-terminal lectin protein and having approximately 95% sequence identity to SEQ ID NO: 12, and / or a C-terminal Cbh1-core protein operably linked to an N-terminal lectin protein and having approximately 95% sequence identity to SEQ ID NO: 12. 24. Recombinant cells of Embodiment 20, comprising an amino acid linker sequence positioned between an N-terminal protein fusion and a C-terminal lectin protein. 25. Recombinant cells of Embodiment 20, comprising an amino acid linker sequence positioned between a C-terminal protein fusion and an N-terminal lectin protein. 26. Recombinant cells of Embodiment 24 or Embodiment 25, wherein the linker sequence has approximately 95% identity with the Kex2 amino acid sequence of Sequence ID No. 20. 27. Recombinant cells of Embodiment 1, comprising gene modifications that cause a loss of production of one or more endogenous enzymes in the cells. 28. Recombinant cells of Embodiment 27, wherein the cells are deficient in the production of one or more secretory lignocellulose-degrading enzymes. 29. Recombinant cells of Embodiment 27, wherein the cells are deficient in the production of one or more secretory proteases. 30. Recombinant cells of Embodiment 1, comprising introduced polynucleotides encoding a protease inhibitor. 31. An expression cassette containing an upstream promoter sequence functional in Ascomycetes cells, which is operably ligated to a downstream nucleic acid encoding a heterologous lectin, which is operably ligated to a downstream nucleic acid encoding a functional secretory (signal peptide) sequence in Ascomycetes cells. 32. A nucleic acid encoding a heterologous lectin is a cassette of Embodiment 31 encoding a lectin monomer. 33. A nucleic acid encoding a heterologous lectin is used in the cassette of Embodiment 31, which encodes a lectin dimer. 34. A nucleic acid encoding a heterologous lectin is used in the cassette of Embodiment 31, which encodes a lectin fusion protein. 35. A method for producing lectins in filamentous fungal cells, comprising: (a) obtaining filamentous fungal cells and introducing an expression cassette into the cells containing an upstream promoter sequence operably linked to a downstream nucleic acid encoding a secretion (signal peptide) sequence operably linked to a downstream nucleic acid encoding a lectin; and (b) fermenting the modified cells under conditions suitable for lectin production, wherein the lectin is secreted into the fermentation broth. 36. The method of Embodiment 35, wherein the lectin is derived from plant cells, cyanobacteria cells, algal cells, bacterial cells, fungal cells, insect cells, or animal cells. 37. Lectins include natural griffithsin (GRFT) lectin or its derivative variant GRFT lectin, natural citobilin (SVN) lectin or its derivative variant SVN lectin, natural cyanobilin-N (CVN) lectin or its derivative variant CVN lectin, natural K. alvarezii KAA-1 lectin or its derivative variant KAA-1 lectin, natural K. alvarezii KAA-2 lectin or its derivative variant KAA-2 lectin, natural Microcystis viridis (MVL) lectin or its derivative variant MVL lectin, natural DCSIGN lectin or its derivative variant DCSIGN lectin, natural Boodlea coacta agglutinin (BCA) lectin or its derivative variant BCA lectin, and natural parahodo (Artocarpus) Jacarina heterophyllus lectin or its derivative variant, jacarina lectin; Musa acuminata (banana) lectin or its derivative variant, banana lectin; Aaptos papilleta (sponge) lectin or its derivative variant, sponge lectin; Abrus precatorius lectin or its derivative variant, azuki bean lectin; Aegapodium podagraria lectin or its derivative variant, aegapodium podagraria lectin; Agaricus bisporus lectin or its derivative variant, mushroom lectin; Albizzia julibrissin lectin or its derivative variant, albizzia julibrissin lectin; Allomyrina dichotoma lectin or its derivative variant, aloe vera Aloe lectin (Aloe plant) arborescens or variant aloe plant lectin derived therefrom, natural AmphicarpaeaBracteata lectin or its derivative variant, wild eel (Anguilla) lectin or its derivative variant, wild sea hare (Aplysia depilans) lectin or its derivative variant, wild peanut (Arachis hypogaea) lectin or its derivative variant, wild purple sedge (Bauhinia purpurea) lectin or its derivative variant, wild Bryonia diocia (white bryony) lectin or its derivative variant, wild hawk moth (Caragana Arborescens) lectin or its derivative variant, wild horseshoe crab (Carcinoscorpius) Horseshoe crab lectin or its derivative variant, horseshoe crab lectin; natural Microcystis aeruginosa (cyanobacteria) microbilin (MVN) lectin or its derivative variant, MVN lectin; natural Eucheuma serra (red algae) ESA-2 lectin or its derivative variant, ESA-2 lectin; natural Musa acuminate (banana) BanLec lectin or its derivative variant, BanLec lectin; natural Aspidistra elatior AEL lectin or its derivative variant, AEL lectin; natural Chaetopterus variopedatus (marine worm) CVL lectin or its derivative variant, CVL lectin; broad bean (Vicia faba) lectin or its derivative variant, broad bean lectin; natural lentil (Lens Lentil lectins derived from culinaris or the wild pea (Pisum pulcherrimum),The method of Embodiment 35, selected from the group consisting of sativum lectin or a variant pea lectin derived therefrom, jacarine-like lectin or a variant jacarine-like lectin derived therefrom, CVN-like lectin or a variant CVN-like lectin derived therefrom, OAA-like lectin or a variant OAA-like lectin derived therefrom, galectin-1-like lectin or a variant galectin-1-like lectin derived therefrom, and lysine-like lectin or a variant lysine-like lectin derived therefrom. 38. The method of Embodiment 35, wherein the cassette comprises an upstream (5') promoter sequence operably ligated to a downstream nucleic acid encoding a secretion (signal peptide) sequence operably ligated to a downstream (3') nucleic acid encoding a lectin, and optionally comprises a terminator sequence located downstream and operably ligated to a nucleic acid encoding a lectin. 39. The method of embodiment 35, wherein the cassette is incorporated into the genome of a cell. 40. The method of Embodiment 35, comprising at least two introduced, lectin-encoding cassettes. 41. The method of Embodiment 35, wherein the cells are ascomycete filamentous fungal cells. 42. The method of Embodiment 35, wherein the cells are selected from the group consisting of Acremonium species cells, Aspergillus species cells, Emericella species cells, Fusarium species cells, Humicola species cells, Mucor species cells, Myceliophthora species cells, Neurospora species cells, Penicillium species cells, Scytalidium species cells, Thielavia species cells, Tolypocladium species cells, and Trichoderma species cells. 43. The method of Embodiment 38, wherein the promoter is selected from the group consisting of cellobiohydrolase promoter, endoglucanase promoter, β-glucosidase promoter, xylanase promoter, rev3 promoter, bxl promoter, tkl1 promoter, dld1 promoter, axe1 promoter, hxk1 promoter, dic1 promoter, opt promoter, gut1 promoter, and pki1 promoter. 44. The method of Embodiment 38, wherein the promoter is a functional cellobiohydrolase-1 promoter (Pcbh1) having at least 95% sequence identity with respect to SEQ ID NO: 14. 45. The method of Embodiment 38, wherein the nucleic acid encoding the secretory (signal peptide) sequence encodes a cellobiohydrolase-1 secretory sequence (cbh1ss) having at least 95% sequence identity with respect to SEQ ID NO: 15. 46. ​​The method of Embodiment 38, wherein the nucleic acid encoding the secretory (signal peptide) sequence encodes a functional aspartate endopeptidase secretory sequence (pep1ss) having at least 95% sequence identity with respect to SEQ ID NO: 16. 47. The method of Embodiment 35, wherein the lectin is a monomer. 48. The method of Embodiment 35, wherein the lectin is a dimer. 49. The method of Embodiment 35, wherein the lectin is a fusion protein. 50. Lectin dimers are the first (1 st ) Lectin protein sequence and second (2 nd The method of Embodiment 48, comprising an amino acid linker sequence between the lectin protein sequence and the lectin protein sequence, wherein the linker sequence comprises approximately 4 to 6 amino acid residues. 51. The method of Embodiment 50, wherein the linker amino acid sequence includes SEQ ID NO: 22. 52. The method of Embodiment 49, wherein the lectin fusion protein comprises an N-terminal protein fusion and / or a C-terminal protein fusion. 53. The method of Embodiment 52, comprising an N-terminal starch-binding domain (SBD) protein operably linked to a C-terminal lectin protein, and / or a C-terminal SBD protein operably linked to an N-terminal lectin protein. 54. The method of Embodiment 53, wherein the SBD protein exhibits approximately 95% identity with the Rhizopus arrhizus SBD protein of Sequence ID No. 52. 55. The method of Embodiment 52, comprising an N-terminal cellobiohydrolase-1 core (Cbh1-core) protein operably linked to a C-terminal lectin protein and having approximately 95% sequence identity to SEQ ID NO: 12, and / or a C-terminal Cbh1-core protein operably linked to an N-terminal lectin protein and having approximately 95% sequence identity to SEQ ID NO: 12. 56. The method of Embodiment 52, comprising an amino acid linker sequence positioned between an N-terminal protein fusion and a C-terminal lectin protein. 57. The method of Embodiment 52, comprising an amino acid linker sequence positioned between a C-terminal protein fusion and an N-terminal lectin protein. 58. The method of Embodiment 56 or Embodiment 57, wherein the linker sequence has approximately 95% identity with the Kex2 amino acid sequence of SEQ ID NO: 20. 59. The method of Embodiment 35, comprising gene modifications that cause a defect in the production of one or more endogenous enzymes in a cell. 60. The method of Embodiment 59, wherein the cells are deficient in the production of one or more secretory lignocellulose-degrading enzymes. 61. The method of Embodiment 59, wherein the cells are deficient in the production of one or more secretory proteases. 62. The method of Embodiment 35, comprising an introduced polynucleotide encoding a protease inhibitor. 63. The method of Embodiment 35, further comprising harvesting the final fermented broth containing secreted lectins. 64. The harvested broth is subjected to a clarification process, according to the method of Embodiment 63. 65. The clarified broth is subjected to a concentration process, according to the method of Embodiment 64. 66. The method of Embodiment 65, wherein the lectin is recovered from the clarified and concentrated broth. 67. The method of Embodiment 63, wherein the pH of the harvested broth is adjusted to approximately 2.0 to approximately 2.5. [Examples]

[0187] Specific aspects of the present invention may be further understood in light of the following examples, but these examples should not be construed as limiting. Modifications of materials and methods will be obvious to those skilled in the art. The standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).

[0188] Example 1 Expression and secretion of heterologous lectin proteins As briefly described above, the applicant has conceived, designed, and constructed recombinant (modified) filamentous fungal cells capable of producing heterologous lectin proteins. In one or more specific embodiments, recombinant polynucleotides encoding lectin proteins (e.g., expression cassettes) are introduced into the filamentous fungal cells of the Disclosure. For example, in certain embodiments, an expression cassette encoding a secreted lectin (fusion) protein comprises, in the 5' to 3' direction, a promoter (pro) region sequence, a nucleic acid encoding a preprotein (signal) secretion sequence operably linked thereto (sig-seq), a nucleic acid encoding the lectin protein of interest (lectin CDS), etc.

[0189] A. Construction of a vector for the expression of griffithsin monomer as a secreted protein. To establish the expression of heterologous lectin proteins in filamentous fungal strains, lectin gene coding sequences (CDS) were operably combined (linked) with DNA encoding signal (secretion) sequences and operably linked with strong promoter DNA sequences, such as the T. reesei cellobiohydrolase I (cbh1) gene promoter (abbreviated as Pcbh1). In a specific embodiment, the lectin gene CDS (e.g., the griffisin gene, GI:90101331) encodes the griffisin (GRFT) protein, and the lectin gene CDS was codon-optimized for expression in T. reesei (SEQ ID NO: 3). For example, a synthetic DNA sequence containing a codon-optimized Griffithsin gene (named "GRFT.op1" herein) that includes a natural Cbh1 signal sequence (SEQ ID NO: 15, Cbh1ss) or a natural Pep1 signal sequence (SEQ ID NO: 16, Pep1ss) was synthesized using GeneArt Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific, Carlsbad, CA) and assembled into the Griffithsin expression vector pLH1061 as follows (SEQ ID NO: 38) (Twist Biosciences, San Francisco, CA).

[0190] The backbone of the expression vector pLH1061 was amplified from a plasmid containing the following features: a 1kb upstream (5') flanking homology sequence (5'-flank) suitable for integration into the genomic locus of T. reesei strain, a cbh1 promoter (Pcbh1) sequence (SEQ ID NO: 14), a Cbh1 protein secretion sequence (SEQ ID NO: 15, Cbh1ss) or a Pep1 protein secretion sequence (SEQ ID NO: 16, Pep1ss), a cbh1 terminator (Tcbh1) region sequence (SEQ ID NO: 17), a T. reesei pyr2 gene marker sequence (SEQ ID NO: 18) for transformation in T. reesei strain, a 1kb downstream (3') flanking homology sequence (3'-flank) suitable for integration into the genomic locus of T. reesei strain, and a bacterial vector sequence for plasmid selection and maintenance in E. coli. The synthetic DNA encoding the Griffith sin protein (GRFT.op1) contains a 25 bp 5' flanking sequence that overlaps with the cbh1 promoter (Pcbh1) region, and a 25 bp 3' flanking sequence that overlaps with the cbh1 terminator (Tcbh1) region.

[0191] In particular, this construction produced a griffithsin expression vector named "pLH1061" ([pI1-Pcbh1-GRFT.op1], SEQ ID NO: 38), as shown in Table 2 (Example 2). Similarly, the plasmid named "pLH1063" ([pI1-Pcbh1-GRFT.op2], SEQ ID NO: 77) was constructed in the same manner as for the expression of a second codon-optimized wild-type griffithsin gene under the control of the Pcbh1 promoter, while the plasmid named "pLH1065" ([pI1-Pcbh1-GRFT.M78Q-op1], SEQ ID NO: 39) was constructed in the same manner as for the secretory expression of the Q-GRFT (M78Q) variant protein. Both codon-optimized GRFT sequences (op1 vs. op2) were generated using an internal algorithm with the aim of maximizing the use of the most frequently used codons in T. reesei.

[0192] B. Construction of a vector for the expression of the Griffith sin dimer as a secreted fusion protein. The natural griffithsin (GRFT) protein from red algae (Griffithsia species) forms a stable dimer structure from two identical GRFT monomers. To evaluate and / or improve the stability of the GRFT protein expressed in T. reesei, a dimerized GRFT sequence was designed based on a codon optimization scheme for the GRFT.op1 gene. More specifically, synthetic DNA encoding the GRFT dimer ([GRFT.op1-dimer], SEQ ID NO: 7) was synthesized (Twist Biosciences), and an expression vector containing the embedded cassette was constructed by Seamless Assembly. The expression vector includes the following features: a 1kb upstream (5') flanking homologous sequence (5'-flank) suitable for integration into the genomic locus of the T. reesei strain, a cbh1 promoter (Pcbh1) sequence (SEQ ID NO: 14), a Cbh1 protein secretion sequence (Cbh1ss, SEQ ID NO: 15), a Cbh1-core protein sequence (CBH1-core, SEQ ID NO: 13), a Kex2 linker sequence (SEQ ID NO: 19), followed by a tandem dimer of the GRFT.op1 gene (between two tandem copies of the GRFT.op1 gene). The vector contains the linker DNA sequence (SEQ ID NO: 21), SEQ ID NO: 7), the cbh1 terminator (Tcbh1) region sequence (SEQ ID NO: 17), the T. reesei pyr2 gene marker sequence (SEQ ID NO: 18) for transformation in T. reesei, a 1kb downstream (3') flanking homologous sequence (3'-flank) suitable for integration into the genomic locus of the T. reesei strain, and a bacterial vector sequence for plasmid selection and maintenance in E. coli. This vector was constructed to produce a GRFT dimer expression vector named "pLH1098" ([pI1-Pcbh1-CBH1core-Kex2-GRFT.op1-linker-GRFT.op1], SEQ ID NO: 40) as shown in Table 2 (Example 2).

[0193] C. Construction of a vector for the expression of KAA-2 as a secretory fusion protein. The red alga (Kappaphycus alvarezii) KAA-2 protein is another lectin with antiviral activity. In this example, the KAA-2 gene was codon-optimized and expressed as a fusion protein to the CBH1 core protein in T. reesei. More specifically, synthetic DNA encoding the KAA-2 lectin was designed, codon-optimized for expression in T. reesei, and the expression vector was constructed in the same way as the GRFT dimer described above. This resulted in the creation of an expression vector named "pLH1080" ([pI1-Pcbh1-CBH1core-KEX2-KAA2], SEQ ID NO: 41), as shown in Table 2 (Example 2).

[0194] Example 2 Vector structure for the expression of additional lectins as secretory fusion proteins Several additional target lectin proteins, including Banlec lectin, sunflower (H. annuus) lectin, AYR06195 lectin, jacarin lectin, CV-N lectin, MVL lectin, MVN lectin, and DCSIGN lectin, were codon-optimized and subcloned for expression in T. reesei (Table 1). Specifically, each expression vector contained the target codon-optimized lectin gene and cbh1 terminator (Tcbh1) region sequence (SEQ ID NO: 17), followed by the Pcbh1 promoter, Cbh1 protein secretion sequence (cbh1ss, SEQ ID NO: 15), starch-binding domain peptide sequence (CBM21, SEQ ID NO: 52), kex2 linker sequence (SEQ ID NO: 19), and T. reesei pyr2 gene marker sequence (SEQ ID NO: 18) for transformation in T. reesei. For the expression of the starch-binding domain (CBM21)-lectin fusion protein, each expression vector was directly transformed into a T. reesei host strain, and stable transformants were selected on Vogel agar. In addition, the AFP signal peptide sequence (AFPss, SEQ ID NO: 68) was used in place of the Cbh1 protein secretion sequence, followed by the target codon optimization gene, the cbh1 terminator, and the pyr2 selection marker.

[0195] [Table 1]

[0196] [Table 2]

[0197] Example 3 Cas9-induced targeting integration of lectin expression cassettes into the T. reesei genome. In this example, a lectin-expressing T. reesei strain was generated by Cas9-induced targeted integration into the genome via homologous recombination (HR) or non-homologous end ligation (NHEJ) mechanisms. The Cas9-ribonucleoprotein complex (Cas9RNP) contains the Cas9 protein and single-stranded guide RNA (sgRNA). During protoplast transformation of T. reesei cells with the Cas9RNP complex and targeted integration cassette, Cas9RNP enters the nucleus via the nuclear localization signal (NLS) at the C-terminus of the Cas9 protein. The guide RNA then directs Cas9RNP to a targeted genomic locus, where it performs a double-strand break, which is subsequently repaired by one of various DNA repair mechanisms, such as an integration cassette containing homologous sequences for both ends of the break site or NHEJ. For targeting via homologous recombination approaches, the integration cassette typically contains sequences of 50 bp to 1000 bp that are homologous to both the 5' and 3' ends of the integration locus. For integration cassettes constructed for lectins, typically 1 kb of homologous sequences for the 5' and 3' ends are included to improve the efficiency of chromosomal integration and homology-based recombination at the desired locus.

[0198] To construct lectin-expressing strains, specific linear expression cassettes were amplified by PCR using primers OT4268 and OT4269 (see Table 3 below) to produce DNA fragments containing 1kb flanking sequences at the 5' and 3' ends for Cas9-targeted chromosomal integration at chromosomal loci.

[0199] [Table 3]

[0200] PCR reaction solutions were prepared in 6× PCR strips. Each strip contained 8 PCR tubes, with a total volume of 1.2 mL, and 50 μL of reaction solution per tube. 5' PCR primers (OT4268) and 3' PCR primers (OT4269) were added together with 0.5 μg / mL of template DNA plasmid to a final concentration of 0.5 μM (Table 3). The PCR reaction was performed using NEB-NEXT PCR Master Mix (New England Biolabs, MA) under the following conditions: 98°C, 30 sec, 35 cycles (98°C, 10 sec, 70°C, 30 sec, 72°C, 4 min), 72°C, 4 min. The final PCR products (HRD1-HRD4, Table 2) were digested with DpnI enzyme (New England Biolabs) to remove the plasmid template DNA. The reaction mixture was purified using Zymo DNA Clean and Concentrator according to the manufacturer's protocol (Zymo Research, Irvine, CA) and dissolved in the elution buffer provided in the kit to a final concentration of 0.5–1.0 μg / μL.

[0201] The Cas9RNP complex used for targeting chromosome integration of the previously described expression cassette was generated as follows: sgRNA targeting the TrC114F locus (SEQ ID NO: 39, not containing the protospacer adjacent motif (PAM) sequence "GGG") in the T. reesei genome was obtained from Synthego (South San Francisco, CA) along with the RNA sequence of SEQ ID NO: 37, and dissolved to 100 μM in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The Cas9RNP assembly reaction mixture contains 12 μM Cas9 protein (New England Biolabs) and 12 μM sgRNA-EclipseA in 1 × NEB 3.1 buffer (New England Biolabs). The reaction mixture was incubated at room temperature for 10 minutes and stored on ice until protoplast transformation was performed. Protoplast transformation of T. reesei host was performed according to standard procedures, using 5 μL of Cas9RNP complex, 4 μg of linear integration cassette PCR product, and 300 μL of T. reesei protoplasts (10 per 1 mL). 8 The transformation was carried out by combining the following: The transformation reaction mixture was placed on Vogel agar and incubated at 32°C for 5 days. Single colonies were picked from the Vogel agar plate and transferred to a fresh Vogel agar plate and incubated at 32°C for 3 days.

[0202] Next, the colonies were screened for the presence or absence of lectin expression, as described in Example 4 below. The integration of linear expression cassettes at the desired gene locus was screened and verified by colony PCR amplification from T. reesei transformants using OT4333 and OT4334 as primers.

[0203] Example 4 Screening of lectin expression clones in small-scale cultures Lectin expression in transformed T. reesei strains was screened by inoculating 1 mL of NREL medium and growing it in a 24-well microtiter plate at 28°C for 5 days. SDS-PAGE analysis was performed to identify the presence of target proteins, such as griffith sin protein (12.7 kDa), griffith sin dimer protein (25.8 kDa), and KAA-2 protein (27.9 kDa). In particular, Figure 4 shows the expression analysis of T. reesei host strains transformed with the embedded cassette HRD1 (pI1-Pcbh1-GRFT.op1, Table 2). Five microliters of culture supernatant from each clone were loaded onto an SDS-PAGE gel. Lanes 1-5 contained codon-optimized griffithsin-derived clones (GRFT-1-GRFT-5, op2), lanes 6-8 contained codon-optimized griffithsin-derived clones (GRFT-6-GRFT-8, op1), lane 9 contained the codon-optimized M78Q variant of griffithsin, and lane 10 contained an empty host strain, T. reesei. As indicated by the red arrows in the gel image (Figure 4), lanes 1-9 each contained a protein band of approximately 14 kDa, which is similar to the molecular weight of the griffithsin protein (approximately 12.7 kDa), while lane 10 did not contain any protein expressed at this molecular weight.

[0204] As shown in Figure 5, a portion of the small cell cultures was treated with pH 2 to purify the lectins based on their stability in a lower pH range. As shown in Figure 5, small cultures of GRFT (codon-optimized sequence: op1), GRFT (codon-optimized sequence: op2), and the variant GRFT-M78Q (codon-optimized sequence: op1) were incubated in 130 mM NaH2PO4, pH 2.3 at 28°C for 2 hours (0.5 mL of supernatant from each culture was added to 1 mL of 200 mM NaH2PO4, pH 2.3 buffer). The cultures were centrifuged, and the supernatant was analyzed by SDS-PAGE. As indicated by the red arrows (Figure 5), lanes 1-11 contained a protein band of approximately 14 kDa, similar in molecular weight to the griffith sin protein (approximately 12.7 kDa), while lane 3 (empty host T. reesei strain) did not contain any protein expressed at this molecular weight. The intensity of the 14kDa band in Figure 5 is generally higher than that of the 14kDa band in Figure 4 (no pH treatment), indicating that the Griffiths sin protein can be partially purified by low pH treatment (e.g., around pH 2). In addition, GRFT-op1 codon-optimized clones exhibit better expression than GRFT-op2 codon-optimized clones. For example, clone GRFT-op1 was selected from microtiter plate expression screening for further study in 2 liters of ferment. This GRFT-op1 strain was named BFZ25. In a similar process, screening of T. reesei transformants from the HRD3 embedded cassette (pI1-Pcbh1-CBH1 core-KEX2-GRFT.op1-linker-GRFT.op1) resulted in the selection of a top expression clone named BFZ72, and screening of T. reesei transformants from the HRD4 embedded cassette (pI1-Pcbh1-CBH1 core-KEX2-KAA2) resulted in the generation of a top expression clone named BFZ27.

[0205] In particular, Figures 6 and 7 show the results from additional small-scale lectin expression screening in T. reesei. As shown in the table on the right side of Figure 6 (GRFT, Q-GRFT, CV-N, MVL, SVN, DCSIGN, KAA-1, EDA2, MPA-2, ConA, Jacarin, BanLec, OAA, ESA-2, MVN, and Galectin-1), the listed lectins were expressed as fusions with the CBM21 protein under the control of the Pcbh1 promoter. As shown in the table on the right side of Figure 7 (Lectin Name), the listed lectins were further expressed as fusion proteins (CBM21) under the control of the same Pcbh1 promoter. In addition, the tables on the right side of Figure 6 (Expression Level) and Figure 7 (Fusion and CBM21 Expression Level) present semi-quantitative expression data from strains expressing lectins with and without CBM21 fusion (Figure 6). The lectin expression levels in each strain are indicated as low (+), intermediate (++), and high (+++).

[0206] Example 5 Production of secretory lectins by Fed batch fermentation Small-scale fed-batch fermentation was performed in a 2L bioreactor for the following three T. reesei strains: (1) BFZ25 (Pcbh1-GRFT.op1) for direct expression of the griffithsin (GRFT) monomer under the cbh1 promoter, (2) BFZ72 (Pcbh1-cbh1 core-KEX2-GRFT.op1 dimer) for expression of the griffithsin dimer as a fusion protein to the Cbh1 core protein and Kex2 linker under the cbh1 promoter, and (3) BFZ27 (Pcbh1-cbh1 core-KEX2-KAA) for expression of KAA-2 lectin as a fusion protein to the Cbh1 core protein and Kex2 linker under the cbh1 promoter.

[0207] Cells were initially grown in minimal medium containing 75 g / L glucose until glucose depletion. To induce protein expression under the control of the cbh1 promoter (Pcbh1), the production phase was initiated by a combined supply of glucose and sophorose at pH 4.5. 10 mL total broth samples were collected every 24 hours and frozen at -20°C, with a total fermentation time of 188 hours. For example, as shown in Figure 8, the total soluble protein secreted in each fermentation run is plotted against the effective fermentation time (EFT, hours) for the three fermentation runs described earlier (BFZ25, BFZ27, and BFZ72 strains). The fermentation supernatant was analyzed by SDS-PAGE, as shown in Figure 9. Major protein bands were detected at 12.7 kDa (Griffiths sin protein), 25.8 kDa (Griffiths sin dimer protein), and 27.9 kDa (KAA-2 protein). For example, lanes 1, 3, and 5 (Figure 9) contain fermentation samples purified by lowering the pH to 2. The griffiscin monomer appears to form a dimer structure resistant to denaturation conditions before loading onto the SDS gel. Lanes 2, 4, and 6 contain samples purified from the pH 2 treatment. These were further treated with EndoH enzyme (endoglycosidase, internally prepared from T. reesei) after neutralization of the supernatant to pH 7.0. A decrease in molecular weight was observed for all three proteins (griffiscin, griffiscin dimer, and KAA-2), indicating that these proteins are produced and glycosylated by T. reesei, and that the glycosylated groups are completely or partially removed by EndoH enzyme. The presence of griffiscin, griffiscin dimer, and KAA-2 was further confirmed by protein mass spectrometry of total protein secreted into the fermentation culture supernatant. The total protein secretion titer after the 188-hour fermentation run is shown in Table 4 below.

[0208] [Table 4]

[0209] Example 6 Expression of jacarin-like, CVN-like, OAA-like, and DC-SIGN lectins in fungal cells In this example, the applicant designed, constructed, and evaluated exemplary fungal host cells for their ability to express / produce heterologous (exotic) lectins. More specifically, as described herein, it was observed that recombinant filamentous fungal cells (e.g., Trichoderma species cells) were surprisingly capable of expressing / producing significant amounts of heterologous (i.e., eukaryotic) lectins known as jacalin-like lectins, CVN-like lectins, and OAA-like lectins. Polynucleotides (expression cassettes) encoding griffisin, jacalin-like, CVN-like, OAA-like, lysine-like, galectin-like, SVN, DC-SIGN, and ConA lectins were constructed and evaluated in 96-well plates and DASGIP fermenters, as shown and illustrated in the following sections.

[0210] A. Construction of expression plasmids and Trichoderma strains expressing Griffithsin, Jacarin-like, CVN-like, OAA-like, Lysine-like, Galectin-like, SVN, DC-SIGN, and ConA lectin. To facilitate high-throughput cloning and expression, telomere (TrTEL)-based expression constructs were created for lectin-coding genes. TrTEL-based expression vectors can be independently replicated in T. reesei and are likely incorporated at random locations in the genome. Under certain circumstances, multiple copies of the expression cassette may be incorporated into the genome. Lectin expression cassettes were subcloned into plasmid pGX256 (Figure 10). Expression vectors were constructed using restriction digestion and ligation methods as follows: (1) synthesize the target codon-optimized gene (GOI), (2) fuse the AFP signal peptide sequence or CBM21 coding sequence using PCR, (3) digest the synthesized GOI and pGX256 using AvrII and NotI, and (4) ligate the enzymatically digested product. All elements in the plasmid are shown in Biological Sequences SEQ ID NOs. 70–77. Immediately after sequence verification, the expression plasmid was directly transformed into T. reesei without vector linearization. All clones on the transformation plate were scraped for expression without individual clone PCR analysis.

[0211] Sixteen expression plasmids encoding griffithsin, jacalin-like, CVN-like, OAA-like, lysine-like, galectin-like, SVN, DC-SIGN, and ConA lectins (see, for example, Figure 11) were constructed for the expression of mature lectins in Trichoderma reesei strains. Similarly, Table 5 below provides additional details of the specific lectins cloned and expressed herein. More specifically, sixteen lectin-producing strains were created in this example. These were based on the trophic-demanding T. reesei strain StarAGX. Typically, lectin gene coding sequences (containing an AFP signal peptide or fused with a cbm1 domain) were codon-optimized based on Trichoderma reesei codon preference and cloned into NotI and AvrII restriction enzymes in the pGX256 vector using a double digestion and ligation method (Figure 11). The plasmid contained an AFP signal peptide or CBM21 domain following the cbh1 promoter, a codon-optimized nucleotide sequence encoding a lectin protein, and acetamidase (amdS) and orotate phosphoribosyltransferase (pyr2) as selection markers for transformation. The constructed vector was transformed into T. reesei strain StarAGX protoplasts and plated onto AMDS plates containing acetamide as the sole nitrogen source. After culturing for 6 days at 28°C in an illuminated incubator, the spores on the plates were scraped and spread onto new AMDS plates, where they were cultured for another 6 days in an illuminated incubator.

[0212] [Table 5]

[0213] B. Evaluation of secretory lectins generated in 96-well plates and DASGIP. Therefore, as generally described in this specification, the expression of a specific lectin protein in the T. reesei strain StarAGX was evaluated and recovered in 96-well plates and 1 L bioreactors. For the evaluation of lectin expression, the lectin-producing strain was grown in a fermentation medium (glycine medium) containing cell growth nutrients such as a carbon source, e.g., sugar, alcohol, organic acid, and amino acid, a nitrogen source such as ammonium salts and nitrates, phosphates, magnesium salts, potassium salts, and sodium salts, trace metal salts containing iron, manganese, zinc, copper, cobalt, molybdate, calcium, boron, etc. to increase the amount of cell mass. Slices of the recombinant strain on AMDS plates were planted into 96-well plates containing 300 μl of glycine medium in each well. The strain was cultured in an oxygen chamber at 28 °C with an oxygen concentration of 80% for 6 days. An equal volume of 100 mM sodium phosphate (pH 2.3) was added to the broth after fermentation and incubated at room temperature for 1 hour with shaking to release the lectin bound to the cell wall.

[0214] C. Fermentation and Recovery The lectin-producing strain was grown in a fermentation medium (glycine medium) containing cell growth nutrients such as a carbon source like sugar, alcohol, organic acid, and amino acid, a nitrogen source such as ammonium salts and nitrates, phosphates, magnesium salts, potassium salts, and sodium salts, trace metal salts containing iron, manganese, zinc, copper, cobalt, molybdate, calcium, boron, etc. to increase the amount of cell mass. The cell bank of the recombinant strain was maintained in a frozen state. First, slices of the strain were planted on AMDS plates, and then spores and hyphae were planted in glycine medium to prepare a seed for DASGIP fermenter fermentation. The seed cells were further grown in a batch of fermentation medium. The fermentation temperature was maintained at 28 °C for 6 hours.

[0215] In this example, the fermentation broth was recovered and made into a clarified concentrate. This can typically be accomplished through a variety of methods beginning with broth treatment including dissolution (e.g., natural or chemically induced dissolution, in this example natural), heat treatment, pH and temperature control, water or buffer dilution, and optionally flocculation. For example, cell separation can be performed by a variety of methods including, but not limited to, centrifugation, depth filtration, or membrane-based operations. Concentration is performed via an ultrafiltration membrane operation. In certain embodiments, the resulting clarified concentrate is further purified as described in subsequent examples. Additional details regarding the recovery process can be found in subsequent examples of the present disclosure and herein.

[0216] D. Expression evaluation More specifically, as shown in Figures 12 and 13, the expression of griffiscin, jacalin-like, CVN-like, OAA-like, lysine-like, galectin-like, SVN, DC-SIGN, and ConA lectin in the broth supernatant was evaluated via SDS-PAGE. 15 microliters (15 μL) of sample were evaluated by SDS-PAGE with gel staining and destaining using standard molecular biology procedures, following Invitrogen broad-spectrum molecular weight standards. As shown in the SDS-PAGE gel (Figures 12 and 13), griffiscin protein (SEQ ID NO: 1, Figure 1A and SEQ ID NO: 4, Figure 1B) appears as a single band with a molecular weight of approximately 10 kDa–16 kDa, with all lanes being supernatant broth. As shown in the SDS-PAGE gel (Figures 12 and 13), jacarin-like proteins (SEQ ID NO: 23, Figure 2A and SEQ ID NO: 27, Figure 2B) appear as single bands with molecular weights of approximately 10 kDa to 16 kDa, and all lanes contain supernatant broth derived from small cultures. The molecular weight of CVN-like lectin (SEQ ID NO: 29) is approximately 10 to 16 kDa, as shown in the SDS-PAGE. As shown in the SDS-PAGE gel (Figures 12 and 13), OAA-like proteins (SEQ ID NO: 10) appear as single bands with molecular weights of approximately 10 kDa to 40 kDa, and all lanes are supernatant broth. As shown in the SDS-PAGE gel (Figures 12 and 13), DC-SIGN-like proteins (SEQ ID NO: 35) appear as single bands with molecular weights of approximately 10 kDa to 16 kDa, and the lanes are supernatant broth.

[0217] Example 7 Recovery and selective purification of griffiscin, jacarin-like, CVN-like, OAA-like, and DC-SIGN lectins produced in fungal cells. This example describes a method for recovering griffiscin, jacarin-like, CVN-like, OAA-like, and DC-SIGN lectins in substantially pure form from fermentation broth obtained from recombinant host cells (e.g., bacterial cells, plant cells, and insect cells). More specifically, such exemplary purification methods include acid treatment by adding 200 mM NaPi (pH 2.3), ion affinity separation via a chromatographic column, and separation into two or more fractions, one of which contains the lectins. Prior to purification, lectins fused with CBM21 were digested with protease, and CBM21 was captured with dextrin-containing resin.

[0218] In particular, as illustrated herein, equal volumes of 200 mM NaPi (pH 2.3) were added to the fermentation broth of the OAA-like lectin (SEQ ID NO: 62), and the mixture was centrifuged at 10,000 g to remove background proteins. The final concentrated sample of the OAA-like lectin was formulated with 20 mM sodium citrate, pH 3.5, 150 mM NaCl buffer, and 40 w / w% glycerol.

[0219] In particular, as illustrated herein, equal volumes of 200 mM NaPi (pH 2.3) were added to the fermentation broth of jacalin-like lectin (SEQ ID NO: 27), and the mixture was centrifuged at 10,000 g to remove background proteins. Subsequently, ammonium sulfate was added to a final concentration of 1 mole. The solution was loaded onto a HIC column pre-equilibriumated with 20 mM NaAC, pH 5.0, supplemented with 1 M ammonium sulfate. The target lectin protein was eluted by a 0–1 M ammonium sulfate gradient. The fraction containing the target protein was pooled, buffered with 20 mM Tris-HCl (pH 7.5), and incubated overnight at 4°C to promote isoelectroprecipitation. The resulting target protein pellet was resuspended in 20 mM sodium citrate, pH 3.5, 150 mM NaCl buffer, and 40 w / w% glycerol.

[0220] In particular, as illustrated herein, equal volumes of 200 mM NaPi (pH 2.3) were added to fermentation broths of griffiscin (SEQ ID NO: 1, SEQ ID NO: 4), jacarin (SEQ ID NO: 27), CVN (SEQ ID NO: 29), and DC-SIGN (SEQ ID NO: 35) lectins, and the mixture was centrifuged at 10,000 g to remove background proteins. The pH of the lectins collected in the supernatant was adjusted to neutral by adding 2 moles of sodium hydroxide. Subsequently, the buffer of the lectin-containing samples was converted to 20 mM sodium citrate, pH 3.5, 150 mM NaCl using a 3 kDa filter. The final concentrated samples were formulated with 20 mM Tris, pH 7.5, 150 mM NaCl buffer, and 40 w / w% glycerol.

[0221] Example 8 Assay of lectin hemagglutination activity This embodiment describes a method for screening the binding activity of one or more purified lectins and / or crude lectins described above using a hemagglutination assay. The principle is that active lectins react with specific carbohydrate moieties on the surface of red blood cells, resulting in the formation of a diffusion matrix, while inactive lectins are unable to bind to red blood cells, resulting in the formation of prominent agglutinations. This allows for clear visual differentiation between active and inactive lectin samples. More specifically, lectin hemagglutination ability (activity) was evaluated using red blood cells from 15 different animal sources (i.e., dogs, rabbits, guinea pigs, mice, rats, humans, chickens, turkeys, ducks, geese, pigs, cattle, horses, sheep, and goats), processed according to the materials and methods described herein.

[0222] A. Material The materials included: (i) animal erythrocytes in Alsever solution (Sbjbio company, Nanjing, China) (including dog, rabbit, guinea pig, mouse, rat, human, chicken, turkey, duck, goose, pig, cattle, horse, sheep, and goat erythrocytes), (ii) 200 mM phosphate buffer (PB, pH 7.4), and (iii) a 96-well round-bottom microwell plate (Nunc, Thermo Scientific, USA).

[0223] B. Method (i) Lectins were detected in both purified and crude samples using a hemagglutination test. Purified lectin samples were diluted to initial concentrations of 1–800 μg / ml using PB buffer, and 50 μL of each was distributed into the sample wells of a 96-well round-bottom microplate. Crude samples were diluted by estimating protein expression levels based on SDS-PAGE gel. (ii) 50 μL of host strain supernatant was distributed into the control wells of the microplate above as a negative control. A column of 50 μL of PB buffer was also included in the microplate above to assess the possible buffer effect. (iii) Animal erythrocytes were diluted to an initial concentration of 2% (v / v) using PB buffer, and 50 μL of each was added to the samples above. (iv) The mixtures were pipetted together for 30 seconds, and the plates were allowed to settle at room temperature for 60 minutes. (v) Hemagglutination activity was determined by visual testing. Active lectins were characterized by the formation of diffusion networks, while inactive lectins were observed to form precipitate buttons at the bottom of the wells.

[0224] C. Results As shown in Figures 14 and 15, the hemagglutinating ability of lectin samples could be easily determined by visually identifying a precipitate button at the bottom of the well for hemagglutinating-negative samples, even for suspensions showing no signs of agglutination in the well for hemagglutinating-positive samples. PB buffer and host strain supernatant did not exhibit hemagglutinating ability (Figure 14). A dose-response pattern of hemagglutinating activity for active samples was detected at various doses of lectin on 1% mouse erythrocytes. This indicates that lectin hemagglutinating ability was dose-dependent (Figure 14). These results demonstrate that the hemagglutination assay is a viable method for distinguishing between "strongly positive" (SEQ ID NO: 27 at 400 μg / ml for canine and rabbit erythrocytes in Figure 14), "weakly positive" (SEQ ID NO: 27 at 50 and 200 μg / ml for canine erythrocytes in Figure 14), "intermediately positive" (SEQ ID NO: 27 at 400 μg / ml for guinea pig erythrocytes in Figure 14), and negative results (SEQ ID NO: 27 for chicken, turkey, and duck erythrocytes in Figure 14). Lectin hemagglutination is stable at room temperature for approximately 1–2 hours, and observations and interpretations may vary from test to test depending on the batch of animal erythrocytes, the type and concentration of lectin, and the incubation time and temperature used in the assay. Therefore, in this embodiment, the hemagglutination assay was not intended to be used as a quantitative method, but rather to detect samples with positive hemagglutination activity. To ensure data accuracy, at least two repeat plates were prepared for each test, and all results showed good reproducibility. The most important positive results are recorded in Figure 15. The corresponding effective animal erythrocytes are summarized in Table 6 below.

[0225] [Table 6]

[0226] References International Publication No. 2002 / 14490 Pamphlet International Publication No. 2003 / 083125 Pamphlet International Publication No. 2005 / 118627 Pamphlet International Publication No. 2007 / 064844 Pamphlet International Publication No. 2008 / 022303 Pamphlet International Publication No. 2010 / 014248 Brochure International Publication No. 2014 / 197650 Pamphlet International Publication No. 2016 / 130628 Brochure International Publication No. 2019 / 108656 Brochure U.S. Patent Application Publication No. 20020127675 U.S. Patent Application Publication No. 20040204365 U.S. Patent Application Publication No. 20110189105 U.S. Patent Application Publication No. 20110263485 Ausubel et al., “Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Ayouba et al., “Legume lectins interact with muramic acid and N-acetylmuramic acid”, FEBS, 289(1):12-104, 1991. Cole et al., “Differentiation of Bacillus anthracis and Other Bacillus Species by Lectins”, J. Clinical Microbiol., 19(1):48-54, 1984. El-Araby et al., “Characterization and antimicrobial activity of lectins purified from three Egyptian leguminous seeds”, AMB Express, 10:90(pages 1-14), 2020. Gengenbach et al.,“Comparison of microbial and transient expression(tobacco plants and plant‐cell packs)for the production and purification of the anticancer mistletoe lectin viscumin”,Biotechnology and Bioengineering,116,pages 2236-2249,2019. Hirayama et al.,“High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120”,Mar.Biotechnol.,Vol 18,pages 144-160,2016. Lagarda-Diaz et al.,“Legume Lectins:Proteins with Diverse Applications”,Int.J.Mol.Sci.,18,1242,2017. O’Keefe et al.,“Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component”,PNAS,Vol.106,No.15,pages 6099-6104,2009. Petrova et al.,“Engineering Lactobacillus rhamnosus GG and GR-1 to express HIV-inhibiting griffithsin”,International Journal of Antimicrobial Agents,volume 52,issue 5,pages 599-607,2018. Petrova et al.,“The lectin-like protein 1 in Lactobacillus rhamnosus GR-1 mediates tissue-specific adherence to vaginal epithelium and inhibits urogenital pathogens”,Scientific Reports 6,Article No.37437,2016. Peumans and Van Damme,“Plant Lectins:Versatile Proteins with Important Perspectives in Biotechnology”,Biotechnology and Genetic Engineering Reviews,15:1,199-228,1998. Pevzner et al.,“Differences in amino acid sequences of mistletoe lectin I and III B-subunits determining carbohydrate binding specificity”,Biochimica et Biophysica Acta 1675:155-164,2004. Sambrook et al.,“Molecular Cloning:A Laboratory Manual” Cold Spring Harbor Laboratory:Cold Spring Harbor,N.Y.(1989),(2001)and(2012). Singh and Sarathi,“Insights of Lectins-A review”,International J.Scientific&Engineering Res.,Volume 3,Issue 4,2012. Whitley et al.,“Burkholderia oklahomensis agglutinin is a canonical two-domain OAA-family lectin:structures,carbohydrate binding,and anti-HIV activity”,FEBS J.,280(9):2056-2067,2013.

Claims

1. Recombinant filamentous fungal cells expressing polynucleotides encoding heterologous lectins.

2. The recombinant cell according to claim 1, wherein the lectin is secreted into the broth when fermented under conditions suitable for the production of the lectin.

3. The recombinant cell according to claim 1, wherein the lectin is derived from plant cells, cyanobacteria cells, algal cells, bacterial cells, fungal cells, insect cells, or animal cells.

4. The recombinant cell according to claim 1, wherein the transexpression cassette includes an upstream promoter sequence operably ligated to a downstream nucleic acid encoding a signal sequence operably ligated to a downstream nucleic acid encoding the lectin.

5. The recombinant cell according to claim 1, wherein the polynucleotide encoding the lectin is incorporated into the cell's genome.

6. Recombinant cells according to claim 1, which are filamentous fungal cells of ascomycetes.

7. The recombinant cell according to claim 1, wherein the lectin is a monomer.

8. The recombinant cell according to claim 1, wherein the lectin is a dimer.

9. The recombinant cell according to claim 1, wherein the lectin is a fusion protein.

10. Recombinant cells according to claim 1, comprising gene modifications that cause the production of one or more endogenous enzymes in the cells.

11. An expression cassette containing an upstream promoter sequence functional in ascomycetes cells, which is operably ligated to a downstream nucleic acid encoding a heterologous lectin, which is operably ligated to a downstream nucleic acid encoding a secretory sequence functional in ascomycetes cells.

12. The cassette according to claim 11, wherein the encoded lectin is a monomer or a dimer.

13. The cassette according to claim 11, wherein the encoded lectin is a fusion protein.

14. A method for inducing the production of heterologous lectins in filamentous fungal cells, (a) Obtaining filamentous fungal cells and introducing an expression cassette into the cells that includes an upstream promoter operably linked to a downstream nucleic acid encoding a secretion sequence operably linked to a downstream nucleic acid encoding the lectin, (b) Fermenting the modified cells under conditions suitable for the production of the lectin. A method comprising the lectin being secreted into the fermentation broth.

15. The method according to claim 14, wherein the lectin is derived from plant cells, cyanobacteria cells, algal cells, bacterial cells, fungal cells, insect cells, or animal cells.

16. The method according to claim 14, wherein the cassette is incorporated into the genome of a cell.

17. The method according to claim 14, wherein the cells are filamentous fungal cells of ascomycetes.

18. The method according to claim 14, wherein the lectin is a monomer.

19. The method according to claim 14, wherein the lectin is a dimer.

20. The method according to claim 14, wherein the lectin is a fusion protein.

21. The method according to claim 19, wherein the lectin dimer includes an amino acid linker sequence between a first lectin sequence and a second lectin sequence, and the linker sequence includes about 4 to about 6 amino acid residues.

22. The method according to claim 20, wherein the lectin fusion protein comprises an N-terminal protein fusion and / or a C-terminal protein fusion.

23. The method according to claim 14, wherein the cells include gene modifications that cause a defect in the production of one or more endogenous enzymes in the cells.

24. The method according to claim 14, comprising harvesting the final fermented broth containing the secreted lectin.

25. The method according to claim 24, wherein the harvested broth is subjected to a clarification process.

26. The method according to claim 25, wherein the clarified broth is subjected to a concentration process.

27. The method according to claim 26, wherein the lectin is recovered from the clarified and concentrated broth.

28. A lectin protein preparation obtained by the method described in claim 14.

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