WO2023245100A1 - Peptides variants de ncr13 antimicrobiens - Google Patents

Peptides variants de ncr13 antimicrobiens Download PDF

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Publication number
WO2023245100A1
WO2023245100A1 PCT/US2023/068490 US2023068490W WO2023245100A1 WO 2023245100 A1 WO2023245100 A1 WO 2023245100A1 US 2023068490 W US2023068490 W US 2023068490W WO 2023245100 A1 WO2023245100 A1 WO 2023245100A1
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nvp
amino acid
seq
acid sequence
sequence
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PCT/US2023/068490
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English (en)
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Lin BAO
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Vestaron Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • Microbial infections e.g., iungal infections
  • iungal infections are a particular problem in damp climates, and are of major concern during crop storage; indeed, the degree and severity of iungal infections can be exacerbated by modern growing methods — as harvesting and storage systems frequently provide a favorable environment for these plant pathogens.
  • pathogenic microbes e.g., fiingi, bacteria, oomycetes, etc.
  • World-wide international travel has aided in spreading these deleterious microbes to parts of the planet where native plants have evolved no defenses.
  • the emphasis on intensive monoculture practices of commercially relevant crops in concert with traditional disease-mitigation strategies has allowed pathogenic microbes to become resistant and thrive.
  • NVP engineered antimicrobial NCR13 variant peptide having antimicrobial activity against one or more microbes, said NVP comprising an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid is any natural or non-natural amino acid other than glycine or alanine; or an agriculturally acceptable salt thereof.
  • NVP antimicrobial NCR13 variant peptide
  • the present disclosure describes a polynucleotide operable to encode an antimicrobial NCR13 variant peptide (NVP) having antimicrobial activity against one or more microbes, said NVP comprising an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid is any natural or non-natural amino acid other than glycine or alanine; or a complementary nucleotide sequence thereof.
  • NVP antimicrobial NCR13 variant peptide
  • composition comprising an antimicrobial NCR13 variant peptide (NVP) having antimicrobial activity against one or more microbes, or an agriculturally acceptable salt thereof, said NVP comprising an N- terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid is any natural or non-natural amino acid other than glycine or alanine; and an excipient.
  • NVP antimicrobial NCR13 variant peptide
  • the present disclosure describes a method of combating, controlling, or inhibiting a microbe comprising applying a antimicrobially effective amount of: (1) an antimicrobial NCR13 variant peptide (NVP) having antimicrobial activity against one or more microbes, or an agriculturally acceptable salt thereof, said NVP comprising an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid is any natural or non-natural amino acid other than glycine or alanine; or (2) a composition comprising the NVP and an excipient; to the microbe, a locus of the microbe, a food supply of the microbe, a habitat of the microbe, or a breeding ground of the microbe; a plant, a seed, a plant part, a locus of a plant, or an environment of a plant that is susceptible to an attack by the microbe; an animal, a locus of an animal, or an environment of an animal susceptible
  • FIG. 1 shows an example of a representative plasmid map for a vector comprising a polynucleotide encoding an NCR13 variant peptide (NVP) of the present disclosure.
  • the vectors comprise the following elements: a Kex2 cleavage site (not shown); a multiple cloning site; a LAC4 terminator; and ADH1 promoter; a 0-lactamase (bla) gene; and an origin of replication site.
  • FIG. 2 shows the results of the deep-well expression assay.
  • the vectors pLB602 (operable to encode NCR13a, SEQ ID NO: 2), pLB603 (operable to encode WT- NCR13, SEQ ID NO: 1), and pLB602Ml-M17 (encoding the NCR13M1 having SEQ ID NO: 3, and NVPs having SEQ ID NOs: 4-19), were then linearized, and transformed into electrocompetent Kluyveromyces lactis host cells, and the expression of the peptides were analyzed.
  • the vector names are shown below, with the N-terminus addition for a given NVP shown in parenthesis.
  • FIG. 3 shows the results of a qPCR assay evaluating yield-per-copy number.
  • the Y-axis shows relative quantification (“RQ”).
  • the numbers above each bar shows the number of integrated gene copies.
  • YCT YCT H12 and YCT 220104 are untransformed control strains.
  • URA3 was used as the endogenous control gene. Error bars show minimum and maximum number of copies.
  • FIG. 4 shows the results of a qPCR assay evaluating yield-per-copy number.
  • the Y-axis shows relative quantification (“RQ”).
  • the numbers above each bar shows the number of integrated gene copies.
  • YCT YCT H12 and YCT 220104 are untransformed control strains.
  • YCT YCT H12 (shown in a box) was the calibration strain.
  • URA3 was used as the endogenous control gene. Error bars show minimum and maximum number of copies.
  • FIG. 5 shows the results of a qPCR assay evaluating yield-per-copy number.
  • the Y-axis shows relative quantification (“RQ”).
  • the numbers above each bar shows the number of integrated gene copies.
  • YCT306 Hl 8 and YCT 16-77 are untransformed control strains.
  • YCT306 Hl 8 (shown in the box) was the calibration strain.
  • URA3 was used as the endogenous control gene. Error bars show minimum and maximum number of copies.
  • FIG. 6 shows the results of a qPCR assay evaluating yield-per-copy number.
  • the Y-axis shows relative quantification (“RQ”).
  • the numbers above each bar shows the number of integrated gene copies.
  • YCT 220104 H6 and YCT 16-778 are untransformed control strains.
  • YCT 16- 778 (shown in the box) was the calibration strain.
  • URA3 was used as the endogenous control gene. Error bars show minimum and maximum number of copies.
  • FIG. 7 shows the results of a qPCR assay evaluating yield-per-copy number.
  • the Y-axis shows relative quantification (“RQ”).
  • the numbers above each bar shows the number of integrated gene copies.
  • YCT 220104 and YCT 16-77 are untransformed control strains.
  • FIG. 8 shows a graph depicting the liner relationship between peptide yield and numbers of integrated gene copies. The slope of lines is defined as gene productivity, which indicates the yield of a given peptide per integrated gene copy.
  • FIG. 9 shows the slopes of the lines shown in FIG. 8.
  • the horizontal line shows NCR13a.
  • FIG. 10 shows the results of the antimicrobial bioassay.
  • the antimicrobial effect of the NCR13 variant peptides (NVPs) of the present disclosure were evaluated against the ftingal species, Botrytis cinerea. Briefly, NVPs showing superior yield relative to NCR13a (i.e., NVPs having an amino acid sequence as set forth in SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, and 17), were incubated at varying concentrations with Botrytis cinerea. The growth of Botrytis cinerea was then assessed via OD600nm after incubation with a given NVP at room temperature after 96 hours.
  • FIG. 11 shows a graph depicting the overall improvement of a given NVP as determined by yield improvement times the activity improvement.
  • the N-terminus amino acid addition of each NVP is shown on the X-axis. Asterisks show the NVP with the greatest improvement.
  • 5’ -end and “3 ’-end” refers to the directionality, i.e., the end-to-end orientation of a nucleotide polymer (e.g., DNA).
  • the 5’-end of a polynucleotide is the end of the polynucleotide that has the fifth carbon.
  • “5’- and 3 ’-homology arms” or “5’ and 3’ arms” or “left and right arms” refers to the polynucleotide sequences in a vector and/or targeting vector that homologously recombine with the target genome sequence and/or endogenous gene of interest in the host organism in order to achieve successfiil genetic modification of the host organism’s chromosomal locus.
  • Affect refers to how a something influences another thing, e.g., how a peptide, polypeptide, protein, drug, or chemical influences a microbe.
  • Agriculturally-acceptable carrier covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in agricultural formulation technology; these are well known to those skilled in agricultural formulations.
  • Agriculturally acceptable salt is synonymous with pharmaceutically acceptable salt, and as used herein refers to a compound that is modified by making acid or base salts thereof.
  • Alignment refers to a method of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide, or protein sequences) for the purpose of determining their relationship to each other. Alignments are typically performed by computer programs that apply various algorithms, however, it is also possible to perform an alignment by hand. Alignment programs typically iterate through potential alignments of sequences and score the alignments using substitution tables, employing a variety of strategies to reach a potential optimal alignment score. Commonly-used alignment algorithms include, but are not limited to, CLUSTALW (see Thompson J. D., Higgins D. G., Gibson T.
  • Exemplary programs that implement one or more of the foregoing algorithms include, but are not limited to, MegAlign from DNAStar (DNAStar, Inc. 3801 Regent St. Madison, Wis. 53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio from Accelrys (Accelrys, Inc., 10188 Telesis Ct, Suite 100, San Diego, Calif. 92121).
  • an alignment will introduce “phase shifts” and/or “gaps” into one or both of the sequences being compared in order to maximize the similarity between the two sequences, and scoring refers to the process of quantitatively expressing the relatedness of the aligned sequences.
  • Alpha-MF signal or “aMF secretion signal” refers to a protein that directs nascent recombinant polypeptides to the secretory pathway.
  • “Ameliorate” or “amelioration” includes the arrest, prevention, decrease, or improvement in one or more the symptoms, signs, and features of the disease being treated, both temporary and long-term.
  • antimicrobial is generally used to refer to the ability of a combination or composition of the present disclosure, to increase mortality or inhibit growth rate of microbes.
  • Antimicrobial effect refers to inhibition or stoppage of the normal metabolic processes required for continued life, or continued growth of a microbe. “Antimicrobial effect” includes killing of any individual or group of microbes.
  • Antimicrobial activity means that upon or after exposing the microbe to the combinations or compositions of the present disclosure, the microbe either dies, stops, or slows its cellular processes; stops or slows its maintenance; stops or slows its growth; fails to reproduce; and the like.
  • Antimicrobial composition refers to a composition comprising a NVP, or an agriculturally acceptable salt thereof; and an excipient.
  • Antimicrobially effective amount refers to an amount of (1) an NVP, or an agriculturally acceptable salt thereof, or (2) an antimicrobial composition comprising: an NVP, or an agriculturally acceptable salt thereof; and an excipient; that is sufficient to: inhibit a microbe, bring about the death of at least one microbe; noticeably reduce or decrease microbe growth, feeding, or normal physiological development; inhibit or decrease the normal microbe cellular processes, including maintenance and growth; and/or attenuate or decrease the severity of a microbial infection. This amount will vary depending on such factors including but not limited to: the specific target microbe to be controlled; the specific environment, location, plant, crop, or agricultural site to be treated; the environmental conditions, method, rate, concentration, stability, and quantity applied.
  • antimicrobially-effective amount may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of microbe infestation.
  • Antimicrobially-effective amounts can be measured by use of assays that measure the reduction in growth or decline in their populations of a microbe.
  • One measure of reduction can be to express the decrease in population in logarithmic scale typical of a specific microbial species. That is, a 1 log reduction is equivalent to a 90% reduction versus a control, a 2 log reduction is a 99% reduction, etc.
  • applying means to dispense and/or otherwise provide, and refers to any method of application or route of administration.
  • applying can refer to, e.g., application of a of an engineered, non-naturally occurring antimicrobial peptide (e.g., an NVP), or an agriculturally acceptable salt thereof, or an antimicrobial composition comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and one or more excipients, e.g., a sprayable composition, a foam; a burning formulation; a fabric treatment; a surface-treatment; a dispersant; or a microencapsulation.
  • an engineered, non-naturally occurring antimicrobial peptide e.g., an NVP
  • an antimicrobial composition comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and one or more excipients, e.g., a sprayable composition, a foam; a burning formulation; a
  • co-application or “co-administer” it is meant that a combination or composition described herein is applied or administered at the same time, just prior to, or just after the application of: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and optionally, one or more additional agents or excipients, also referred to herein as a “additional agent.”
  • the NVP, or an agriculturally acceptable salt thereof, of the present disclosure, and optionally one or more excipients can be administered alone or can be co -administered to the locus of a microbe.
  • Co-application or coadministration is meant to include simultaneous or sequential application, e.g., one or more NVPs and/or one or more NVPs and one or more excipients.
  • bp or “base pair” refers to a molecule comprising two chemical bases bonded to one another.
  • a DNA molecule consists of two winding strands, wherein each strand has a backbone made of an alternating deoxyribose and phosphate groups. Attached to each deoxyribose is one of four bases, i.e., adenine (A), cytosine (C), guanine (G), or thymine (T), wherein adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • C -terminus or “C-terminal” refers to the free carboxyl group (i.e., -COOH) that is positioned on the terminal end of a polypeptide.
  • “Combination” refers to the result of combining two or more separate components.
  • a “combination” refers to an association of two or more separate components, e.g., an NVP and/or an NVP-antimicrobial protein of the present disclosure, and a chelating agent of the present disclosure.
  • a combination can refer to the association of (1) an NVP and/or an NVP- antimicrobial protein of the present disclosure, and (2) a chelating agent of the present disclosure, e.g., as a mixture, which may or may not be part of a composition fiirther comprising one or more excipients.
  • a combination can refer to the simultaneous, separate, or sequential application of two or more separate components (e.g., an NVP and/or an NVP-antimicrobial protein, and a chelating agent of the present disclosure).
  • a “combination” refers to the result of a simultaneous application of both an NVP and/or an NVP-antimicrobial protein of the present disclosure, and a chelating agent of the present disclosure.
  • a “combination” refers to the result of a separate application of an NVP and/or an NVP- antimicrobial protein of the present disclosure, and a chelating agent of the present disclosure.
  • a “combination” refers to the result of a sequential application of two or more separate components, e.g., a first application of an NVP and/or an NVP-antimicrobial protein of the present disclosure, followed by a second application of a chelating agent of the present disclosure, or vice versa. Where the application is sequential or separate, the delay in applying the second component should not be such as to lose the beneficial effect of the combination.
  • “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides as understood by those of skill in the art. Thus, two sequences are “complementary” to one another if they are capable of hybridizing to one another to form a stable anti-parallel, double-stranded nucleic acid structure.
  • a first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.
  • the polynucleotide whose sequence 5 ’-TAT AC-3’ is complementary to a polynucleotide whose sequence is 5’- GT ATA-3’.
  • the term “conservative amino acid substitutions” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic, acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); small aliphatic, nonpolar or slightly polar residues (e.g., Alanine, serine, threonine, proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine, is
  • amino acid substitutions may be made in nonconserved regions that retain fimction. In general, such substitutions would not be made for conserved amino acid residues or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that are identical in an alignment of homologs).
  • residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that have only conservative substitutions between all proteins contained in the alignment of the homologs).
  • residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments e.g., residues that have only conservative substitutions between all proteins contained in the alignment of the homologs.
  • residues that have only conservative substitutions between all proteins contained in the alignment of the homologs e.g., residues that have only conservative substitutions between all proteins contained in the alignment of the homologs.
  • Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic iunction on a protein is generally understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(l):105-32). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological iunctionally equivalent protein.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, ibid).
  • the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+0.1); alanine (-0.5); histidine (-0.5); cysteine (- 1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • “Culture” or “cell culture” refers to the maintenance of cells in an artificial, in vitro environment.
  • “Culturing” refers to the propagation of organisms on or in various kinds of media.
  • the term “culturing” can mean growing a population of cells under suitable conditions in a liquid or solid medium.
  • culturing refers to fermentative recombinant production of a peptide of interest (e.g., an NVP) and/or other desired end products (typically in a vessel or reactor).
  • a peptide of interest e.g., an NVP
  • other desired end products typically in a vessel or reactor.
  • “Decreasing” or “decrease” or “decreased” or “reducing” or “reduced” or “a reduction” or “inhibiting” or “stopping” or “combatting” or “controlling” or any variation of these terms refers to making something (e.g., the number of microbes and/or degree or severity of a microbe infection or disease) less in size, amount, intensity, or degree.
  • the application of a antimicrobially-effective amount of an NVP of the present disclosure, or an agriculturally acceptable salt thereof, and/or an antimicrobial composition comprising: an NVP, or an agriculturally acceptable salt thereof, and an excipient; to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe can result in the following effect: a decrease or reduction in the number of microbes and/or a decrease or reduction in the degree or severity of a microbe infection or disease, relative to the number of microbes and/or degree or severity of a microbe infection or disease that has not been treated with, or had applied thereto, an antimicrobially-effective amount of an NVP, or an agriculturally acceptable salt thereof, and/or an antimicrobial composition comprising: an NVP, or an agriculturally acceptable salt thereof, and an excipient, as described herein.
  • reducing or decreasing e.g., the number of microbes and/or the degree or severity of a microbe infection or disease
  • reducing or decreasing includes any measurable decrease or complete inhibition to achieve a desired result.
  • the terms “reduction in the number of microbes and/or degree or severity of a microbe infection or disease,” refers to a decrease or reduction in the number of microbes and/or degree or severity of a microbe infection or disease by a plant or animal susceptible to an attack by the microbe that has received an antimicrobially effective amount of a combination of the present disclosure, or an agricultural composition thereof, that is at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 2.25%, at least about 2.5%, at least about 2.75%, at least about 3%, at least about 3.25%, at least about 3.5%
  • DNA refers to deoxyribonucleic acid, comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form.
  • deoxyribonucleic acid comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form.
  • nucleotides i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]
  • one or more nucleotides creates a polynucleotide.
  • Excipient refers to any agriculturally acceptable additive, carrier, surfactant, emulsifier, thickener, preservative, solvent, disintegrant, glidant, lubricant, diluent, filler, bulking agent, binder, emollient, stiffening agent, stabilizer, solubilizing agents, dispersing agent, suspending agent, antioxidant, antiseptic, wetting agent, humectant, fragrant, suspending agents, pigments, colorants, isotonic agents, viscosity enhancing agents, mucoadhesive agents, and/or any combination thereof, that can be added to a composition, preparation, and/or formulation, which may be usefiil in achieving a desired modification to the characteristics of the composition, preparation, and/or formulation.
  • excipients can be formulated alongside an NVP or an NVP-antimicrobial protein of the present disclosure, when preparing a composition, e.g., for the purpose of bulking up compositions (thus often referred to as bulking agents, fillers or diluents).
  • an excipient can be used to provide stability, or prevent contamination (e.g., microbial contamination).
  • an excipient can be used to confer a physical property to a composition (e.g., a composition that is a dry granular, or dry flowable powder physical form). Reference to an excipient includes both one and more than one such excipients. Suitable excipients are described in Remington's Pharmaceutical Sciences, by E.W. Martin, the disclosure of which is incorporated herein by reference in its entirety.
  • “Expression cassette” refers to (1) a DNA sequence of interest, e.g., a polynucleotide operable to encode an NVP; and one or more of the following: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post -transcriptional regulatory elements.
  • the combination (1) with at least one of (2)-(6) is called an “expression cassette.”
  • there can be a first expression cassette comprising a polynucleotide operable to encode an NVP.
  • a double expression cassette can be generated by subcloning a second expression cassette into a vector containing a first expression cassette.
  • a triple expression cassette can be generated by subcloning a third expression cassette into a vector containing a first and a second expression cassette.
  • “Fermentation beer” refers to spent fermentation medium, i.e., fermentation medium supernatant after removal of organisms, that has been inoculated with and consumed by a transformed host cell (e.g., a yeast cell operable to express an NVP of the present disclosure).
  • fermentation beer refers to the solution that is recovered following the fermentation of the transformed host cell.
  • the term “fermentation” refers broadly to the enzymatic and anaerobic or aerobic breakdown of organic substances (e.g., a carbon substrate) nutrient substances by microorganisms under controlled conditions (e.g., temperature, oxygen, pH, nutrients, and the like) to produce fermentation products (e.g., one or more peptides of the present disclosure). While fermentation typically describes processes that occur under anaerobic conditions, as used herein it is not intended that the term be solely limited to strict anaerobic conditions, as the term “fermentation” used herein may also occur processes that occur in the presence of oxygen.
  • GFP green fluorescent protein from the jellyfish, Aequorea victoria.
  • Crowth medium refers to a nutrient medium used for growing cells in vitro.
  • Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a ftinction of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared xl00.
  • the term “homologous” refers to the sequence similarity between two polypeptide molecules, or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the homology between two sequences is a ftinction of the number of matching or homologous positions shared by the two sequences. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology.
  • sequence identity refers to a measure of relatedness between two or more nucleic acid sequences or two or more polypeptide sequences, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues or amino acid residues that are identical and in the same relative positions in their respective larger sequences.
  • “Homologous recombination” refers to the event of substitution of a segment of DNA by another one that possesses identical regions (homologous) or nearly so.
  • “homologous recombination” refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Briefly, homologous recombination is most widely used by cells to accurately repair harmftil breaks that occur on both strands of DNA, known as double-strand breaks.
  • homologous recombination varies widely among different organisms and cell types, most forms involve the same basic steps: after a double-strand break occurs, sections of DNA around the 5' ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3' end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. After strand invasion, the fiirther sequence of events may follow either of two main pathways, i.e., the double strand break repair pathway, or the synthesis -dependent strand annealing pathway.
  • homologous recombination is conserved across all three domains of life as well as viruses, suggesting that it is a nearly universal biological mechanism.
  • homologous recombination can occur using a site-specific integration (SSI) sequence, whereby there is a strand exchange crossover event between nucleic acid sequences substantially similar in nucleotide composition.
  • SSI site-specific integration
  • crossover events can take place between sequences contained in the targeting construct of the invention (i.e., the SSI sequence) and endogenous genomic nucleic acid sequences (e.g., the polynucleotide encoding the peptide subunit).
  • Hybridize refers to the annealing of one single-stranded polynucleotide to another polynucleotide based on the well-understood principle of sequence complementarity.
  • the other polynucleotide is a single-stranded polynucleotide.
  • the propensity for hybridization between polynucleotides depends on the temperature and ionic strength of their milieu, the length of the polynucleotides, and the degree of complementarity. The effect of these parameters on hybridization are well known in the art.
  • Hybridization refers to any process by which a strand of polynucleotide binds with a complementary strand through base pairing.
  • Two single-stranded polynucleotides “hybridize” when they form a double-stranded duplex.
  • the term “hybridize” refers to the annealing of one single-stranded polynucleotide to another polynucleotide based on the well-understood principle of sequence complementarity.
  • the other polynucleotide is a single-stranded polynucleotide.
  • the propensity for hybridization between polynucleotides depends on the temperature and ionic strength of their milieu, the length of the polynucleotides, and the degree of complementarity. The effect of these parameters on hybridization are well known in the art.
  • the region of double- strandedness can include the lull-length of one or both of the single-stranded polynucleotides, or all of one single stranded polynucleotide and a subsequence of the other single stranded polynucleotide, or the region of double-strandedness can include a subsequence of each polynucleotide.
  • Hybridization also includes the formation of duplexes which contain certain mismatches, provided that the two strands are still forming a double stranded helix. See “Stringent hybridization conditions ” below.
  • IC50 or “IC50” refers to half-maximal inhibitory concentration, which is a measurement of how much of an agent is needed to inhibit a biological process by half, thus providing a measure of potency of said agent.
  • Identity refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing said sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity can be readily calculated by any one of the myriad methods known to those having ordinary skill in the art, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990), the disclosures of which are incorporated herein by reference in their entireties.
  • in vivo refers to in the living body of a plant or animal (e.g., an animal, plant or a cell) and to processes or reactions that occur within the living body of a plant or animal.
  • “Inactive” refers to a condition wherein something is not in a state of use, e.g., lying dormant and/or not working.
  • inactive when used in the context of a gene or when referring to a gene, the term inactive means said gene is no longer actively synthesizing a gene product, having said gene product translated into a protein, or otherwise having the gene perform its normal function.
  • the term inactive can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with noncoding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and/or inhibition and/or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.
  • RNA processing e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications
  • interference with noncoding RNA maturation e.g., from the nucleus to the cytoplasm
  • interference with RNA export e.g., from the nucleus to the cytoplasm
  • interference with translation e.g., from the nucleus to the
  • “Inhibiting” or “inhibit” or “combating” or “combat” or “controlling” or “control,” or any variation of these terms refers to making something (e.g., the number of microbes, the ftinctions and/or activities of the microbe, and/or the deleterious effect of the microbe on a plant or animal susceptible to attack thereof less in size, amount, intensity, or degree.
  • vital building blocks e.g. nucleic acids, amino acids, biochemical metabolites
  • growth, reproduction, and/or any other parameter that is essential to the microbe’s survival and/or reproduction relative to the number of microbes or activities thereof that had not been exposed to an antimicrobially effective amount of an NVP or agriculturally acceptable salt thereof, or an agricultural composition comprising an NVP or agriculturally acceptable salt thereof.
  • combating, controlling, or inhibiting a microbe includes any measurable decrease or complete inhibition to achieve a desired result.
  • About as used herein means within ⁇ 10%, preferably ⁇ 5% of a given value.
  • the terms “combating, controlling, or inhibiting a microbe,” refers to: a decrease in the number of microbes; an increase in the susceptibility of the microbe to one or more antimicrobial agents; and/or inhibition or impairment of one or more of the microbe’s activities or ftinctions, such as any physiological fiinction required for normal physiological maintenance and/or survival and/or reproduction (e.g. respiration, membrane integrity, energy utilization, synthesis of vital building blocks e.g.
  • “Inoperable” refers to the condition of a thing not ftmctioning, malfunctioning, or no longer able to function.
  • inoperable when used in the context of a gene or when referring to a gene, the term inoperable means said gene is no longer able to operate as it normally would, either permanently or transiently.
  • inoperable in some embodiments, means that a gene is no longer able to synthesize a gene product, having said gene product translated into a protein, or is otherwise unable to gene perform its normal iunction.
  • the term inoperable can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post -transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and/or inhibition and/or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.
  • RNA processing e.g., pre-mRNA processing; RNA splicing; or other post -transcriptional modifications
  • interference with non-coding RNA maturation e.g., from the nucleus to the cytoplasm
  • interference with RNA export e.g., from the nucleus to the cytoplasm
  • interference with translation e.g., from the
  • isolated refers to separating a thing and/or a component from its natural environment, e.g., WT NCR13 peptide isolated from an organism means that peptide is separated from its natural environment.
  • kb refers to kilobase, i.e., 1000 bases.
  • the term “kb” means a length of nucleic acid molecules.
  • 1 kb refers to a nucleic acid molecule that is 1000 nucleotides long.
  • a length of double-stranded DNA that is 1 kb long contains two thousand nucleotides (i.e., one thousand on each strand).
  • a length of singlestranded RNA that is 1 kb long contains one thousand nucleotides.
  • kDa refers to kilodalton, a unit equaling 1,000 daltons; a “Dalton” is a unit of molecular weight (MW).
  • Knockdown dose 50 or “KD50” refers to the median dose required to cause paralysis or cessation of movement in 50% of a population.
  • LC50 or “lethal concentration 50%” refers to the concentration of an agent required to kill 50% of a population.
  • “Locus of a microbe” refers to the habitat of a microbe; food supply of a microbe; breeding ground of a microbe; area inhabited or colonized by a microbe; material infested, eaten, or used by a microbe; and/or any environment in which a microbe inhabits, uses, is present in, or is expected to be.
  • the locus of a microbe includes, without limitation, a microbe habitat; a microbe food supply; a microbe breeding ground; a microbe area; a microbe environment; any surface or location that may be frequented and/or infested by a microbe; any plant or animal, or a locus of a plant or animal, susceptible to attack by a microbe; and/or any surface or location where a microbe may be found, may be expected to be found, or is likely to be attacked by a microbe.
  • “Medium” refers to a nutritive solution for culturing cells in cell culture.
  • Metal ion refers to an atom or compound that has an electric charge.
  • metal ions are cations, e.g., a positively charged ion, and can be represented as M z+ , where z is the electrical charge.
  • Metal ions may be dissolved in water, can be referred to as “metal ion in aqueous solution” or “aqua ion,” and may be represented by the formula [M(H2O) n ] z+ , where n is the solvation number and z is the electrical charge.
  • Metal-ion chelating agent refers to a chelating agent operable to chelate a metal ion, e.g., a chelating agent operable to chelate one of the following metal ions: Al 3+ ; Ag + ; AS 3+ ; AU + ; AU 3+ ; Ba 2+ ; Be 2+ ; Ca 2+ ; Cd 2+ ; Co 2+ ; Co 3+ ; Cr 2+ ; Cr 3+ ; Cs + ; Cu + ; Cu 2+ ; Fe 2+ ; Fe 3+ ; Ga 3+ ; Hg 2+ ; Hg 2 2+ ; In 3+ ; K + ; Li + ; Mg 2+ ; Mn 2+ ;Na + ; Ni 2+ ; Pb 2+ ; Pb 4+ , Sn 2+ ; Sn 4+ ; Sr 2+ ; and/or Zn 2+ .
  • “Microbe” refers to any microscopic organism, e.g., any multi-cellular or unicellular microorganism, or a virus, including all of the prokaryotes, namely the eubacteria and archaeabacteria, and various forms of eukaryote, comprising the protozoa, ftmgi (e.g., yeast).
  • “microbe” refers to all bacteria, all archaea, unicellular protista, unicellular animals, unicellular plants, unicellular ft gi, unicellular algae, all protozoa, and all chromista.
  • a microbe can be a pathogenic microbe, wherein the microbe causes an infection or disease in a living organism when introduced into said organism; or wherein the presence of the microbe is deleterious to the organism.
  • a microbe can be a pathogen to plants, e.g., a phytopathogen, such as a bacterium, a protozoan, or a ftingus.
  • MO A refers to mechanism of action.
  • MW refers to the mass or weight of a molecule, and for proteins is typically measured in “daltons (Da)” or kilodaltons (kDa).
  • MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), gel chromatography, analytical ultracentriftigation, mass spectrometry, or light scattering.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • the SDS-PAGE method is as follows: the sample of interest is separated on a gel with a set of molecular weight standards. The sample is run, and the gel is then processed with a desired stain, followed by destaining for about 2 to 14 hours.
  • the next step is to determine the relative migration distance (Rf) of the standards and protein of interest.
  • the logarithm of the MW can be determined based on the values obtained for the bands in the standard; e.g., in some embodiments, the logarithm of the molecular weight of an SDS- denatured polypeptide and its relative migration distance (Rf) is plotted into a graph. After plotting the graph, interpolating the value derived will provide the molecular weight of the unknown protein band.
  • “Motif’ refers to a polynucleotide or polypeptide sequence that is implicated in having some biological significance and/or exerts some effect or is involved in some biological process.
  • “Mutant” refers to an organism, DNA sequence, amino acid sequence, peptide, polypeptide, or protein, that has an alteration or variation (for example, in the nucleotide sequence or the amino acid sequence), which causes said organism and/or sequence to be different from the naturally occurring or wild-type organism, wild-type sequence, and/or reference sequence with which the mutant is being compared. In some embodiments, this alteration or variation can be one or more nucleotide and/or amino acid substitutions or modifications (e.g., deletion or addition).
  • the one or more amino acid substitutions or modifications can be conservative; here, such a conservative amino acid substitution and/or modification in a “mutant” does not substantially diminish the activity of the mutant in relation to its non-mutant form.
  • a “mutant” possesses one or more conservative amino acid substitutions when compared to a peptide with a disclosed and/or claimed sequence, as indicated by a SEQ ID NO.
  • N-terminus refers to the free amine group (i.e., -NFb) that is positioned on beginning or start of a polypeptide.
  • N-terminus addition refers to the addition of an amino acid to the N-terminus of a peptide.
  • an NVP can comprise an amino acid sequence according to Formula (I): Xi-T-K-P-C-Q-S-D-K-D-C-K-K-F-A-C-R-K-P-K-V-P-K-C-I-N-G-F-C-K-C-V-R, wherein Xi represents the amino acid added to the N-terminus of SEQ ID NO: 1, wherein X
  • “Native” refers to items found in nature in their natural, unmodified state.
  • Natural amino acid refers to one of the 20 amino acids typically found in proteins in nature, and used for protein biosynthesis as well as other amino acids which can be incorporated into proteins during translation (including pyrrolysine and selenocysteine).
  • the 20 natural amino acids include histidine, alanine, valine, glycine, leucine, iso leucine, aspartic acid, glutamic acid, serine, glutamine, asparagine, threonine, arginine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine and lysine.
  • Non-natural amino acid refers to an organic compound that is not among those amino acids encoded by the standard genetic code, or incorporated into proteins during translation. Therefore, non-natural amino acids include amino acids or analogs of amino acids, but are not limited to, the D-isostereomers of amino acids, the beta-amino-analogs of amino acids (0-amino acids, 0 3 and 0 2 ), citrulline, homocitrulline, homoarginine, hydroxyproline, homoproline, ornithine, 4-amino-phenylalanine, cyclohexylalanine, a- aminoisobutyric acid, N-methyl-alanine, N-methyl-glycine, norleucine, N-methyl-glutamic acid, tert-butylglycine, a-aminobutyric acid, tert -butylalanine, 2-aminoisobutyric acid, a- aminoisobutyric acid, 2-a
  • NCBI refers to the National Center for Biotechnology Information.
  • NCR refers to nodule-specific cysteine-rich.
  • NVP or “NCR13 variant peptide” refers to peptides having antimicrobial activity against one or more microbes, said NVP comprising an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid is any natural or non-natural amino acid other than glycine or alanine; or an agriculturally acceptable salt thereof.
  • an NVP of the present disclosure comprises a recombinant peptide having an amino acid sequence that is at least 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence according to Formula (I): Xi-T-K-P-C-Q-S-D-K-D-C-K-K-F-A-C-R-K- P-K-V-P-K-C-I-N-G-F-C-K-C-V-R, wherein Xi represents the amino acid added to the N- terminus, wherein Xi is V, L, I, M, F, W, P, S, T, Y, N, Q, D, E, K, R, or H; and wherein Xi is not G or A.
  • Formula (I) Xi-T-K-P-C-Q-S-D-K-D-C-K-K-F-A-C-R-K- P-K-V-P-K-C-
  • an NVP can comprise a peptide having an amino sequence that is at least 95%, 96%, 97%, 97%, 98%, 99% or 100% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 4-19, or 66.
  • an “NCR13 variant peptide” includes a peptide that comprises, consists essentially of, or consists of an amino acid sequence that is at least 80%, 85%, 90%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical across the entire length of the peptide to the amino acid sequence as set forth in SEQ ID NO: 1.
  • an illustrative “NCR13 variant peptide” includes an antimicrobial peptide that has 1, 2, 3, 4, 5, 6, or 7 conservative amino acid substitutions at any amino acid position within the full length sequence as set forth in SEQ ID NO: 1.
  • NVP expression cassette refers to one or more regulatory elements such as promoters; enhancer elements; iriRNA stabilizing polyadenylation signal; an internal ribosome entry site (IRES); introns; post-transcriptional regulatory elements; and a polynucleotide operable to encode an NVP, e.g., an NVP ORF.
  • an NVP expression cassette is one or more segments of DNA that contains a polynucleotide segment operable to express an NVP, a ADH1 promoter, a LAC4 terminator, and an alpha- MF secretory signal.
  • An NVP expression cassette contains all of the nucleic acids necessary to encode an NVP or an NVP-antimicrobial protein.
  • NVP ORF refers to a polynucleotide operable to encode an NVP, or an NVP- antimicrobial protein.
  • NVP ORF diagram refers to the composition of one or more NVP ORFs, as written out in diagram or equation form.
  • a “NVP ORF diagram” can be written out as using acronyms or short -hand references to the DNA segments contained within the expression ORF.
  • a “NVP ORF diagram” may describe the polynucleotide segments encoding the ERSP, LINKER, STA, and NVP, by diagramming in equation form the DNA segments as “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide); “linker” or “Z” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and “nvp” (i.e., the polynucleotide sequence encoding an NVP), respectively.
  • An example of an NVP ORF diagram is “ersp-sta-(linker-nvp ) ⁇ ” or “ersp-fnvpj-linkerjN- sta” and/or any combination of the DNA segments thereof.
  • NVP-antimicrobial protein or “NVP-antimicrobial polypeptide” or “antimicrobial protein” or “antimicrobial polypeptide” refers to any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, comprising: (1) at least one NVP, or two or more NVPs; and (2) additional peptides, polypeptides, or proteins.
  • these additional peptides, polypeptides, or proteins have the ability to increase the mortality and/or inhibit the growth of microbes when the microbes are exposed to an NVP-antimicrobial protein, relative to an NVP alone; increase the expression of said NVP-antimicrobial protein, e.g., in a host cell or an expression system; and/or affect the post-translational processing of the NVP-antimicrobial protein.
  • an NVP-antimicrobial protein can be a polymer comprising two or more NVPs.
  • an NVP-antimicrobial protein can be a polymer comprising two or more NVPs, wherein the NVPs are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker.
  • an NVP-antimicrobial protein can refer to a one or more NVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); a cleavable or non-cleavable linker (L); and/or any other combination thereof.
  • STA stabilizing domain
  • ERSP endoplasmic reticulum signaling protein
  • L cleavable or non-cleavable linker
  • an NVP-antimicrobial protein can be a non-naturally occurring protein comprising (1) an NVP; and (2) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.
  • OD600nm or “ODeoonm” refers to optical densities of the liquid sample measured (for example, a microbial cell culture) when measured in a spectrophotometer at 600 nanometers (nm).
  • OD660nm or “ODeeonm” refers to optical densities of the liquid sample measured (for example, a microbial cell culture) when measured in a spectrophotometer at 660 nanometers (nm).
  • a pathogenic microbe refers to any microbe that is deleterious or pathogenic to an organism; e.g., any microbe that causes or exacerbates an infection or disease in a living organism.
  • a pathogenic microbe can be a pathogen to plants, e.g., a phytopathogen, such as a bacterium, a protozoan, or a fungus; in other embodiments, a pathogenic microbe can be a pathogen to animals.
  • “Pharmaceutically acceptable salt” is synonymous with agriculturally acceptable salt, and as used herein refers to a compound that is modified by making acid or base salts thereof.
  • Polynucleotide refers to a polymeric-form of nucleotides (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof of any length; e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides.
  • the term “polynucleotide” includes double- and single-stranded DNA, as well as double- and singlestranded RNA; it also includes modified and unmodified forms of a polynucleotide (modifications to and of a polynucleotide, for example, can include methylation, phosphorylation, and/or capping).
  • a polynucleotide can be one of the following: a gene or gene fragment (for example, a probe, primer, EST, or SAGE tag); genomic DNA; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA; ribosomal RNA; ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probe; primer or amplified copy of any of the foregoing.
  • a gene or gene fragment for example, a probe, primer, EST, or SAGE tag
  • genomic DNA for example, genomic DNA fragment; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA; ribosomal RNA; ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of
  • a polynucleotide can refer to cDNA.
  • polynucleotides can have any three-dimensional structure and may perform any ftinction, known or unknown.
  • the structure of a polynucleotide can also be referenced to by its 5’- or 3’- end or terminus, which indicates the directionality of the polynucleotide.
  • Adjacent nucleotides in a single-strand of polynucleotides are typically joined by a phosphodiester bond between their 3’ and 5’ carbons.
  • intemucleotide linkages could also be used, such as linkages that include a methylene, phosphoramidate linkages, etc.
  • a polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with nonnatural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • modified nucleotides such as methylated nucleotides and nucleotide analogs (including nucleotides with nonnatural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • RNA molecule refers to a polynucleotide having a ribose sugar rather than deoxyribose sugar and typically uracil rather than thymine as one of the pyrimidine bases.
  • An RNA molecule of the disclosure is generally single-stranded, but can also be double-stranded.
  • the RNA molecule can include the single-stranded molecules transcribed from DNA in the cell nucleus, mitochondrion or chloroplast, which have a linear sequence of nucleotide bases that is complementary to the DNA strand from which it is transcribed.
  • a polynucleotide can fiirther comprise one or more heterologous regulatory elements.
  • the regulatory element is one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; or combinations thereof.
  • Ratio refers to the quantitative relation between two amounts showing the number of times one value contains or is contained within the other.
  • Recombinant DNA or “rDNA” refers to DNA that is comprised of two or more different DNA segments.
  • Stringent hybridization conditions are sequence- and length-dependent, and will be different in different circumstances. Similarly, stringent hybridization conditions depend on % (percent) -identity (or %-mismatch) over a certain length of nucleotide residues. Generally, longer sequences hybridize specifically at higher temperatures than shorter sequences. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 or 500 nucleotides in length
  • moderate stringency hybridization conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5x to IxSSC at 55-60°C.
  • high stringency hybridization conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C., and a final wash in O.lxSSC at 60 to 65°C. for at least about 20 minutes.
  • wash buffers may comprise about 0.1% to about 1% SDS.
  • the duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
  • T m thermal melting point
  • T m 81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, “% form” is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Washes are typically performed at least until equilibrium is reached and a low background level of hybridization is achieved, such as for 2 hours, 1 hour, or 30 minutes.
  • the T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C.
  • stringent hybridization conditions are selected to be about 5 °C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • stringent hybridization conditions can utilize a hybridization and/or wash temperature that is about 1°C, 2°C, 3 °C, or 4°C lower than the thermal melting point (T m ).
  • a polynucleotide of the present disclosure can stringently hybridize to a polynucleotide segment of a polynucleotide encoding an NVP, or a complementary nucleotide sequence thereof, e.g., a polynucleotide operable to encode an NVP having an amino acid sequence as set forth in any one of SEQ ID NOs: 4-19, or 66
  • “Susceptible to attack by a microbe (or microbes)” or “susceptible to a microbial infection” or “susceptible to microbial disease” and the like refer to plants, or human or animal patients or subjects, susceptible to a microbe pathogen or microbial infections.
  • Beneficial or desired results can include, but are not limited to, death of at least one microbe; alleviation or amelioration of one or more symptoms or conditions caused by a pathogenic microbe; diminishment of extent of condition, disorder or disease caused by a pathogenic microbe; stabilization of the state of condition, disorder or disease caused by a pathogenic microbe; prevention of development of condition, disorder or disease caused by a pathogenic microbe; prevention of spread of condition, disorder or disease caused by a pathogenic microbe; delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset caused by a pathogenic microbe; amelioration or palliation of the condition, disorder or disease state, and remission of a disease or disease state caused by a pathogenic microbe; whether partial or total.
  • treating can also mean prolonging survival of an organism beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the disease or disorder, slowing the progression of disorder or disease temporarily, although in some instances, it involves halting the progression of the disorder or disease permanently.
  • treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition caused by a pathogenic microbe.
  • treatment can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the number of pathogenic microbes, and/or the severity of an established disease, condition, or symptom of the disease or condition caused by a pathogenic microbe. It is understood that treatment does not necessarily refer to the death of all pathogenic microbes and/or the cure or complete ablation of the disease, condition, or symptoms of the disease or condition caused by a pathogenic microbe.
  • var.” refers to varietas or variety.
  • the term “var.” is used to indicate a taxonomic category that ranks below the species level and/or subspecies (where present).
  • the term “var.” represents members differing from others of the same subspecies or species in minor but permanent or heritable characteristics.
  • Vector refers to the DNA segment that accepts a foreign gene of interest (e.g., nvp).
  • the gene of interest is known as an “insert” or “transgene.”
  • Wild type or “WT” refers to the phenotype and/or genotype (i.e., the appearance or sequence) of an organism, polynucleotide sequence, and/or polypeptide sequence, as it is found and/or observed in its naturally occurring state or condition.
  • Wild-type NCR13 or “WT-NCR13” or “WT NCR13” or “NCR13 peptide” or “NCR13” refers to a wild-type nodule-specific cysteine-rich 13 (NCR13) peptide.
  • An exemplary NCR13 peptide is proved herein, having an amino acid sequence (designated as single letter amino acid sequence) consisting of: TKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 1) (NCBI Accession No. DAA64987).
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • NCR13 Variant Peptides NCP13 Variant Peptides
  • Bio antimicrobial agents e.g., antimicrobial agents based on, or derived from, natural sources (such as an organism or product therefrom), may confer crop protection by suppressing disease incidence, and/or reducing the number of pathogenic microbes or the severity of symptoms caused by the same.
  • biological antimicrobial agents represent a desirable replacement of traditional agrochemicals, and can be used to avoid or remediate the adverse effects thereof.
  • NCR nodule-specific cysteine-rich
  • NCR peptides have been identified in legumes including, but not limited to, Vicia faba, Medicago sativa, Trifolium repens, Galega orientalis, Pisum sativum, Astragalus sinicus, Cicer arietinum and Glycyrrhiza lepidota.
  • NCR peptides have a structure that resembles antimicrobial defensin peptides, which are effectors of innate immunity in plants and animals, including humans. See Kereszt et al., Impact of Plant Peptides on Symbiotic Nodule Development and Functioning. FRONT PLANT SCI. 2018; 9: 1026.
  • NCRs have been shown to exhibit antimicrobial activity, e.g., against gram-negative and gram-positive bacteria as well as unicellular and filamentous fiingi. See Maroti and Kondorosi, Nitrogen-fixing Rhizobium-Zeguwe symbiosis: are polyploidy and host peptide-govemed symbiont differentiation general principles of endosymbiosis? FRONT MICROBIOL. 2014; 5: 326; Maroti et al., Natural roles of antimicrobial peptides in microbes, plants and animals. RES MICROBIOL. 2011 May; 162(4): 363 -74.
  • NCR13 variant peptides NCP13 variant peptides
  • the present disclosure provides an engineered, non-naturally occurring antimicrobial peptides called “NCR13 variant peptides (NVPs)”, agriculturally acceptable salts thereof; agricultural compositions thereof, farther comprising an excipient; and methods of making and using the same.
  • an antimicrobial NCR13 variant peptide can be a recombinant peptide having antimicrobial activity against one or more microbes, or an agriculturally acceptable salt thereof, wherein the NVP comprises at least one mutation comprising an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid added to the N-terminus can be any natural or non-natural amino acid other than glycine or alanine.
  • an antimicrobial NCR13 variant peptide can be a recombinant peptide having antimicrobial activity against one or more microbes, or an agriculturally acceptable salt thereof, wherein the NVP comprises an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in Formula (I): Xi-T-K-P-C- Q-S-D-K-D-C-K-K-F-A-C-R-K-P-K-V-P-K-C-I-N-G-F-C-K-C-V-R, wherein Xi represents the amino acid added to the N-terminus of SEQ ID NO: 1, wherein Xi is any natural or nonnatural amino acid other than glycine or alanine; or an agriculturally acceptable salt thereof.
  • an NVP can be a mutant or variant that differs from a WT-NCR13 (SEQ ID NO: 1), wherein the resulting NVP comprises at least one mutation comprising an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid added to the N-terminus can be any natural or non-natural amino acid other than glycine or alanine.
  • an “NCR13 variant peptide” includes a peptide that comprises, consists essentially of, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical across the entire length of the peptide to the amino acid sequence of WT- NCR13 as set forth in SEQ ID NOs: 4-19, and 66, and comprises at least one mutation comprising an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid added to the N-terminus can be any natural or non-natural amino acid other than glycine or alanine.
  • an illustrative “NCR13 variant peptide” includes an antimicrobial peptide that has 1, 2, 3, 4, 5, 6, or 7 conservative amino acid substitutions at any amino acid position within the fall length sequence as set forth in any one of the amino acid sequences set forth in SEQ ID NOs: 4-19, and 66, or an agriculturally acceptable salt thereof, wherein the amino acid added to the N- terminus can be any natural or non-natural amino acid other than glycine or alanine.
  • Table 1 Summary of NVPs possessing mutations that confer novel and unexpected properties relative to WT-NCR13. Table 1 provides a summary of NVPs that confer at least one novel property relative to WT-NCR13.
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NCR13 variant peptide (NVP) having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99% identical, at least 99
  • an NCR13 variant peptide (NVP) having antimicrobial activity against one or more microbe species comprises, consists essentially of, or consists of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence having an N-terminus addition
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “VTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR” (SEQ ID NO
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “LTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR” (SEQ ID NO
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “STKPCQSDKDCKKFACRKPKVPKCINGFCKCVR” (SEQ ID NO
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence: “TTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR” (SEQ ID NO
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • NCP antimicrobial NCR13 variant peptide
  • An antimicrobial NCR13 variant peptide (NVP) of the present invention does not have an N-terminus addition of a glycine (e.g., SEQ ID NO: 2) or an alanine (e.g., SEQ ID NO: 3).
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, a homopolymer or heteropolymer of two or more NVPs, wherein the amino acid sequence of each NVP is the same or different.
  • an NVP of the present disclosure can comprise, consist essentially of, or consist of, an NVP that is a ftised protein comprising two or more NVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each NVP may be the same or different.
  • the linker is a cleavable linker.
  • the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 39-51.
  • the linker is cleavable inside at least one of (i) the gut or hemolymph of an invertebrate, and (ii) cleavable inside the gut of a mammal.
  • an NCR13 variant peptide (NVP) of the present disclosure comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • conservative amino acid substitutions refers to amino acid substitutions to a molecule that do not affect the functional and/or chemical characteristics of the molecule (i.e., the NVP). Accordingly, conservative amino acid substitutions are generally therefore based on the relative similarity of the amino acid side- chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary conservative amino acid substitutions are well known to those having ordinary skill in the art. For example, in some embodiments, conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • polar, negatively charged residues and their amides e.g., aspartic acid, asparagine, glutamic, acid, glutamine
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • small aliphatic, nonpolar or slightly polar residues e.g., alanine, serine, threonine, proline, glycine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • large aliphatic, nonpolar residues e.g., methionine, leucine, isoleucine
  • the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NCR13 variant peptide (NVP).
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NCR13 variant peptide (NVP) having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical,
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical,
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical,
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical,
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical,
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical,
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical,
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical,
  • a polynucleotide of the present disclosure comprises, consists essentially of, or consists of, a polynucleotide operable to encode an NVP that can comprise, consist essentially of, or consist of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9%
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • LTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR SEQ ID NO: 5
  • SEQ ID NO: 5 a complementary nucleotide sequence thereof.
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • MTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 7), or a complementary nucleotide sequence thereof.
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:
  • FKPCQSDKDCKKFACRKPKVPKCINGFCKCVR SEQ ID NO: 8
  • a complementary nucleotide sequence thereof SEQ ID NO: 8
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:
  • PTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR SEQ ID NO: 10
  • a complementary nucleotide sequence thereof SEQ ID NO: 10
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • TTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR SEQ ID NO: 12
  • a complementary nucleotide sequence thereof SEQ ID NO: 12
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • YTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 13), or a complementary nucleotide sequence thereof.
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:
  • QTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 15), or a complementary nucleotide sequence thereof.
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence:
  • DTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR SEQ ID NO: 16
  • SEQ ID NO: 16 a complementary nucleotide sequence thereof.
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • KTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR SEQ ID NO: 18
  • a complementary nucleotide sequence thereof SEQ ID NO: 18
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • a polynucleotide of the present disclosure is operable to encode an NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • RTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 66), or a complementary nucleotide sequence thereof.
  • the polynucleotide comprises, consists essentially of, or consists of, a nucleotide sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the polynucleotide comprises, consists essentially of, or consists of, a nucleotide sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the polynucleotide is operable to encode an NVP that can comprise, consist essentially of, or consist of, a homopolymer or heteropolymer of two or more NVPs, wherein the amino acid sequence of each NVP is the same or different.
  • the polynucleotide is operable to encode an NVP that can comprise, consist essentially of, or consist of, an NVP that is a fiised protein comprising two or more NVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each NVP may be the same or different.
  • the linker is a cleavable linker.
  • the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 39-51.
  • the linker is cleavable inside at least one of (i) the gut or hemolymph of an invertebrate, and (ii) cleavable inside the gut of a mammal.
  • a polynucleotide of the present disclosure can stringently hybridize to a polynucleotide encoding an NVP, or a complementary sequence thereof, said NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • the present disclosure provides a method comprising contacting a sample of nucleic acids with a nucleic acid probe that hybridizes under stringent hybridization conditions with a polynucleotide comprising a polynucleotide segment encoding an NVP or fragment thereof as provided herein, and does not hybridize under such hybridization conditions with a polynucleotide that does not comprise the segment, wherein the probe is homologous or complementary to a polynucleotide encoding any one of SEQ ID NOs: 23-38, or a polynucleotide encoding an OVP comprising an amino acid sequence having at least 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NOs: 4-19, or 66.
  • the method may ftirther comprise (a) subjecting the sample and probe to stringent hybridization conditions; and (b) detecting hybridization of the probe with polynucle
  • an NVP-antimicrobial protein can be any protein, peptide, polypeptide, amino acid sequence, configuration, construct, or arrangement, comprising: (1) at least one NVP, or two or more NVPs; and (2) one or more additional non- NVP peptides, polypeptides, or proteins.
  • these additional non-NVP peptides, polypeptides, or proteins may have the ability to increase the mortality and/or inhibit the growth of microbes exposed to the NVP-antimicrobial protein, relative to the NVP alone; increase the expression of the NVP-antimicrobial protein, e.g., in a host cell; and/or affect the post -translational processing of the NVP-antimicrobial protein.
  • an NVP-antimicrobial protein can be a polymer comprising two or more NVPs.
  • an NVP-antimicrobial protein can be a polymer comprising two or more NVPs, wherein the NVPs are operably linked via a linker peptide, e.g., a cleavable and/or a non-cleavable linker.
  • the linker peptide falls under the category of the additional non-NVP peptide described above.
  • an NVP-antimicrobial protein can refer to a one or more NVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an microbe cleavable or microbe non- cleavable linker (L); and/or any other combination thereof.
  • STA stabilizing domain
  • ERSP endoplasmic reticulum signaling protein
  • L microbe cleavable or microbe non- cleavable linker
  • an NVP-antimicrobial protein can be a polymer of amino acids that, when properly folded or in its most natural thermodynamic state, exerts an antimicrobial activity against one or more microbes.
  • an NVP-antimicrobial protein can be a polymer comprising two or more NVPs that are different.
  • an antimicrobial protein can be a polymer of two or more NVPs that are the same.
  • an NVP-antimicrobial protein can comprise one or more NVPs, and one or more peptides, polypeptides, or proteins, that may assist in the NVP- antimicrobial protein’s folding.
  • an NVP-antimicrobial protein can comprise one or more NVPs, and one or more peptides, polypeptides, or proteins, wherein the one or more peptides, polypeptides, or proteins are protein tags that help stability or solubility.
  • the peptides, polypeptides, or proteins can be protein tags that aid in affinity purification.
  • an NVP-antimicrobial protein can refer to a one or more NVPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an microbe cleavable or microbe non- cleavable linker; one or more heterologous peptides; one or more additional polypeptides; and/or any other combination thereof.
  • an antimicrobial protein can comprise a one or more NVPs as disclosed herein.
  • an NVP-antimicrobial protein can comprise an NVP homopolymer, e.g., two or more NVP monomers that are the same NVP.
  • the antimicrobial protein can comprise an NVP heteropolymer, e.g., two or more NVP monomers, wherein the NVP monomers are different.
  • an NVP-antimicrobial protein can comprise, consist essentially of, or consist of one or more NVPs having an amino acid sequence set forth in SEQ ID NOs: 4-19 and 66, or an agriculturally acceptable salt thereof.
  • an NVP-antimicrobial protein can comprise, consist essentially of, or consist of one or more NVPs having an amino acid sequence set forth in SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, and 17, or an agriculturally acceptable salt thereof.
  • the NVP-antimicrobial protein may comprise an NVP having an amino acid sequence having at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 4-19 or 66, or an agriculturally acceptable salt thereof.
  • the NVP-antimicrobial protein may comprise an NVP having an amino acid sequence having at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, and 17, or an agriculturally acceptable salt thereof.
  • linkers include, but not limited to, the following sequences: IGER (SEQ ID NO: 39), EEKKN, (SEQ ID NO:40), and ETMFKHGL (SEQ ID NO:41), or combinations thereof.
  • the linker can be one or more of the following: ALKFLV (SEQ ID NO: 42), ALKLFV (SEQ ID NO: 43), IFVRLR (SEQ ID NO: 44), LFAAPF (SEQ ID NO: 45), ALKFLVGS (SEQ ID NO: 46), ALKLFVGS (SEQ ID NO: 47), IFVRLRGS (SEQ ID NO: 48), LFAAPFGS (SEQ ID NO: 49), LFVRLRGS (SEQ ID NO: 50), and/or LGERGS (SEQ ID NO: 51).
  • the NVP, or an agriculturally acceptable salt thereof of the present disclosure can be produced and/or obtained via any method well known in the art of peptide synthesis.
  • the engineered, non-naturally occurring antimicrobial peptide of the present disclosure can be produced using recombinant methods.
  • an engineered, non-naturally occurring antimicrobial peptide of the present disclosure can be created using any known method for producing a peptide or protein.
  • an engineered, non-naturally occurring antimicrobial peptide of the present disclosure can be created using a recombinant expression system, such as yeast expression system or a bacterial expression system.
  • a recombinant expression system such as yeast expression system or a bacterial expression system.
  • an NVP of the present disclosure can be produced using a recombinant expression system.
  • the host cell can be any host cell that satisfies the requirements of the end-user.
  • a user may desire to use one specific type of host cell (e.g., a yeast cell or a bacteria cell) as opposed to another; the preference of a given host cell can range from desired codon usage, availability and cost, among other considerations.
  • a polynucleotide operable to encode an NVP of the present disclosure as provided in Table 1 can be cloned into a vector using a variety of cloning strategies, and commercial cloning kits and materials readily available to those having ordinary skill in the art.
  • a polynucleotide which encodes the NVP can be cloned into a vector using such strategies as the SnapFast; Gateway; TOPO; Gibson; LIC; InFusionHD; or Electra strategies.
  • a polynucleotide encoding an NVP of the present disclosure can be cloned into a vector such as a plasmid, cosmid, virus (bacteriophage, animal viruses, and plant viruses), and/or artificial chromosome (e.g., YACs).
  • a vector such as a plasmid, cosmid, virus (bacteriophage, animal viruses, and plant viruses), and/or artificial chromosome (e.g., YACs).
  • a polynucleotide encoding an NVP of the present disclosure can be inserted into other commercially available plasmids and/or vectors that are readily available to those having skill in the art, e.g., plasmids are available from Addgene (a non-profit plasmid repository); GenScript®; Takara®; Qiagen®; and PromegaTM.
  • Addgene a non-profit plasmid repository
  • GenScript® a non-profit plasmid repository
  • Takara® Takara®
  • Qiagen® and PromegaTM.
  • the polynucleotide sequence encoding an NVP of the present disclosure can be chemically synthesized using commercially available polynucleotide synthesis services, such as those offered by GENEWIZ® (e.g., TurboGENETM; PriorityGENE; and FragmentGENE), or SIGMA-ALDRICH® (e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos).
  • GENEWIZ® e.g., TurboGENETM; PriorityGENE; and FragmentGENE
  • SIGMA-ALDRICH® e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos.
  • Exemplary method for generating DNA and or custom chemically synthesized polynucleotides are well known in the art, and are illustratively provided in U.S. Patent No. 5,736,135, Serial No. 08/389,615, filed on Feb.
  • Chemically synthesizing polynucleotides allows for a DNA sequence to be generated that is tailored to produce a desired peptide based on the arrangement of nucleotides within said sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A] or thymine [T] molecules); the iriRNA sequence that is transcribed from the chemically synthesized DNA polynucleotide can be translated to a sequence of amino acids, each amino acid corresponding to a codon in the iriRNA sequence.
  • Obtaining an NVP of the present disclosure from a chemically synthesized DNA polynucleotide sequence and/or a wild-type DNA polynucleotide sequence can be achieved by cloning the DNA sequence into an appropriate vector.
  • the vector can be a plasmid, which can introduce a heterologous gene and/or expression cassette into yeast cells to be transcribed and translated.
  • the term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a vector may contain “vector elements” such as an origin of replication (ORI); a gene that confers antibiotic resistance to allow for selection; multiple cloning sites; a promoter region; a selection marker for non-bacterial transfection; and a primer binding site.
  • vector elements such as an origin of replication (ORI); a gene that confers antibiotic resistance to allow for selection; multiple cloning sites; a promoter region; a selection marker for non-bacterial transfection; and a primer binding site.
  • a nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • viruses bacteriophage, animal viruses, and plant viruses
  • artificial chromosomes e.g., YACs
  • One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al., 1989 and Ausubel et al., 1996, both incorporated herein by reference.
  • a vector may also encode a targeting molecule.
  • a targeting molecule is one that directs the desired nucleic acid to a particular tissue, cell, or other location.
  • Peptide synthesis or the chemical synthesis of peptides and/or polypeptides can be used to produce or synthesize an NVP of the present disclosure. These methods can be performed by those having ordinary skill in the art, and/or through the use of commercial vendors (e.g., GenScript®; Piscataway, New Jersey). For example, in some embodiments, chemical peptide synthesis can be achieved using Liquid phase peptide synthesis (LPPS), or solid phase peptide synthesis (SPPS).
  • LPPS Liquid phase peptide synthesis
  • SPPS solid phase peptide synthesis
  • peptide synthesis can generally be achieved by using a strategy wherein the coupling the carboxyl group of a subsequent amino acid to the N- terminus of a preceding amino acid generates the nascent polypeptide chain — a process that is opposite to the type of polypeptide synthesis that occurs in nature.
  • Peptide deprotection is an important first step in the chemical synthesis of polypeptides. Peptide deprotection is the process in which the reactive groups of amino acids are blocked through the use of chemicals in order to prevent said amino acid’s ftinctional group from taking part in an unwanted or non-specific reaction or side reaction; in other words, the amino acids are “protected” from taking part in these undesirable reactions.
  • the amino acids Prior to synthesizing the peptide chain, the amino acids must be “deprotected” to allow the chain to form (i.e., amino acids to bind).
  • Chemicals used to protect the N-termini include 9-fluorenylmethoxycarbonyl (Fmoc), and tert-butoxycarbonyl (Boc), each of which can be removed via the use of a mild base (e.g., piperidine) and a moderately strong acid (e.g., trifluoracetic acid (TFA)), respectively.
  • a mild base e.g., piperidine
  • a moderately strong acid e.g., trifluoracetic acid (TFA)
  • the C-terminus protectant required is dependent on the type of chemical peptide synthesis strategy used: e.g., LPPS requires protection of the C-terminal amino acid, whereas SPPS does not owing to the solid support which acts as the protecting group.
  • Side chain amino acids require the use of several different protecting groups that vary based on the individual peptide sequence and N-terminal protection strategy; typically, however, the protecting group used for side chain amino acids are based on the tert-butyl (tBu) or benzyl (Bzl) protecting groups.
  • Amino acid coupling is the next step in a peptide synthesis procedure.
  • the incoming amino acid’s C-terminal carboxylic acid must be activated: this can be accomplished using carbodiimides such as diisopropylcarbodiimide (DIC), or dicyclohexylcarbodiimide (DCC), which react with the incoming amino acid’s carboxyl group to form an O-acylisourea intermediate.
  • DIC diisopropylcarbodiimide
  • DCC dicyclohexylcarbodiimide
  • the O-acylisourea intermediate is subsequently displaced via nucleophilic attack via the primary amino group on the N- terminus of the growing peptide chain.
  • the reactive intermediate generated by carbodiimides can result in the racemization of amino acids.
  • reagents such as 1 -hydroxybenzotriazole (HOBt) are added in order to react with the O- acylisourea intermediate.
  • HOBt 1 -hydroxybenzotriazole
  • Other couple agents include 2-(lH-benzotriazol-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), and benzotriazol- 1-yl-oxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP), with the additional activating bases.
  • HBTU 2-(lH-benzotriazol-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate
  • BOP benzotriazol- 1-yl-oxy- tris(dimethylamino)phosphonium hexafluorophosphate
  • peptides can be purified based on the peptide’s physiochemical characteristics (e.g., charge, size, hydrophobicity, etc.).
  • Techniques that can be used to purify peptides include Purification techniques include Reverse-phase chromatography (RPC); Size-exclusion chromatography; Partition chromatography; High- performance liquid chromatography (HPLC); and Ion exchange chromatography (IEC).
  • any of the methods described herein can be used to generate an NVP described herein, e.g., an engineered, non-naturally occurring antimicrobial peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 4-19, or 66, e.g., SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, or 17, or encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 24, 26, 27, 28, 30, 32, 35, or 36.
  • an NVP described herein e.g., an engineered, non-naturally occurring antimicrobial peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 4-19, or 66, e.g., SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, or 17, or encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 24, 26, 27, 28, 30, 32, 35, or 36.
  • transformation and “transfection” both describe the process of introducing exogenous and/or heterologous polynucleotide (e.g., DNA or RNA) to a host organism.
  • exogenous and/or heterologous polynucleotide e.g., DNA or RNA
  • those having ordinary skill in the art sometimes reserve the term “transformation” to describe processes where exogenous and/or heterologous polynucleotide (e.g., DNA or RNA) are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and/or heterologous polynucleotide (e.g., DNA or RNA) into eukaryotic cells.
  • transformation and “transfection” are used synonymously, regardless of whether a process describes the introduction exogenous and/or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).
  • a prokaryote e.g., bacteria
  • a eukaryote e.g., yeast, plants, or animals
  • a host organism can be transformed with a polynucleotide operable to encode an NVP.
  • the host organism can be an microorganism, e.g., a cell.
  • a vector comprising an NVP expression cassette can be cloned into an expression plasmid and transformed into a host cell.
  • the host cell can be selected from any host cell described herein.
  • a host cell can be transformed using the following methods: electroporation; cell squeezing; microinjection; impalefection; the use of hydrostatic pressure; sonoporation; optical transfection; continuous infiision; lipofection; through the use of viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus; the chemical phosphate method; endocytosis via DEAE- dextran or polyethylenimine (PEI); protoplast ftision; hydrodynamic deliver; magnetofection; nucleoinfection; and/or others.
  • viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus
  • viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus
  • viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus,
  • Electroporation is an exemplary method for transforming host cells. Electroporation is a technique in which electricity is applied to cells causing the cell membrane to become permeable; this in turn allows exogenous DNA to be introduced into the cells. Electroporation is readily known to those having ordinary skill in the art, and the tools and devices required to achieve electroporation are commercially available (e.g., Gene Pulser XcellTM Electroporation Systems, Bio-Rad®; Neon® Transfection System for Electroporation, Thermo -Fisher Scientific; and other tools and/or devices). Exemplary methods of electroporation are illustrated in Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol.
  • electroporation can be used transform a cell with one or more vectors containing a polynucleotide operable to encode one or more NVPs or NVP- antimicrobial proteins.
  • electroporation can be used transform a cell with one or more vectors containing one or more NVP expression cassettes.
  • electroporation can be used transform a yeast cell with one or more vectors containing one or more NVP expression cassettes, which can produce NVP in a yeast culture with a yield of: at least 70 mg/L, at least 80 mg/L, at least 90 mg/L, at least 100 mg/L, at least 110 mg/L, at least 120 mg/L, at least 130 mg/L, at least 140 mg/L, at least 150 mg/L, at least 160 mg/L, at least 170 mg/L, at least 180 mg/L, at least 190 mg/L 200 mg/L, at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, at least 1,250 mg/L, at least 1,500 mg/L, at least 1,750 mg/L, at least 2,000 mg/L, at least 2,500 mg/L, at least 3,000 mg/L, at least 3,500 mg/L, at least 4,000 mg/L, at least 4,500 mg/L, at least 5,000 mg/L, at least
  • electroporation can be used to introduce a vector containing a polynucleotide encoding an NVP into yeast, for example, in some embodiments, an NVP expression cassette cloned into a plasmid, and transformed into yeast cells via electroporation.
  • an NVP expression cassette cloned into a plasmid, and transformed a host cell (e.g., a yeast cell) via electroporation can be accomplished by inoculating about 10-200 mL of yeast extract peptone dextrose (YEPD) with a suitable yeast species, for example, Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, etc., and incubate on a shaker at 30°C until the early exponential phase of yeast culture (e.g.
  • galactose, maltose, latotriose, sucrose, fructose or glucose and/or sugar alcohol for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, followed by spinning down at 3,000 rpm for 5 minutes; resuspending the cells with proper volume of ice cold IM fermentable sugar, e.g.
  • galactose maltose, latotriose, sucrose, fructose or glucose and/or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol mixture, and then spreading onto selective plates.
  • a sugar alcohol for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol mixture, and then spreading onto selective plates.
  • electroporation can be used to introduce a vector containing a polynucleotide encoding an NVP into yeast, for example, an NVP cloned into a plasmid, and transformed into K. lactis cells via electroporation, can be accomplished by inoculating about 10-200 mL of yeast extract peptone dextrose (YEPD) incubating on a shaker at 30°C until the early exponential phase of yeast culture (e.g.
  • yeast extract peptone dextrose YEPD
  • galactose, maltose, latotriose, sucrose, fructose or glucose and/or sugar alcohol for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, followed by spinning down at 3,000 rpm for 5 minutes; resuspending the cells with proper volume of ice cold IM fermentable sugar, e.g.
  • a sugar alcohol for example, erythritol, hydrogenated starch hydrolysates, iso
  • galactose maltose, latotriose, sucrose, fructose or glucose and/or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol mixture, and then spreading onto selective plates.
  • a sugar alcohol for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol mixture, and then spreading onto selective plates.
  • NVP NVP in amounts of: at least 70 mg/L, at least 80 mg/L, at least 90 mg/L, at least 100 mg/L, at least 110 mg/L, at least 120 mg/L, at least 130 mg/L, at least 140 mg/L, at least 150 mg/L, at least 160 mg/L, at least 170 mg/L, at least 180 mg/L, at least 190 mg/L 200 mg/L, at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, at least 1,250 mg/L, at least 1,500 mg/L, at least 1,750 mg/L, at least 2,000 mg/L, at least 2,500 mg/L, at least 3,000 mg/L, at least 3,500 mg/L, at least 4,000 mg/L, at least 4,500 mg/L, at least 5,000 mg/L, at least 5,500 mg//
  • electroporation can be used to introduce a vector containing a polynucleotide encoding an NVP into plant protoplasts by incubating sterile plant material in a protoplast solution (e.g., around 8 mL of 10 mM 2-[7V- morpholino]ethanesulfonic acid (MES), pH 5.5; 0.01% (w/v) pectylase; 1% (w/v) macerozyme; 40 mM CaCh; and 0.4 M mannitol) and adding the mixture to a rotary shaker for about 3 to 6 hours at 30°C to produce protoplasts; removing debris via 80-pm-mesh nylon screen filtration; rinsing the screen with about 4 ml plant electroporation buffer (e.g., 5 mM CaCh; 0.4 M mannitol; and PBS); combining the protoplasts in a sterile 15 mL conical centriftige tube, and then
  • a protoplast solution
  • the methods, compositions, NVPs, and NVP-antimicrobial proteins of the present disclosure may be implemented in any host organism.
  • the host organism can be a cell.
  • the cell can be, e.g., a eukaryotic or prokaryotic cell.
  • the host cell used to produce a NVP or NVP- antimicrobial protein can be eukaryote.
  • the host cell used to produce a NVP or NVP- antimicrobial protein may be any species within the genera, Kluyveromyces.
  • the host cell used to produce a NVP or NVP- antimicrobial protein may be a species in the genera, Kluyveromyces, e.g., the host cell may be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans, or Kluyveromyces wickerhamii.
  • the host cell used to produce a NVP or NVP- antimicrobial protein may be a species within the Pichia genus.
  • the host cell may be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica, or Pichia subpelliculosa.
  • the host cell used to produce a NVP or NVP- antimicrobial protein may be a species within the Saccharomyces genus.
  • the host cell may be one of the following: Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis,
  • the procedures and methods described here can be accomplished using a host cell that is a Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, or Hansenula anomala.
  • a host cell that is a Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, or Hansenula anomala.
  • the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Hansenula species including any species of Hansenula and preferably Hansenula polymorpha. In some embodiments, the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Yarrowia species for example, Yarrowia lipolytica. In some embodiments, the procedures and methods described here can be accomplished with any species of yeast, including but not limited to any species of Schizosaccharomyces species including any species of Schizosaccharomyces and preferably Schizosaccharomyces pombe.
  • yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as a host organism.
  • Yeast cell culture techniques are well known to those having ordinary skill in the art. Exemplary methods of yeast cell culture can be found in Evans, Yeast Protocols. Springer (1996); Bill, Recombinant Protein Production in Yeast. Springer (2012); Hagan et al., Fission Yeast: A Laboratory Manual, CSH Press (2016); Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture. Biosci Biotechnol Biochem.
  • a host cell can be transformed with a polynucleotide operable to encode an NVP (e.g., by using any of the vectors described herein).
  • that host cell can be yeast strain.
  • a yeast strain can be produced by preparing a vector comprising a first expression cassette comprising a polynucleotide operable to express a NVP or complementary nucleotide sequence thereof.
  • the present disclosure comprises, consists essentially of, or consists of, a yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an NVP, said NVP comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence having an N-terminus addition of an amino acid to a WT-NCR13 amino acid sequence as set forth in SEQ ID NO: 1, wherein the amino acid is any natural or non-natural amino acid other than glycine or alanine; or a complementary nucleotide sequence thereof.
  • the present disclosure comprises, consists essentially of, or consists of, a yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an NVP, said NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99
  • the present disclosure comprises, consists essentially of, or consists of, a yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an NVP, said NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical,
  • the present disclosure comprises, consists essentially of, or consists of, a yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an NVP, said NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical,
  • the present disclosure comprises, consists essentially of, or consists of, a yeast strain comprising: a first expression cassette comprising a polynucleotide operable to encode an NVP, said NVP comprising, consisting essentially of, or consisting of, an amino acid sequence that is at least is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical,
  • the yeast strain comprises a polynucleotide which enables the synthesis of an NVP, wherein the NVP comprises, consists essentially of, or consists of, an amino sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% as the amino acid sequence set forth in any one of SEQ ID NOs: 4-19 or 66, or a complementary nucleotide sequence thereof.
  • the yeast strain comprises a polynucleotide which enables the synthesis of an NVP, wherein the NVP comprises, consists essentially of, or consists of, an amino sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% as the amino acid sequence set forth in any one of SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, and 17, or a complementary nucleotide sequence thereof.
  • the yeast strain is selected from any species belonging to the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces.
  • the yeast cell is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.
  • the yeast cell is Kluyveromyces lactis or Kluyveromyces marxianus.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the NVP is a homopolymer or heteropolymer of two or more NVPs, wherein the amino acid sequence of each NVP is the same or different.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the NVP is a fiised protein comprising two or more NVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each NVP may be the same or different.
  • the linker is a cleavable linker.
  • the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 39-51.
  • the linker is cleavable inside at least one of (i) the gut or hemolymph of an invertebrate, and (ii) cleavable inside the gut of a mammal.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the vector is a plasmid comprising an alpha-MF signal.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the vector is transformed into a yeast strain.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the yeast strain is selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the yeast strain is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the yeast strain is Kluyveromyces lactis.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein expression of the NVP provides a yield of at least: 70 mg/L, 80 mg/L, 90 mg/L, 100 mg/L, 110 mg/L, 120 mg/L, 130 mg/L, 140 mg/L, 150 mg/L, 160 mg/L, 170 mg/L, 180 mg/L, 190 mg/L 200 mg/L, 500 mg/L, 750 mg/L, 1,000 mg/L, 1,250 mg/L, 1,500 mg/L, 1,750 mg/L or at least 20,000 mg/L, or more, of NVP per liter of medium.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein expression of the NVP provides a yield of at least 100 mg/L of NVP per liter of medium.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein expression of the NVP in the medium results in the expression of a single NVP in the medium.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein expression of the NVP in the medium results in the expression of an NVP polymer comprising two or more NVP polypeptides in the medium.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the vector comprises two or three expression cassettes, each expression cassette operable to encode the NVP of the first expression cassette.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the vector comprises two or three expression cassettes, each expression cassette operable to encode the NVP of the first expression cassette, or an NVP of a different expression cassette.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the expression cassette is operable to encode an NVP as set forth in any one of SEQ ID NOs: 4-19, or 66.
  • a yeast strain can be operable to express an NVP or NVP-antimicrobial protein, wherein the expression cassette is operable to encode an NVP as set forth in any one of SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, and 17.
  • any of the aforementioned methods, and/or any of the methods described herein, can be used to produce one or more of the NVPs or NVP-antimicrobial proteins as described herein.
  • any of the methods described herein can be used to produce one or more of the NVPs described in the present disclosure, e.g., an NCR13 variant peptide (NVP) of the present disclosure which comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93%
  • An exemplary method of yeast transformation is as follows: first, expression vectors carrying an NVP ORF are transformed into yeast cells; the expression vectors are usually linearized by specific restriction enzyme cleavage to facilitate chromosomal integration via homologous recombination. The linear expression vector is then transformed into yeast cells by a chemical or electroporation method of transformation and integrated into the targeted locus of the yeast genome by homologous recombination. The integration can happen at the same chromosomal locus multiple times; therefore, the genome of a transformed yeast cell can contain multiple copies of NVP expression cassettes.
  • the successfiilly transformed yeast cells can be identified using growth conditions that favor a selection marker engineered into the expression vector and co-integrated into yeast chromosomes with the NVP ORF; examples of such markers include, but are not limited to, acetamide prototrophy, zeocin resistance, geneticin resistance, nourseothricin resistance, and uracil prototrophy.
  • Selection markers are well known in the art, and any of these well-known selection markers can be implemented in the present disclosure.
  • a selection marker can be a positive selection marker, or negative selection marker.
  • Positive selection markers permit the selection for cells in which the gene product of the marker is expressed. This generally comprises contacting cells with an appropriate agent that, but for the expression of the positive selection marker, kills or otherwise selects against the cells.
  • An exemplary method of using selection markers is disclosed in U.S. Patent No. 5,464,764, the disclosure of which is incorporated herein by reference in its entirety. Additional exemplary descriptions and methods concerning selection markers are provided in Wigler et al., Cell 11 :223 (1977); Szybalska & Szybalski, Proc. Natl.
  • transgenic yeast colonies carrying the NVP transgenes should be screened for high yield strains.
  • Two effective methods for such screening each dependent on growth of small-scale cultures of the transgenic yeast to provide conditioned media samples for subsequent analysis — use reverse-phase HPLC or housefly injection procedures to analyze conditioned media samples from the positive transgenic yeast colonies.
  • the transgenic yeast cultures can be obtained, e.g., using 14 mL round bottom polypropylene culture tubes with 5 to 10 mL defined medium added to each tube, or in 48- well deep well culture plates with 2.2 mL defined medium added to each well.
  • the defined medium not containing crude proteinaceous extracts or by-products such as yeast extract or peptone, is used for the cultures to reduce the protein background in the conditioned media harvested for the later screening steps.
  • the cultures are performed at the optimal temperature, for example, 23.5°C for K. lactis, for about 5-6 days, until the maximum cell density is reached.
  • NVPs will now be produced by the transformed yeast cells and secreted out of cells to the growth medium.
  • To prepare samples for the screening cells are removed from the cultures by centrifiigation and the supernatants are collected as the conditioned media, which are then cleaned by filtration through 0.22 pm filter membrane and then made ready for strain screening.
  • positive yeast colonies transformed with NVP can be screened via reverse-phase HPLC (rpHPLC) screening of putative yeast colonies.
  • rpHPLC reverse-phase HPLC
  • an HPLC analytic column with bonded phase of Cl 8 can be used.
  • Acetonitrile and water are used as mobile phase solvents, and a UV absorbance detector set at 220 nm is used for the peptide detection.
  • Appropriate amounts of the conditioned medium samples are loaded into the rpHPLC system and eluted with a linear gradient of mobile phase solvents.
  • the corresponding peak area of the antimicrobial peptide in the HPLC chromatograph is used to quantify the NVP concentrations in the conditioned media.
  • Known amounts of pure NVP are run through the same rpHPLC column with the same HPLC protocol to confirm the retention time of the peptide and to produce a standard peptide HPLC curve for the quantification.
  • An exemplary reverse-phase HPLC screening process of positive K. lactis cells is as follows: an NVP ORF can be inserted into the expression vector, pKLACl, and transformed into the K. lactis strain, YCT306, from New England Biolabs, Ipswich, MA, USA.
  • pKLACl vector is an integrative expression vector.
  • the resulting transformed colonies produced pre-propeptides comprising an a-mating factor signal peptide, a Kex2 cleavage site and mature NVPs.
  • the a-Mating factor signal peptide guides the pre-propeptides to enter the endogenous secretion pathway, and mature NVPs are released into the growth media.
  • codon optimization for NVP expression can be performed in two rounds, for example, in the first round, based on some common features of high expression DNA sequences, multiple variants of the NVP ORF, expressing an a-Mating factor signal peptide, a Kex2 cleavage site and the NVP, are designed and their expression levels are evaluated in the YCT306 strain of K. lactis, resulting in an initial K. lactis expression algorithm; in a second round of optimization, additional variant NVP ORFs can be designed based on the initial K. lactis expression algorithm to fiirther fine-tuned the K. lactis expression algorithm, and identify the best ORF for NVP expression in K. lactis.
  • the resulting DNA sequence from the foregoing optimization can have an open reading frame encoding an a-MF signal peptide, a Kex2 cleavage site and an NVP, which can be cloned into the pKLACl vector using Hind III and Not I restriction sites, resulting in NVP expression vectors.
  • the yeast, Pichia pastoris can be transformed with an NVP expression cassette.
  • An exemplary method for transforming P. pastoris is as follows: yeast vectors can be used to transform an NVP expression cassette into P. pastoris.
  • the vectors can be obtained from commercial vendors known to those having ordinary skill in the art.
  • the vectors can be integrative vectors, and may use the uracil phosphoribosyltransferase promoter (pUPP) to enhance the heterologous transgene expression.
  • pUPP uracil phosphoribosyltransferase promoter
  • the vectors may offer different selection strategies; e.g., in some embodiments, the only difference between the vectors can be that one vector may provide G418 resistance to the host yeast, while the other vector may provide Zeocin resistance.
  • pairs of complementary oligonucleotides, encoding the NVP may be designed and synthesized for subcloning into the two yeast expression vectors.
  • Hybridization reactions can be performed by mixing the corresponding complementary oligonucleotides to a final concentration of 20 pM in 30 mM NaCl, 10 mM Tris-Cl (all final concentrations), pH 8, and then incubating at 95°C for 20 min, followed by a 9-hour incubation starting at 92°C and ending at 17°C, with 3°C drops in temperature every 20 min.
  • the hybridization reactions will result in DNA fragments encoding NVP.
  • the two P. pastoris vectors can be digested with Bsal-HF restriction enzymes, and the double stranded DNA products of the reactions are then subcloned into the linearized P. pastoris vectors using standard procedures.
  • plasmid aliquots can be transfected by electroporation into a P. pastoris strain (e.g., Bg08).
  • the resulting transformed yeast can be selected based on resistance (e.g., in this example, to Zeocin or G418) conferred by elements engineered into the vectors.
  • Methods of protein purification are well-known in the art, and any known method can be employed to purify and/or recover NVPs of the present disclosure.
  • the following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica, or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and the like.
  • proteins of the present disclosure can be purified using one of the following; affinity chromatography; ion exchange chromatography; filtration; electrophoresis; hydrophobic interaction chromatography; gel filtration chromatography; reverse phase chromatography; concanavalin A chromatography; and differential solubilization.
  • Peptide yield can be determined by any of the methods known to those of skill in the art (e.g., capillary gel electrophoresis (CGE), Western blot analysis, and the like). Activity assays, as described herein and known in the art, can also provide information regarding peptide yield. In some embodiments, these or any other methods known in the art can be used to evaluate peptide yield. [0343] Quantification assays
  • NVP peptide yield can be measured using: HPLC; Mass spectrometry (MS) and related techniques; LC/MS/MS; reverse phase protein arrays (RPPA); immunohistochemistry; ELISA; suspension bead array, mass spectrometry; dot blot; SDS-PAGE; capillary gel electrophoresis (CGE); Western blot analysis; Bradford assay; measuring UV absorption at 260nm; Lowry assay; Smith copper/bicinchoninic assay; a secretion assay; Pierce protein assay; Biuret reaction; and the like. Exemplary methods of protein quantification are provided in Stoscheck, C.
  • NVP peptide yield can be quantified and/or assessed using methods that include, without limitation: recombinant protein mass per volume of culture (e.g., gram or milligrams protein per liter culture); percent or fraction of recombinant protein insoluble precipitate obtained after cell lysis determined in (e.g., recombinant protein extracted supernatant in an amount/amount of protein in the insoluble components); percentage or fraction of active protein (e.g., an amount/analysis of the active protein for use in protein amount); total cell protein (tcp) percentage or fraction; and/or the amount of protein/cell and the dry biomass of a percentage or ratio.
  • recombinant protein mass per volume of culture e.g., gram or milligrams protein per liter culture
  • percent or fraction of recombinant protein insoluble precipitate obtained after cell lysis determined in e.g., recombinant protein extracted supernatant in an amount/amount of protein in the insoluble components
  • percentage or fraction of active protein
  • the culture cell density may be taken into account, particularly when yields between different cultures are being compared.
  • the present disclosure provides a method of producing a heterologous polypeptide that is at least about 5%, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or greater of total cell protein (tcp).
  • Percent total cell protein is the amount of heterologous polypeptide in the host cell as a percentage of aggregate cellular protein. The determination of the percent total cell protein is well known in the art.
  • Total cell protein (tcp)” or “Percent total cell protein (% tcp)” is the amount of protein or polypeptide in the host cell as a percentage of aggregate cellular protein. Methods for the determination of the percent total cell protein are well known in the art.
  • HPLC can be used to quantify peptide yield.
  • peptide yield can be quantified using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, Cl 8 reverse-phase analytical HPLC column and an auto-injector.
  • lactis cells are analyzed using Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, Cl 8 reverse-phase analytical HPLC column and an autoinjector by analyzing HPLC grade water and acetonitrile containing 0.1% trifluoroacetic acid, constituting the two mobile phase solvents used for the HPLC analyses; the peak areas of both the NVP or NVP-antimicrobial protein are analyzed using HPLC chromatographs, and then used to calculate the peptide concentration in the conditioned media, which can be birther normalized to the corresponding final cell densities (as determined by OD600 measurements) as normalized peptide yield.
  • positive yeast colonies transformed with NVP or NVP- antimicrobial protein can be screened using a housefly injection assay.
  • NVP or NVP- antimicrobial protein can paralyze/kill houseflies when injected in measured doses through the body wall of the dorsal thorax.
  • the efficacy of the NVP or NVP-antimicrobial protein can be defined by the median paralysis/lethal dose of the peptide (PD50/LD50), which causes 50% knock-down ratio or mortality of the injected houseflies respectively.
  • the pure NVP or NVP- antimicrobial protein is normally used in the housefly injection assay to generate a standard dose-response curve, from which a PD50/LD50 value can be determined.
  • quantification of the NVP or NVP-antimicrobial protein produced by the transformed yeast can be achieved using a housefly injection assay performed with serial dilutions of the corresponding conditioned media.
  • An exemplary housefly injection bioassay is as follows: conditioned media is serially diluted to generate full dose-response curves from the housefly injection bioassay. Before injection, adult houseflies (Musca domesticd) are immobilized with CO2, and 12-18 mg houseflies are selected for injection. A microapplicator, loaded with a 1 cc syringe and 30-gauge needle, is used to inject 0.5 pL per fly, doses of serially diluted conditioned media samples into houseflies through the body wall of the dorsal thorax.
  • Peptide yield means the peptide concentration in the conditioned media in units of mg/L.
  • peptide yields are not always sufficient to accurately compare the strain production rate. Individual strains may have different growth rates, hence when a culture is harvested, different cultures may vary in cell density. A culture with a high cell density may produce a higher concentration of the peptide in the media, even though the peptide production rate of the strain is lower than another strain which has a higher production rate.
  • normalized yield is created by dividing the peptide yield with the cell density in the corresponding culture and this allows a better comparison of the peptide production rate between strains.
  • the cell density is represented by the light absorbance at 600 nm with a unit of “A” (Absorbance unit).
  • Screening yeast colonies that have undergone a transformation with a polynucleotide operable to encode an NVP or NVP-antimicrobial protein can identify the high yield yeast strains from hundreds of potential colonies. These strains can be fermented in bioreactor to achieve at least up to 4 g/L or at least up to 3 g/L or at least up to 2 g/L yield of the NVP or NVP-antimicrobial protein when using optimized fermentation media and fermentation conditions described herein.
  • the higher rates of production can be anywhere from about 100 mg/L to about 100,000 mg/L; or from about 100 mg/L to about 90, 000 mg/L; or from about 100 mg/L to about 80,000 mg/L; or from about 100 mg/L to about 70,000 mg/L; or from about 100 mg/L to about 60,000 mg/L; or from about 100 mg/L to about 50,000 mg/L; or from about 100 mg/L to about 40,000 mg/L; or from about 100 mg/L to about 30,000 mg/L; or from about 100 mg/L to about 20,000 mg/L; or from about 100 mg/L to about 17,500 mg/L; or from about 100 mg/L to about 15,000 mg/L; or from about 100 mg/L to about 12,500 mg/L; or from about 100 mg/L to about 10,000 mg/L; or from about 100 mg/L to about 9,000 mg/L; or from about 100 mg/L to about 8,000 mg/L; or from about 100 mg/L to about 7,000 mg/L; or
  • Cell culture techniques are well-known in the art.
  • the culture method and/or materials will necessarily require adaption based on the host cell selected; and, such adaptions (e.g., modifying pH, temperature, medium contents, and the like) are well known to those having ordinary skill in the art.
  • any known culture technique may be employed to produce an NVP or NVP-antimicrobial protein of the present disclosure.
  • yeast cell culture techniques are well known to those having ordinary skill in the art. Exemplary methods of yeast cell culture can be found in Evans, Yeast Protocols. Springer (1996); Bill, Recombinant Protein Production in Yeast. Springer (2012); Hagan et al., Fission Yeast: A Laboratory Manual, CSH Press (2016); Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in preculture. Biosci Biotechnol Biochem. 2014; 78(6): 1090-3; Dymond, Saccharomyces cerevisiae growth media. Methods Enzymol.
  • yeast can be cultured in a variety of media, e.g., in some embodiments, yeast can be cultured in minimal medium; YPD medium; yeast synthetic drop-out medium; Yeast Nitrogen Base (YNB with or without amino acids); YEPD medium; ADE D medium; ADE DS" medium; LEU D medium; HIS D medium; or Mineral salts medium.
  • yeast can be cultured in minimal medium.
  • minimal medium ingredients can comprise: 2% Sugar; Phosphate Buffer, pH 6.0; Magnesium Sulfate; Calcium Chloride; Ammonium Sulfate; Sodium Chloride;
  • yeast can be cultured in YPD medium.
  • YPD medium comprises a bacteriological peptone, yeast extract, and glucose.
  • yeast can be cultured in yeast synthetic drop-out medium, which can be used to differentiate auxotrophic mutant strains that cannot grow without a specific medium component transformed with a plasmid that allows said transformant to grow on a medium lacking the required component.
  • yeast can be cultured using Yeast Nitrogen Base (YNB with or without amino acids), which comprises nitrogen, vitamins, trace elements, and salts.
  • the medium can be YEPD medium, e.g., a medium comprising 2% D-glucose, 2% BACTO Peptone (Difco Laboratories, Detroit, MI), 1% BACTO yeast extract (Difco), 0.004% adenine, and 0.006% L-leucine; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol
  • the medium can be ADE D medium, e.g., a medium comprising 0.056%-Ade-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, and 0.5% 200x tryptophan, threonine solution; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol
  • the medium can be ADE DS" medium, e.g., a medium comprising 0.056%-Ade-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, 0.5% 200x tryptophan, threonine solution, and 18.22% D-sorbitol; or, a variation thereof, wherein the carbon source is entirely a sugar alcohol, e.g., glycerol or sorbitol
  • the medium can be LEU D medium e.g., a medium comprising 0.052%-Leu-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, and 0.5% 200x tryptophan, threonine solution; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol.
  • LEU D medium e.g., a medium comprising 0.052%-Leu-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, and 0.5% 200x tryptophan, threonine solution
  • the carbon source is a sugar alcohol, e.g., glycerol or sorbitol.
  • the medium can be HIS D medium, e.g., a medium comprising 0.052%-His-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, and 0.5% 200x tryptophan, threonine solution; or, a variation thereof, wherein the carbon source is a sugar alcohol, e.g., glycerol or sorbitol.
  • HIS D medium e.g., a medium comprising 0.052%-His-Trp-Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, and 0.5% 200x tryptophan, threonine solution
  • the carbon source is a sugar alcohol, e.g., glycerol or sorbitol.
  • a mineral salts medium can be used.
  • Mineral salts media consists of mineral salts and a carbon source such as, e.g., glucose, sucrose, or glycerol.
  • Examples of mineral salts media include, e.g., M9 medium, Pseudomonas medium (ATCC 179), and Davis and Mingioli medium. See, Davis & Mingioli (1950) J. Bact. 60:17- 28.
  • the mineral salts used to make mineral salts media include those selected from among, e.g., potassium phosphates, ammonium sulfate or chloride, magnesium sulfate or chloride, and trace minerals such as calcium chloride, borate, and sulfates of iron, copper, manganese, and zinc.
  • no organic nitrogen source such as peptone, tryptone, amino acids, or a yeast extract, is included in a mineral salts medium.
  • an inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia.
  • a mineral salts medium will typically contain glucose or glycerol as the carbon source.
  • minimal media can also contain mineral salts and a carbon source, but can be supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients, though these are added at very minimal levels.
  • Media can be prepared using the methods described in the art, e.g., in U.S. Pat. App. Pub. No. 2006/0040352, the disclosure of which is incorporated herein by reference in its entirety. Details of cultivation procedures and mineral salts media usefiil in the methods of the present disclosure are described by Riesenberg, D et al., 1991, “High cell density cultivation of Escherichia coli at controlled specific growth rate,” J. Biotechno 1. 20 (1): 17-27.
  • Kluyveromyces lactis are grown in minimal media supplemented with 2% glucose, galactose, sorbitol, or glycerol as the sole carbon source. Cultures are incubated at 30°C until mid-log phase (24-48 hours) for 0-galactosidase measurements, or for 6 days at 23.5°C for heterologous protein expression.
  • yeast cells can be cultured in 48-well Deep-well plates, sealed after inoculation with sterile, air-permeable cover.
  • Colonies of yeast, for example, K. lactis cultured on plates can be picked and inoculated the deep-well plates with 2.2 mL media per well, composed of DMSor.
  • Inoculated deep-well plates can be grown for 6 days at 23.5°C with 280 rpm shaking in a refrigerated incubator-shaker.
  • conditioned media should be harvested by centrifiigation at 4000 rpm for 10 minutes, followed by filtration using filter plate with 0.22 pM membrane, with filtered media are subject to HPLC analyses.
  • yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as a host organism, and/or the yeast to be modified using the methods described herein.
  • Temperature and pH conditions will vary depending on the stage of culture and the host cell species selected. Variables such as temperature and pH in cell culture are readily known to those having ordinary skill in the art.
  • the pH level is important in the culturing of yeast.
  • the culturing process includes not only the start of the yeast culture but the maintenance of the culture as well.
  • the yeast culture may be started at any pH level, however, since the media of a yeast culture tends to become more acidic (i.e., lowering the pH) over time, care must be taken to monitor the pH level during the culturing process.
  • the yeast is grown in a medium at a pH level that is dictated based on the species of yeast used, the stage of culture, and/or the temperature.
  • the pH level can fall within a range from about 2 to about 10.
  • the pH can range from 2 to 6.5.
  • the pH can range from about 4 to about 4.5.
  • fimgal species can grow in a pH of from about 2 to about 8.5, but favor an acid pH. See Mountney & Gould, Practical food microbiology and technology. 1988. Ed. 3; and Pena et al., Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae. FEMS Yeast Res. 2015 Mar;15(2):fou005. [0377] In other embodiments, the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to
  • the pH of the medium can be at least 5.5. In other aspects, the medium can have a pH level of about 5.5. In other aspects, the medium can have a pH level of between 4 and 8. In some cases, the culture is maintained at a pH level of between 5.5 and 8. In other aspects, the medium has a pH level of between 6 and 8. In some cases, medium has a pH level that is maintained at a pH level of between 6 and 8. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.1 and 8.1. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.2 and
  • the yeast is grown and/or maintained at a pH level of between 6.3 and 8.3. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.4 and 8.4. In some embodiments, the yeast is grown and/or maintained at a pH level of between 5.5 and 8.5. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.5 and 8.5. In some embodiments, the yeast is grown at a pH level of about
  • the yeast is grown at a pH level of about 6. In some embodiments, the yeast is grown at a pH level of about 6.5. In some embodiments, the yeast is grown at a pH level of about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In some embodiments, the yeast is grown at a pH level of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,
  • the yeast is grown at a level of above 8.
  • the pH of the medium can range from a pH of 2 to 8.5. In certain embodiments, the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,
  • Exemplary methods of yeast culture can be found in U.S. Patent No. 5,436,136, entitled “Repressible yeast promoters” (filed 12/20/1991; assignee Ciba-Geigy Corporation); U.S. Patent No. 6,645,739, entitled “Yeast expression systems, methods of producing polypeptides in yeast, and compositions relating to same” (filed 07/26/2001; assignee Phoenix Pharmacologies, Inc., Lexington, KY); and U.S. Patent No. 10,023,836, entitled “Medium for yeasts” (filed 08/23/2013; assignee Yamaguchi University); the disclosures of which are incorporated herein by reference in their entireties.
  • Fermentation [0382] The present disclosure contemplates the culture of host organisms in any fermentation format. For example, batch, fed-batch, semi-continuous, and continuous fermentation modes may be employed herein.
  • Fermentation may be performed at any scale.
  • the methods and techniques contemplated according to the present disclosure are useiul for recombinant protein expression at any scale.
  • microliter-scale, milliliter scale, centiliter scale, and deciliter scale fermentation volumes may be used, and 1 Liter scale and larger fermentation volumes can be used.
  • the fermentation volume is at or above about 1 Liter.
  • the fermentation volume is about 1 liter to about 100 liters.
  • the fermentation volume is about 1 liter, about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, or about 10 liters.
  • the fermentation volume is about 1 liter to about 5 liters, about 1 liter to about 10 liters, about 1 liter to about 25 liters, about 1 liter to about 50 liters, about 1 liter to about 75 liters, about 10 liters to about 25 liters, about 25 liters to about 50 liters, or about 50 liters to about 100 liters
  • the fermentation volume is at or above 5 Liters, 10 Liters, 15 Liters, 20 Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 500 Liters, 1,000 Liters, 2,000 Liters, 5,000 Liters, 10,000 Liters, or 50,000 Liters.
  • the fermentation medium can be a nutrient solution used for growing and or maintaining cells.
  • this solution ordinarily provides at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbon source, e.g., glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
  • the fermentation medium can be the same as the cell culture medium or any other media described herein. In some embodiments, the fermentation medium can be different from the cell culture medium. In some embodiments, the fermentation medium can be modified in order to accommodate the large-scale production of proteins.
  • the fermentation medium can be supplemented electively with one or more components from any of the following categories: (1) hormones and other growth factors such as, serum, insulin, transferrin, and the like; (2) salts, for example, magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases such as, adenosine, thymidine, etc.; (5) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (6) antibiotics, such as gentamycin; and (7) cell protective agents, for example pluronic polyol.
  • hormones and other growth factors such as, serum, insulin, transferrin, and the like
  • salts for example, magnesium, calcium, and phosphate
  • buffers such as HEPES
  • nucleosides and bases such as, adenosine, thymidine, etc.
  • protein and tissue hydrolysates for example peptone or
  • the pH of the fermentation medium can be maintained using pH buffers and methods known to those of skill in the art. Control of pH during fermentation can also can be achieved using aqueous ammonia. In some embodiments, the pH of the fermentation medium will be selected based on the preferred pH of the organism used. Thus, in some embodiments, and depending on the host cell and temperature, the pH can range from about to 1 to about 10.
  • the pH of the fermentation medium can range from a pH of 2 to 8.5. In certain embodiments, the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
  • the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8
  • the optimal pH range is between 6.5 and 7.5, depending on the temperature.
  • the pH can range from about 4.0 to 8.0.
  • neutral pH i.e., a pH of about 7.0 can be used.
  • the fermentation medium can be supplemented with a buffer or other chemical in order to avoid changes to the pH.
  • a buffer or other chemical for example, in some embodiments, the addition of Ca(OH)2, CaCCh, NaOH, or NH4OH can be added to the fermentation medium to neutralize the production of acidic compounds that occur, e.g., in some yeast species during industrial processes.
  • Temperature is another important consideration in the fermentation process; and, like pH considerations, temperature will depend on the type of host cell selected.
  • the fermentation temperature is maintained at about 4°C. to about 42°C.
  • the fermentation temperature is about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11 °C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C.
  • the fermentation temperature is maintained at about 25°C to about 27°C, about 25°C to about 28°C, about 25°C to about 29°C, about 25°C to about 30°C, about 25°C to about 31 °C, about 25°C to about 32°C, about 25°C to about 33°C, about 26°C to about 28°C, about 26°C to about 29°C, about 26°C to about 30°C, about 26°C to about 31 °C, about 26°C to about 32°C, about 27°C to about 29°C, about 27°C to about 30°C, about 27°C to about 31 °C, about 27°C to about 32°C, about 26°C to about 33°C, about 28°C to about 30°C, about 28°C to about 31 °C, about 28°C to about 32°C, about 29°C to about 31 °C, about 29°C to about 32°C, about 29°C to about 33°C, about 30°C to
  • the temperature is changed during fermentation, e.g., depending on the stage of fermentation.
  • microorganisms for up-scaled production of an NVP or NVP-antimicrobial protein include any microorganism listed herein.
  • non- limiting examples of microorganisms include strains of the genus Saccharomyces spp. (including, but not limited to, S. cerevisiae (baker's yeast), S. distaticus, S. uvarum), or the genus Kluyveromyces, (including, but not limited to, K. marxianus, K. fragilis). See, e.g., Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212.
  • Fermentation medium may be selected depending on the host cell and/or needs of the end-user. Any necessary supplements besides, e.g., carbon, may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • Fermentation methods using yeast are well known to those having ordinary skill in the art.
  • batch fermentation can be used according to the methods provided herein; in other embodiments, continuous fermentation procedures can be used.
  • the batch method of fermentation can be used to produce NVPs of the present disclosure.
  • the batch method of fermentation refers to a type of fermentation that is performed with a closed system, wherein the composition of the medium is determined at the beginning of the fermentation and is not subject to artificial alterations during the fermentation (i.e., the medium is inoculated with one or more yeast cells at the start of fermentation, and fermentation is allowed to proceed, uninterrupted by the user).
  • the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped.
  • yeast cells pass through a static lag phase to a high growth log phase, and, finally, to a stationary phase, in which the growth rate is diminished or stopped. If untreated, yeast cells in the stationary phase will eventually die.
  • yeast cells in log phase generally are responsible for the bulk of synthesis of end product.
  • fed-batch fermentation can be used to produce NVPs of the present disclosure.
  • fed-batch fermentation is similar to typical batch method (described above), however, the substrate in the fed-batch method is added in increments as the fermentation progresses.
  • Fed-batch fermentation is usefiil when catabolite repression may inhibit yeast cell metabolism, and when it is desirable to have limited amounts of substrate in the medium.
  • the measurement of the substrate concentration in a fed-batch system is estimated on the basis of the changes of measurable factors reflecting metabolism, such as pH, dissolved oxygen, the partial pressure of waste gases (e.g., CO2), and the like.
  • the fed-batch fermentation procedure can be used to produce NVPs as follows: culturing a production organism (e.g., a modified yeast cell) in a 10 L bioreactor sparged with an N2/CO2 mixture, using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L com steep liquor, and an initial first and second carbon source concentration of 20 g/L. As the modified yeast cells grow and utilize the carbon sources, additional 70% carbon source mixture is then fed into the bioreactor at a rate approximately balancing carbon source consumption. The temperature of the bioreactor is generally maintained at 30° C.
  • a production organism e.g., a modified yeast cell
  • the heterologous peptides reach a desired concentration, e.g., with the cell density being between about 5 and 10 g/L.
  • the fermenter contents can be passed through a cell separation unit such as a centrifiige to remove cells and cell debris, and the fermentation broth can be transferred to a product separations unit. Isolation of the heterologous peptides can take place by standard separations procedures well known in the art.
  • continuous fermentation can be used to produce NVPs of the present disclosure.
  • continuous fermentation refers to fermentation with an open system, wherein a fermentation medium is added continuously to a bioreactor, and an approximately equal amount of conditioned medium is removed simultaneously for processing.
  • Continuous fermentation generally maintains the cultures at a high density, in which yeast cells are primarily in log phase growth.
  • continuous fermentation methods are performed to maintain steady state growth conditions, and yeast cell loss, due to medium withdrawal, should be balanced against the cell growth rate in the fermentation.
  • the continuous fermentation method can be used to produce NVPs as follows: a modified yeast strain can be cultured using a bioreactor apparatus and a medium composition, albeit where the initial first and second carbon source is about, e.g., 30-50 g/L. When the carbon source is exhausted, feed medium of the same composition is supplied continuously at a rate of between about 0.5 L/hr and 1 L/hr, and liquid is withdrawn at the same rate.
  • the heterologous peptide concentration in the bioreactor generally remains constant along with the cell density. Temperature is generally maintained at 30° C., and the pH is generally maintained at about 4.5 using concentrated NaOH and HC1, as required.
  • the bioreactor when producing NVPs, can be operated continuously, for example, for about one month, with samples taken every day or as needed to assure consistency of the target chemical compound concentration.
  • fermenter contents are constantly removed as new feed medium is supplied.
  • the exit stream, containing cells, medium, and heterologous peptides, can then be subjected to a continuous product separations procedure, with or without removing cells and cell debris, and can be performed by continuous separations methods well known in the art to separate organic products from peptides of interest.
  • a yeast cell operable to express an NVP or NVP- antimicrobial protein can be grown, e.g., using a fed batch process in aerobic bioreactor. Briefly, reactors are filled to about 20% to about 70% capacity with medium comprising a carbon source and other reagents. Temperature and pH is maintained using one or more chemicals as described herein. Oxygen level is maintained by sparging air intermittently in concert with agitation.
  • the present disclosure provides a method of using a fed batch process in aerobic bioreactor, wherein the reactor is filled to about 20%; 21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%; 32%; 33%; 34%; 35%; 36%;
  • the present disclosure provides a fed batch fermentation method using an aerobic bioreactor to produce NVPs, wherein the medium is a rich culture medium.
  • the carbon source can be glucose, sorbitol, or lactose.
  • the amount of glucose can be about 2 g/L; 3 g/L; 4 g/L;
  • the amount of sorbitol can be about 2 g/L; 3 g/L; 4 g/L;
  • the amount of lactose can be about 2 g/L; 3 g/L; 4 g/L;
  • the present disclosure provides a fed batch fermentation method using an aerobic bioreactor, wherein the medium is supplemented with one or more of phosphoric acid, calcium sulfate, potassium sulfate, magnesium sulfate heptahydrate, potassium hydroxide, and/or com steep liquor.
  • the medium can be supplemented with phosphoric acid in an amount of about 2 g/L; 3 g/L; 4 g/L; 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L;
  • the medium can be supplemented with calcium sulfate in an amount of about 0.05 g/L; 0.15 g/L; 0.25 g/L; 0.35 g/L; 0.45 g/L; 0.55 g/L; 0.65 g/L; 0.75 g/L; 0.85 g/L; 0.95 g/L; 1.05 g/L; 1.15 g/L; 1.25 g/L; 1.35 g/L; 1.45 g/L; 1.55 g/L; 1.65 g/L; 1.75 g/L; 1.85 g/L; 1.95 g/L; 2.05 g/L; 2.15 g/L; 2.25 g/L; 2.35 g/L; 2.45 g/L; 2.55 g/L; 2.65 g/L; 2.75 g/L; 2.85 g/L; or 2.95 g/L to the medium.
  • the medium can be supplemented with potassium sulfate in an amount of about 2 g/L; 2.5 g/L; 3 g/L; 3.5 g/L; 4 g/L; 4.5 g/L; 5 g/L; 5.5 g/L; 6 g/L; 6.5 g/L; 7 g/L; 7.5 g/L; 8 g/L; 8.5 g/L; 9 g/L; 9.5 g/L; 10 g/L; 10.5 g/L; 11 g/L; 11.5 g/L; 12 g/L; 12.5 g/L; 13 g/L; 13.5 g/L; 14 g/L; 14.5 g/L; 15 g/L; 15.5 g/L; 16 g/L; 16.5 g/L; 17 g/L; 17.5 g/L; 18 g/L; 18.5 g/L; 19 g/L; 1
  • the medium can be supplemented with magnesium sulfate heptahydrate in an amount of about 0.25 g/L; 0.5 g/L; 0.75 g/L; 1 g/L; 1.25 g/L; 1.5 g/L; 1.75 g/L; 2 g/L; 2.25 g/L; 2.5 g/L; 2.75 g/L; 3 g/L; 3.25 g/L; 3.5 g/L; 3.75 g/L; 4 g/L; 4.25 g/L; 4.5 g/L; 4.75 g/L; 5 g/L; 5.25 g/L; 5.5 g/L; 5.75 g/L; 6 g/L; 6.25 g/L; 6.5 g/L; 6.75 g/L; 7 g/L; 7.25 g/L; 7.5 g/L; 7.75 g/L; 8 g/L; 8.25 g/L;
  • the medium can be supplemented with potassium hydroxide in an amount of about 0.25 g/L; 0.5 g/L; 0.75 g/L; 1 g/L; 1.25 g/L; 1.5 g/L; 1.75 g/L; 2 g/L; 2.25 g/L; 2.5 g/L; 2.75 g/L; 3 g/L; 3.25 g/L; 3.5 g/L; 3.75 g/L; 4 g/L; 4.25 g/L; 4.5 g/L; 4.75 g/L; 5 g/L; 5.25 g/L; 5.5 g/L; 5.75 g/L; 6 g/L; 6.25 g/L; 6.5 g/L; 6.75 g/L; or 7 g/L to the medium.
  • the medium can be supplemented with com steep liquor in an amount of about 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L; 12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L; 29 g/L; 30 g/L; 31 g/L; 32 g/L; 33 g/L; 34 g/L; 35 g/L; 36 g/L;
  • the temperature of the reactor can be maintained between about 15°C and about 45°C.
  • the reactor can have a temperature of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.
  • the pH can have a level of about 3 to about 6.
  • the pH can be 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.
  • the pH can be maintained at a constant level via the addition of one or more chemicals.
  • ammonium hydroxide can be added to maintain pH.
  • ammonium hydroxide can be added to a level of ammonium hydroxide in the medium that is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, of ammonium hydroxide
  • oxygen levels can be maintained by sparging.
  • dissolved oxygen can be maintained at a constant level by sparging air between 0.5 -1.5 volume/volume/min and by increasing agitation to maintain a set point of 10-30%.
  • inoculation of the reactor can be accomplished based on an overnight seed culture comprising from about 2.5 g/L to about 50 g/L of a carbon source, e.g., glucose, sorbitol, or lactose.
  • the overnight seed culture can comprise com steep liquor, e.g., from about 2.5 g/L to about 50 g/L of com steep liquor.
  • the inoculation percentage can range from about 5-20% of initial fill volume.
  • the reactor can be fed with from about a 50% to about an 80% solution of the selected carbon source up until the reactor is filled and/or the desired supernatant peptide concentration is achieved.
  • the time required to fill the reactor can range from about 86 hours to about 160 hours. In some embodiments, the quantity required to reach the desired peptide concentration can range from about 0.8 g/L to about 1.2 g/L.
  • the contents can be passed through a cell separation unit and optionally concentrated, depending on intended use of the material.
  • MSM media recipe 2 g/L sodium citrate dihydrate; 1 g/L calcium sulfate dihydrate (0.79 g/L anhydrous calcium sulfate); 42.9g/L potassium phosphate monobasic; 5.17g/L ammonium sulfate; 14.33 g/L potassium sulfate; 11.7 g/L magnesium sulfate heptahydrate; 2 mL/L PTMltrace salt solution; 0.4 ppm biotin (from 500X, 200 ppm stock); 1 -2% pure glycerol or other carbon source.
  • PTM1 trace salts solution Cupric sulfate-5H2O 6.0 g; Sodium iodide 0.08 g; Manganese sulfate-H2O 3.0 g; Sodium mo lybdate-2H2O 0.2 g; Boric Acid 0.02 g; Cobalt chloride 0.5 g; Zinc chloride 20.0 g; Ferrous sulfate-V LO 65.0 g; Biotin 0.2 g; Sulfiiric Acid 5.0 ml; add Water to a final volume of 1 liter.
  • An illustrative composition for K An illustrative composition for K.
  • lactis defined medium is as follows: 11.83 g/L KH2PO4, 2.299 g/L K2HPO4, 20 g/L of a fermentable sugar, e.g., galactose, maltose, latotriose, sucrose, fructose or glucose and/or a sugar alcohol, for example, erythritol, hydrogenated starch hydrolysates, isomalt, lactitol, maltitol, mannitol, and xylitol, 1 g/L MgSO4.7H2O, 10 g/L (NH4)SO4, 0.33 g/L CaCl 2 .2H 2 O, 1 g/L NaCl, 1 g/L KC1, 5 mg/L CuSO 4 .5H 2 O, 30 mg/L MnSO 4 .H 2 O, 10 mg/L, ZnCl 2 , 1 mg/L KI, 2 mg/L COCI2.6H2O,
  • Proteins, polypeptides, and peptides degrade in both biological samples and in solution (e.g., cell culture and/or during fermentation).
  • Methods of detecting NVP peptide degradation are well known in the art. Any of the well-known methods of detecting peptide degradation (e.g., during fermentation) may be employed here.
  • peptide degradation can be detected using isotope labeling techniques; liquid chromatography/mass spectrometry (LC/MS); HPLC; radioactive amino acid incorporation and subsequent detection, e.g., via scintillation counting; the use of a reporter protein, e.g., a protein that can be detected (e.g., by fluorescence, spectroscopy, luminometry, etc.); fluorescent intensity of one or more bioluminescent proteins and/or fluorescent proteins and/or fiisions thereof; pulse-chase analysis (e.g., pulse-labeling a cell with radioactive amino acids and following the decay of the labeled protein while chasing with unlabeled precursor, and arresting protein synthesis and measuring the decay of total protein levels with time); cycloheximide-chase assays;
  • an assay can be used to detect peptide degradation, wherein a sample is contacted with a non- fluorescent compound that is operable to react with free primary amine in said sample produced via the degradation of a peptide, and which then produces a fluorescent signal that can be quantified and compared to a standard.
  • non- fluorescent compounds that can be utilized as fluorescent tags for free amines according to the present disclosure are 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA), fluorescamine, and o-phthaldialdehyde.
  • the method to determine the readout signal from the reporter protein depends from the nature of the reporter protein.
  • the readout signal corresponds to the intensity of the fluorescent signal.
  • the readout signal may be measured using spectroscopy-, fluorometry-, photometry-, and/or luminometry-based methods and detection systems, for example. Such methods and detection systems are well known in the art.
  • peptide degradation can be detected in a sample using immunoassays that employ a detectable antibody.
  • immunoassays include, for example, agglutination assays, ELISA, Pandex microfluorimetric assay, flow cytometry, serum diagnostic assays, and immunohistochemical staining procedures, all of which are well- known in the art.
  • the levels (e.g., of fluorescence) in one sample can be compared to a standard.
  • An antibody can be made detectable by various means well known in the art.
  • a detectable marker can be directly or indirectly attached to the antibody.
  • Useftd markers include, for example, radionucleotides, enzymes, fluorogens, chromogens and chemiluminescent labels.
  • agriculturally acceptable salts, hydrates, solvates, crystal forms and individual isomers, enantiomers, tautomers, diastereomers and prodrugs of the NVP described herein can be utilized.
  • an agriculturally acceptable salt of the present disclosure possesses the desired pharmacological activity of the parent compound.
  • Such salts include: acid addition salts, formed with inorganic acids; acid addition salts formed with organic acids; or salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, aluminum ion; or coordinates with an organic base such as ethanolamine, and the like.
  • agriculturally acceptable salts include conventional toxic or non-toxic salts.
  • convention non-toxic salts include those such as fiimarate, phosphate, citrate, chlorydrate, and the like.
  • the agriculturally acceptable salts of the present disclosure can be synthesized from a parent compound by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
  • non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is incorporated herein by reference in its entirety.
  • an agriculturally acceptable salt can be one of the following: hydrochloride; sodium; sulfate; acetate; phosphate or diphosphate; chloride; potassium; maleate; calcium; citrate; mesylate; nitrate; tartrate; aluminum; or gluconate.
  • a list of agriculturally acceptable acids that can be used to form salts can be: glycolic acid; hippuric acid; hydrobromic acid; hydrochloric acid; isobutyric acid; lactic acid (DL); lactobionic acid; lauric acid; maleic acid; malic acid (- L); malonic acid; mandelic acid (DL); methanesulfonic acid ; naphthalene -1,5 -disulfonic acid; naphthalene-2-sulfonic acid; nicotinic acid; nitric acid; oleic acid; oxalic acid; palmitic acid; pamoic acid; phosphoric acid; proprionic acid; pyroglutamic acid (- L); salicylic acid; sebacic acid; stearic acid; succinic acid; sulfiiric acid; tartaric acid (+ L); thiocyanic acid; toluenesulfonic acid (p); undecy
  • agriculturally acceptable salt can be any organic or inorganic addition salt.
  • the salt may use an inorganic acid and an organic acid as a free acid.
  • the inorganic acid may be hydrochloric acid, bromic acid, nitric acid, sulfiiric acid, perchloric acid, phosphoric acid, etc.
  • the organic acid may be citric acid, acetic acid, lactic acid, maleic acid, ftimaric acid, gluconic acid, methane sulfonic acid, gluconic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid, ethane sulfonic acid, 4- toluene sulfonic acid, salicylic acid, citric acid, benzoic acid, malonic acid, etc.
  • the salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.).
  • the acid addition salt may include acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisilate, esylate, formate, fiimarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthalate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate,
  • the agriculturally acceptable salt can be a salt with an acid such as acetic acid, propionic acid, butyric acid, formic acid, trifluoroacetic acid, maleic acid, tartaric acid, citric acid, stearic acid, succinic acid, ethylsuccinic acid, lactobionic acid, gluconic acid, glucoheptonic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, 2 -hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, laurylsulfuric acid, malic acid, aspartic acid, glutaminic acid, adipic acid, cysteine, N- acetylcysteine, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfiiric acid, hydroiodic acid, nicotinic acid, ox
  • the agriculturally acceptable salt can be prepared from either inorganic or organic bases.
  • Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, ferrous, zinc, copper, manganous, aluminum, ferric, manganic salts, and the like.
  • Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally-occurring substituted amines, and cyclic amines, including isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and the like.
  • Preferred organic bases are isopropylamine, diethylamine, ethanolamine, piperidine, tromethamine, and choline.
  • agriculturally acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Agriculturally acceptable salts are well known in the art. For example, S. M. Berge, et al. describe agriculturally acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977), the disclosure of which is incorporated herein by reference in its entirety.
  • the salts of the present disclosure can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base ftinction with a suitable organic acid.
  • suitable organic acid examples include agriculturally acceptable, nontoxic acid addition salts.
  • agriculturally acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfiiric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • Other agriculturally acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fiimarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydro iodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pe
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further agriculturally acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • NVPs described herein, and/or an antimicrobial protein comprising at least one NVP as described herein can be incorporated into plants, plant tissues, plant cells, plant seeds, and/or plant parts thereof, for either the stable, or transient expression of an NVP or an NVP-antimicrobial protein, and/or a polynucleotide sequence encoding the same.
  • the NVP or NVP-antimicrobial protein can be incorporated into a plant using recombinant techniques known in the art.
  • the NVP or NVP-antimicrobial protein may be in the form of an antimicrobial protein which may comprise one or more NVP monomers.
  • NVP also encompasses an NVP-antimicrobial protein
  • a “NVP polynucleotide” is similarly also used to encompass a polynucleotide or group of polynucleotides operable to express and/or encode an antimicrobial protein comprising one or more NVPs.
  • the goal of incorporating an NVP into plants is to deliver NVPs and/or NVP- antimicrobial proteins to the pathogenic microbe via the microbe’s consumption of the transgenic NVP expressed in a plant tissue consumed by the microbe.
  • the consumed NVP may have the ability to inhibit the growth, impair the movement, or even kill an microbe.
  • transgenic plants expressing an NVP polynucleotide and/or an NVP polypeptide may express said NVP polynucleotide/polypeptide in a variety of plant tissues, including but not limited to: the epidermis (e.g., mesophyll); periderm; phloem; xylem; parenchyma; collenchyma; sclerenchyma; and primary and secondary meristematic tissues.
  • the epidermis e.g., mesophyll
  • periderm periderm
  • phloem e.g., periderm
  • phloem e.g., phloem
  • xylem e.g., parenchyma
  • collenchyma collenchyma
  • sclerenchyma sclerenchyma
  • primary and secondary meristematic tissues e.g.,
  • a polynucleotide sequence encoding an NVP can be operably linked to a regulatory region containing a phosphoenolpyruvate carboxylase promoter, resulting in the expression of an NVP in a plant’s mesophyll tissue.
  • Transgenic plants expressing an NVP and/or a polynucleotide operable to express NVP can be generated by any one of the various methods and protocols well known to those having ordinary skill in the art; such methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant be used, only that the nucleotide construct gains access to the interior of at least one cell of the plant.
  • Transgenic plants or “transformed plants” or “stably transformed” plants or cells or tissues refers to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not present in the untransformed plant cell, as well as those that may be endogenous, or present in the untransformed plant cell.
  • Heterologous generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.
  • Transformation of plant cells can be accomplished by one of several techniques known in the art.
  • a construct that expresses an exogenous or heterologous peptide or polypeptide of interest e.g., an NVP
  • the design and organization of such constructs is well known in the art.
  • a gene can be engineered such that the resulting peptide is secreted, or otherwise targeted within the plant cell to a specific region and/or organelle.
  • the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum.
  • a plant expression cassette can be inserted into a plant transformation vector.
  • This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation.
  • DNA vectors needed for achieving plant transformation.
  • Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate fiinctions onto separate DNA molecules.
  • Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the NVP are located between the left and right borders.
  • a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells.
  • This plasmid often contains the virulence fiinctions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).
  • Several types of Agrobacterium strains e.g. LBA4404, GV3101, EHA101, EHA105, etc.
  • the second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
  • plant transformation methods involve transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass.
  • Explants are typically transferred to a fresh supply of the same medium and cultured routinely.
  • the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent.
  • the shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet.
  • the transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g.
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation.
  • Generation of transgenic plants may be performed by one of several methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNAby Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, ballistic particle acceleration, aerosol beam transformation, Led transformation, and various other non-particle direct-mediated methods to transfer DNA.
  • Exemplary transformation protocols are disclosed in U.S. Published Application No. 20010026941; U.S. Pat. No. 4,945,050; International Publication No. WO 91/00915; and U.S. Published Application No. 2002015066, the disclosures of which are incorporated herein by reference in their entireties.
  • Chloroplasts can also be readily transformed, and methods concerning the transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606, the disclosure of which is incorporated herein by reference in its entirety.
  • the method of chloroplast transformation relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination.
  • plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase.
  • tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91 :7301 - 7305.
  • heterologous foreign DNA Following integration of heterologous foreign DNA into plant cells, one having ordinary skill may then apply a maximum threshold level of appropriate selection chemical/reagent (e.g., an antibiotic) in the medium to kill the untransformed cells, and separate and grow the putatively transformed cells that survive from this selection treatment by transferring said surviving cells regularly to a fresh medium.
  • appropriate selection chemical/reagent e.g., an antibiotic
  • an artisan identifies and proliferates the cells that are transformed with the plasmid vector.
  • Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.
  • the cells that have been transformed may be grown into plants in accordance with conventional methods known to those having ordinary skill in the art. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84, the disclosure of which is incorporated herein by reference in its entirety. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present disclosure provides transformed seed (also referred to as “transgenic seed”) having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
  • the present disclosure provides an NVP- antimicrobial protein, that act as substrates for microbe proteinases, proteases and peptidases (collectively referred to herein as “proteases”) as described above.
  • transgenic plants or parts thereof, that may be receptive to the expression of NVPs can include: alfalfa, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, com, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet com, sweet gum, sweet potato, switchgrass, tea
  • the transgenic plant may be grown from cells that were initially transformed with the DNA constructs described herein.
  • the transgenic plant may express the encoded NVP in a specific tissue, or plant part, for example, a leaf, a stem a flower, a sepal, a fruit, a root, a seed, or combinations thereof.
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the NVP has the amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least
  • the plant, plant tissue, plant cell, or plant seed can be transformed with an NVP that comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 4-19 or 66.
  • the plant, plant tissue, plant cell, or plant seed can be transformed with an NVP that comprises, consists essentially of, or consists of, an amino sequence as set forth in any one of SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, and 17.
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the polynucleotide is operable to encode a homopolymer or heteropolymer of two or more NVPs, wherein the amino acid sequence of each NVP is the same or different.
  • the plant, plant tissue, plant cell, plant seed, or part thereof can be transformed with an NVP or a polynucleotide encoding the same, wherein the polynucleotide is operable to encode an NVP that is a fiised protein comprising two or more NVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each NVP may be the same or different.
  • the linker is a cleavable linker.
  • the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 39-51.
  • the linker is cleavable inside at least one of (i) the gut or hemolymph of an invertebrate, and (ii) cleavable inside the gut of a mammal.
  • the plant, plant tissue, plant cell, or plant seed can be transformed with an NVP wherein the NVP has an amino acid sequence of any of the aforementioned NVPs (e.g., one or more the NVPs enumerated in Table 1), or a polynucleotide encoding the same.
  • NVP has an amino acid sequence of any of the aforementioned NVPs (e.g., one or more the NVPs enumerated in Table 1), or a polynucleotide encoding the same.
  • the plant, plant tissue, plant cell, or plant seed can be transformed with an NVP having an amino acid sequence selected from the group consisting of SEQ NOs: 4-19 or 66, or a polynucleotide encoding the same.
  • the plant, plant tissue, plant cell, or plant seed can be transformed with an NVP having an amino acid sequence selected from the group consisting of SEQ NOs: 5, 7, 8, 9, 11, 13, 16, and 17, or a polynucleotide encoding the same.
  • the plant, plant tissue, plant cell, or plant seed can be transformed with an NVP wherein the NVP is a homopolymer or heteropolymer of two or more NVP polypeptides, wherein the amino acid sequence of each NVP is the same or different, or a polynucleotide encoding the same.
  • any of the aforementioned methods, and/or any of the methods described herein, can be used to incorporate one or more of the NVPs or NVP-antimicrobial proteins as described herein, into plants or plant parts thereof.
  • any of the methods described herein can be used to incorporate into plants one or more of the NVPs described in the present disclosure, e.g., NVPs having the amino acid sequence of SEQ ID NOs: 4-19 or 66, which are likewise described herein.
  • a challenge regarding the expression of heterogeneous polypeptides in transgenic plants is maintaining the desired effect (e.g., antimicrobial activity) of the introduced polypeptide upon expression in the host organism; one way to maintain such an effect is to increase the chance of proper protein folding through the use of an operably linked Endoplasmic Reticulum Signal Peptide (ERSP).
  • Another method to maintain the effect of a transgenic protein is to incorporate a Translational Stabilizing Protein (STA).
  • Plants can be transiently or stably transfected with the DNA sequence that encodes an NVP or an NVP-antimicrobial protein comprising one or more NVPs, using any of the transfection methods described above.
  • plants can be transfected with a polynucleotide that encodes an NVP, wherein said NVP is operably linked to a polynucleotide operable to encode an Endoplasmic Reticulum Signal Peptide (ERSP); linker, Translational Stabilizing Protein (STA); or combination thereof.
  • ESP Endoplasmic Reticulum Signal Peptide
  • STA Translational Stabilizing Protein
  • a transgenic plant or plant genome can be transformed with a polynucleotide sequence that encodes the Endoplasmic Reticulum Signal Peptide (ERSP); NVP; and/or intervening linker peptide (LINKER or L), thus causing mRNA transcribed from the heterogeneous DNA to be expressed in the transformed plant, and subsequently, said mRNA to be translated into a peptide.
  • ESP Endoplasmic Reticulum Signal Peptide
  • NVP NVP
  • LINKER or L intervening linker peptide
  • the subcellular targeting of a recombinant protein to the ER can be achieved through the use of an ERSP operably linked to said recombinant protein; this allows for the correct assembly and/or folding of such proteins, and the high level accumulation of these recombinant proteins in plants.
  • Exemplary methods concerning the compartmentalization of host proteins into intracellular storage are disclosed in McCormick et al., Proc. Natl. Acad. Sci. USA 96(2):703-708, 1999; Staub et al., Nature Biotechnology 18:333-338, 2000; Conrad et al., Plant Mol. Biol. 38:101-109, 1998; and Stoger et al., Plant Mol. Biol.
  • an endoplasmic reticulum signal peptide (ERSP)
  • a peptide comprising an Endoplasmic Reticulum Signal Peptide can be operably linked to an NVP (designated as ERSP-NVP), wherein said ERSP is the N-terminal of said peptide.
  • the ERSP peptide is between 3 to 60 amino acids in length, between 5 to 50 amino acids in length, between 20 to 30 amino acids in length.
  • NVP ORF starts with an ersp at its 5 ’-end.
  • NVP ersp nucleotide ftised in frame with the polynucleotide encoding an NVP.
  • translated ERSP can direct the NVP being translated to insert into the Endoplasmic Reticulum (ER) of the plant cell by binding with a cellular component called a signal-recognition particle.
  • the ERSP peptide is cleaved by signal peptidase and the NVP is released into the ER, where the NVP is properly folded during the post-translation modification process, for example, the formation of disulfide bonds. Without any additional retention protein signals, the protein is transported through the ER to the Golgi apparatus, where it is finally secreted outside the plasma membrane and into the apoplastic space. NVP can accumulate at apoplastic space efficiently to reach the antimicrobial dose in plants.
  • the ERSP peptide is at the N-terminal region of the plant -translated NVP complex and the ERSP portion is composed of about 3 to 60 amino acids. In some embodiments it is 5 to 50 amino acids. In some embodiments it is 10 to 40 amino acids but most often is composed of 15 to 20; 20 to 25; or 25 to 30 amino acids.
  • the ERSP is a signal peptide so called because it directs the transportation of a protein. Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals.
  • the signal peptides for ER trafficking are often 15 to 30 amino acid residues in length and have a tripartite organization, comprised of a core of hydrophobic residues flanked by a positively charged amino terminal and a polar, but uncharged carboxyterminal region. (Zimmermann, et al, “Protein translocation across the ER membrane,” Biochimica et Biohysica Acta, 2011, 1808: 912-924).
  • ERSPs are known. It is NOT required that the ERSP be derived from a plant ERSP, non-plant ERSPs will work with the procedures described herein. Many plant ERSPs are however well known and we describe some plant derived ERSPs here.
  • the ERSP can be a barley alpha-amylase signal peptide (BAAS), which is derived from the plant, Hordeum vulgare, and has an amino acid sequence as follows: “MANKHLSLSLFLVLLGLSASLASG” (SEQ ID NO:52) [0500]
  • BAAS barley alpha-amylase signal peptide
  • Plant ERSPs which are selected from the genomic sequence for proteins that are known to be expressed and released into the apoplastic space of plants, include examples such as BAAS, carrot extensin, and tobacco PR1.
  • the following references provide ftirther descriptions, and are incorporated by reference herein in their entirety: De Loose, M. et al.
  • the ERSP can include, but is not limited to, one of the following: a BAAS; a tobacco extensin signal peptide; a modified tobacco extensin signal peptide; or a Jun a 3 signal peptide from Juniperus ashei.
  • a plant can be transformed with a nucleotide that encodes any of the peptides that are described herein as Endoplasmic Reticulum Signal Peptides (ERSP), and an NVP.
  • the tobacco extensin signal peptide motif is another exemplary type of ERSP. See Memelink et al, the Plant Journal, 1993, V4: 1011-1022; Pogue GP et al, Plant Biotechnology Journal, 2010, V8: 638-654, the disclosures of which are incorporated herein by reference in their entireties.
  • an NVP ORF can have a nucleotide sequence operable to encode a tobacco extensin signal peptide motif.
  • the NVP ORF can encode an extensin motif according to SEQ ID NO: 53.
  • the NVP ORF can encode an extensin motif according to SEQ ID NO: 54.
  • a DNA sequence encoding an extensin motif is designed (for example, the DNA sequence shown in SEQ ID NO: 55 or SEQ ID NO:56) using oligo extension PCR with four synthetic DNA primers; ends sites such as a restriction site, for example, a Pac I restriction site at the 5 ’-end, and a 5 ’-end of a GFP sequence at the 3 ’-end, can be added using PCR with the extensin DNA sequence serving as a template, and resulting in a fragment; the fragment is used as the forward PCR primer to amplify the DNA sequence encoding an NVP ORF , for example “gfp-l-nvp” contained in a pFECT vector, thus producing an NVP ORF encoding (from N’ to C’ terminal) “ERSP-GFP-L-NVP” wherein the ERSP is extensin.
  • the resulting DNA sequence can then be cloned
  • an illustrative expression system can include the FECT expression vectors containing NVP ORF is transformed into Agrobacterium, GV3101, and the transformed GV3101 is injected into tobacco leaves for transient expression of NVP ORF.
  • a Translational stabilizing protein can increase the amount of NVP in plant tissues.
  • One of the NVP ORFs i.e., ERSP -NVP, may be sufficient to express a properly folded NVP in the transfected plant; however, in some embodiments, effective protection of a plant from pathogenic microbe damage may require that the plant expressed NVP accumulate.
  • ERSP -NVP a transgenic plant can express and accumulate greater amounts of the correctly folded NVP. When a plant accumulates greater amounts of properly folded NVP, it can more easily resist, inhibit, and/or kill the pathogenic microbes that attack and eat the plants.
  • the translational stabilizing protein can be used to significantly increase the accumulation of NVP in plant tissue, and thus increase the efficacy of a plant transfected with NVP with regard to pathogenic microbe resistance.
  • the translational stabilizing protein is a protein with sufficient tertiary structure that it can accumulate in a cell without being targeted by the cellular process of protein degradation.
  • the translational stabilizing protein can be a domain of another protein, or it can comprise an entire protein sequence.
  • the translational stabilizing protein can be between 5 and 50 amino acids, 50 to 250 amino acids (e.g., GNA), 250 to 750 amino acids (e.g., chitinase) and 750 to 1500 amino acids (e.g., enhancin).
  • One embodiment of the translational stabilizing protein can be a polymer of frision proteins comprising at least one NVP.
  • a specific example of a translational stabilizing protein is provided here to illustrate the use of a translational stabilizing protein. The example is not intended to limit the disclosure or claims in any way.
  • Useftd translational stabilizing proteins are well known in the art, and any proteins of this type could be used as disclosed herein. Procedures for evaluating and testing production of peptides are both known in the art and described herein.
  • One example of one translational stabilizing protein is Green- Fluorescent Protein (GFP) (SEQ ID NO: 57; NCBI Accession No. P42212.1).
  • a protein comprising an Endoplasmic Reticulum Signal Peptide can be operably linked to an NVP, which is in turn operably linked to a Translational Stabilizing Protein (STA).
  • STA Translational Stabilizing Protein
  • this configuration is designated as ERSP-STA- NVP or ERSP-NVP-STA, wherein said ERSP is the N-terminal of said protein and said STA may be either on the N-terminal side (upstream) of the NVP, or of the C -terminal side (downstream) of the NVP.
  • a protein designated as ERSP-STA-NVP or ERSP-NVP-STA comprising any of the ERSPs or NVPs described herein, can be operably linked to a STA, for example, any of the translational stabilizing proteins described, or taught by this document including GFP (Green Fluorescent Protein; SEQ ID NO: 57; NCBI Accession No. P42212), or Jun a 3, (Juniperus ashev, SEQ ID NO:58; NCBI Accession No. P81295.1).
  • GFP Green Fluorescent Protein
  • SEQ ID NO: 57 NCBI Accession No. P42212
  • Jun a 3 Jun a 3
  • an NVP ORF can be transformed into a plant, for example, in the tobacco plant, Nicotiana benthamiana, using an NVP ORF that contains a STA.
  • the STA can be Jun a 3.
  • the mature Jun a 3 is a ⁇ 30 kDa plant defending protein that is also an allergen for some people.
  • Jun a 3 is produced by Juniperus ashei trees and can be used in some embodiments as a translational stabilizing protein (STA).
  • the Jun a 3 amino acid sequence can be the sequence shown in SEQ ID NO:58.
  • the Jun a 3 amino acid sequence can be the sequence shown in SEQ ID NO:59.
  • Linker proteins assist in the proper folding of the different motifs composing an NVP ORF.
  • the NVP ORF described in this invention also incorporates polynucleotide sequences encoding intervening linker peptides between the polynucleotide sequences encoding the NVP (nvp) and the translational stabilizing protein st a), or between polynucleotide sequence encoding multiple polynucleotide sequences encoding NVP, i.e., (I- nvp)N or (nvp-l)N, if the expression ORF involves multiple NVP domain expression.
  • the intervening linker peptides separate the different parts of the expressed NVP construct, and help proper folding of the different parts of the complex during the expression process.
  • different intervening linker peptides can be involved to separate different ftinctional domains.
  • the intervening linker peptide can be between 1 and 30 amino acids in length. However, it is not necessarily an essential component in the expressed NVP in plants.
  • the NVP-antimicrobial protein comprises at least one NVP operably linked to a cleavable peptide. In other embodiments, the NVP-antimicrobial protein comprises at least one NVP operably linked to a non-cleavable peptide.
  • a cleavable linker peptide can be designed to the NVP ORF to release the properly NVP from the expressed NVP complex in the transformed plant to improve the protection the NVP affords the plant with regard to pathogenic microbe damage.
  • One type of the intervening linker peptide is the plant cleavable linker peptide. This type of linker peptides can be completely removed from the expressed NVP ORF complex during plant post-translational modification. Therefore, in some embodiments, the properly folded NVP linked by this type of intervening linker peptides can be released in the plant cells from the expressed NVP ORF complex during post -translational modification in the plant.
  • the NVP ORF can contain a cleavable type of intervening linker, for example, the type listed in SEQ ID NO: 39, having the amino acid code of “IGER” (SEQ ID NO: 39).
  • the molecular weight of this intervening linker or LINKER is 473.53 Daltons.
  • the intervening linker peptide (LINKER) can also be one without any type of protease cleavage site, i.e., an uncleavable intervening linker peptide, for example, the linker “EEKKN” (SEQ ID NO: 40) or “ETMFKHGL” (SEQ ID NO:41).
  • the NVP-antimicrobial protein can have two or more cleavable peptides, wherein the antimicrobial protein comprises an microbe cleavable linker (L), the microbe cleavable linker being fiised in frame with a construct comprising (NVP-L) n , wherein “n” is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10.
  • the NVP-antimicrobial protein comprises an endoplasmic reticulum signal peptide (ERSP) operably linked with an NVP, which is operably linked with an microbe cleavable linker (L) and/or a repeat construct (L-NVP) n or (NVP-L) n , wherein n is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10.
  • SRP endoplasmic reticulum signal peptide
  • L microbe cleavable linker
  • L-NVP repeat construct
  • n is an integer ranging from 1 to 200, or from 1 to 100, or from 1 to 10.
  • a protein comprising an Endoplasmic Reticulum Signal Peptide can be operably linked to an NVP and an intervening linker peptide (L or Linker); such a construct is designated as ERSP-L-NVP, or ERSP-NVP-L, wherein said ERSP is the N-terminal of said protein, and said L or Linker may be either on the N-terminal side (upstream) of the NVP, or the C-terminal side (downstream) of the NVP.
  • L or Linker may be either on the N-terminal side (upstream) of the NVP, or the C-terminal side (downstream) of the NVP.
  • a protein designated as ERSP-L-NVP, or ERSP-NVP-L, comprising any of the ERSPs or NVPs described herein, can have a Linker “L” that can be an uncleavable linker peptide, or a cleavable linker peptide, and which may be cleavable in a plant cells during protein expression process.
  • an NVP-antimicrobial protein can comprise any of the intervening linker peptides (LINKER or L) described herein, or taught by this document, including but not limited to following sequences: IGER (SEQ ID NO: 39), EEKKN, (SEQ ID NO:40), and ETMFKHGL (SEQ ID NO:41), or combinations thereof.
  • the linker can be one or more of the following: ALKFLV (SEQ ID NO: 42), ALKLFV (SEQ ID NO: 43), IFVRLR (SEQ ID NO: 44), LFAAPF (SEQ ID NO: 45), ALKFLVGS (SEQ ID NO: 46), ALKLFVGS (SEQ ID NO: 47), IFVRLRGS (SEQ ID NO: 48), LFAAPFGS (SEQ ID NO: 49), LFVRLRGS (SEQ ID NO: 50), and/or LGERGS (SEQ ID NO: 51).
  • an exemplary antimicrobial protein can include a protein construct comprising: (ERSP)-(NVP-L) n ; (ERSP)-(L)-(NVP-L) n ; (ERSP)-(L-NVP) n ; (ERSP)-(L-NVP) n -(L); wherein n is an integer ranging from 1 to 200 or from 1 to 100, or from 1 to 10.
  • an NVP is any NVP described herein, L is a non-cleavable or cleavable peptide, and n is an integer ranging from 1 to 200, preferably an integer ranging from 1 to 100, and more preferably an integer ranging from 1 to 10.
  • the NVP-antimicrobial protein may contain NVP peptides that are the same or different, and microbe cleavable peptides that are the same or different.
  • the C-terminus NVP is operably linked at its C-terminus with a cleavable peptide.
  • the N-terminus NVP is operably linked at its N-terminus with a cleavable peptide.
  • linker peptides can be found in the following references, which are incorporated by reference herein in their entirety: a plant expressed serine proteinase inhibitor precursor was found to contain five homogeneous protein inhibitors separated by six same linker peptides, as disclosed in Heath et al. “Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata” European Journal of Biochemistry, 1995; 230: 250-257. A comparison of the folding behavior of green fluorescent proteins through six different linkers is explored in Chang, H.C. et al.
  • TMV tobacco mosaic virus
  • TMOF trypsin-modulating oostatic factor
  • NVP ORF refers to a nucleotide encoding an NVP, and/or one or more stabilizing proteins, secretory signals, or target directing signals, for example, ERSP or ST A, and is defined as the nucleotides in the ORF that has the ability to be translated.
  • a “NVP ORF diagram” refers to the composition of one or more NVP ORFs, as written out in diagram or equation form.
  • a “NVP ORF diagram” can be written out as using acronyms or short -hand references to the DNA segments contained within the expression ORF.
  • a “NVP ORF diagram” may describe the polynucleotide segments encoding the ERSP, LINKER, STA, and NVP, by diagramming in equation form the DNA segments as “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide); “linked or “Z” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and “nvp” (i.e., the polynucleotide sequence encoding an NVP), respectively.
  • An example of an NVP ORF diagram is “ersp-sta-(linker-nvp ) ⁇ ,” or “ersp-(nvpj-linkeri)N-sta” and/or any combination of the DNA segments thereof.
  • the NVP open reading frame (ORF) described herein is a polynucleotide sequence that will enable the plant to express mRNA, which in turn will be translated into peptides that will folded properly, and/or accumulated to such an extent that said proteins provide a dose sufficient to inhibit and/or kill one or more pathogenic microbes.
  • an example of a protein NVP ORF can be any NVP described herein nvp), an “ersp” (i.e., the polynucleotide sequence that encodes the ERSP polypeptide) a “linked (i.e., the polynucleotide sequence that encodes the LINKER polypeptide), a “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), or any combination thereof, and can be described in the following equation format: ersp-sta-(linkeri-nvp ⁇ n, or ersp-(nvpj-linkeri) n -sta
  • ERSP-STA-(LINKERI-NVPJ)N containing four possible peptide components with dash signs to separate each component.
  • the nucleotide component of ersp is a polynucleotide segment encoding a plant endoplasmic reticulum trafficking signal peptide (ERSP).
  • the component of sta is a polynucleotide segment encoding a translation stabilizing protein (STA), which helps the accumulation of the NVP expressed in plants, however, in some embodiments, the inclusion of sta may not be necessary in the NVP ORF.
  • STA translation stabilizing protein
  • the component of linken is a polynucleotide segment encoding an intervening linker peptide (L OR LINKER) to separate the NVP from other components contained in ORF, and from the translation stabilizing protein.
  • the subscript letter “i” indicates that in some embodiments, different types of linker peptides can be used in the NVP ORF.
  • the component “nvp” indicates the polynucleotide segment encoding the NVP.
  • the subscript “j” indicates different polynucleotides may be included in the NVP ORF.
  • the polynucleotide sequence can encode an NVP with a different amino acid substitution.
  • n indicates that the structure of the nucleotide encoding an intervening linker peptide and an NVP can be repeated “n” times in the same open reading frame in the same NVP ORF , where “n” can be any integrate number from 1 to 10; “n” can be from 1 to 10, specifically “n” can be 1, 2, 3, 4, or 5, and in some embodiments “n” is 6, 7, 8, 9 or 10.
  • the repeats may contain polynucleotide segments encoding different intervening linkers (LINKER) and different NVPs. The different polynucleotide segments including the repeats within the same NVP ORF are all within the same translation frame.
  • the inclusion of a sta polynucleotide in the NVP ORF may not be required.
  • an ersp polynucleotide sequence can be directly be linked to the polynucleotide encoding an NVP variant polynucleotide without a linker.
  • the polynucleotide “nvp” encoding the polypeptide “NVP” can be the polynucleotide sequence that encodes any NVP as described herein, e.g., an NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least
  • NVP comprises an amino acid addition to the N-terminus of a WT-NCR13 amino acid sequence as set forth in SEQ ID NO:1, and wherein Xi is any natural or non-natural amino acid other than glycine or alanine; or a complementary nucleotide sequence thereof.
  • the nvp polynucleotide, or polynucleotide operable to encode an NVP, or complementary nucleotide sequence thereof can encode an NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid
  • the nvp polynucleotide, or polynucleotide operable to encode an NVP, or complementary nucleotide sequence thereof can encode an NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid
  • a polynucleotide is operable to encode an NVP- antimicrobial protein having the following NVP construct orientation and/or arrangement: ERSP-NVP; ERSP-(NVP) N ; ERSP-NVP-L; ERSP-(NVP)N-L; ERSP-(NVP-L) N ; ERSP-L- NVP; ERSP-L-(NVP) N ; ERSP-(L-NVP) N ; ERSP-STA-NVP; ERSP-STA-(NVP) N ; ERSP- NVP-STA; ERSP-(NVP)N-STA; ERSP-(STA-NVP) N ; ERSP-(NVP-STA) N ; ERSP-(NVP-STA) N ; ERSP-(NVP-STA) N ; ERSP-(NVP-STA) N ; ERSP-(NVP-STA) N ; ERS
  • any of the aforementioned methods, and/or any of the methods described herein, can be used to incorporate into a plant or a plant part thereof, one or more polynucleotides operable to express any one or more of the NVPs or NVP -antimicrobial proteins as described herein; e.g., one or more NVPs or NVP -antimicrobial protein having the amino acid sequence of SEQ ID NOs: 4-19, or 66, which are likewise described herein.
  • the present disclosure may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
  • Crops for which a transgenic approach or PEP would be an especially usefiil approach include, but are not limited to: alfalfa, cotton, tomato, maize, wheat, com, sweet com, lucerne, soybean, sorghum, field pea, linseed, safflower, rapeseed, oil seed rape, rice, soybean, barley, sunflower, trees (including coniferous and deciduous), flowers (including those grown commercially and in greenhouses), field lupins, switchgrass, sugarcane, potatoes, tomatoes, tobacco, crucifers, peppers, sugarbeet, barley, and oilseed rape, Brassica sp., rye, millet, peanuts, sweet potato, cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • the NVP ORFs and NVP constructs described above and herein can be cloned into any plant expression vector for NVP to be expressed in plants, either transiently or stably.
  • Transient plant expression systems can be used to promptly optimize the structure of the NVP ORF for some specific NVP expression in plants, including the necessity of some components, codon optimization of some components, optimization of the order of each component, etc.
  • a transient plant expression vector is often derived from a plant virus genome. Plant virus vectors provide advantages in quick and high level of foreign gene expression in plant due to the infection nature of plant viruses. The full length of the plant viral genome can be used as a vector, but often a viral component is deleted, for example the coat protein, and transgenic ORFs are subcloned in that place. The NVP ORF can be subcloned into such a site to create a viral vector.
  • viral vectors can be introduced into plant mechanically since they are infectious themselves, for example through plant wound, spray-on etc. They can also be transfected into plants via agroinfection, by cloning the virus vector into the T-DNA of the crown gall bacterium, Agrobacterium tumefaciens, or the hairy root bacterium, Agrobacterium rhizogenes.
  • the expression of the NVP in this vector is controlled by the replication of the RNA virus, and the virus translation to mRNA for replication is controlled by a strong viral promoter, for example, 35S promoter from Cauliflower mosaic virus.
  • Viral vectors with NVP ORF are usually cloned into T-DNA region in a binary vector that can replicate itself in both E.
  • the transient transfection of a plant can be done by infiltration of the plant leaves with the Agrobacterium cells which contain the viral vector for NVP expression.
  • the foreign protein expression In the transient transformed plant, it is common for the foreign protein expression to be ceased in a short period of time due to the post -transcriptional gene silencing (PTGS).
  • PTGS post -transcriptional gene silencing
  • Sometimes a PTGS suppressing protein gene is necessary to be co -transformed into the plant transiently with the same type of viral vector that drives the expression of with the NVP ORF. This improves and extends the expression of the NVP in the plant.
  • the most commonly used PTGS suppressing protein is P19 protein discovered from tomato bushy stunt virus (TBSV).
  • transient transfection of plants can be achieved by recombining a polynucleotide encoding a NVP with any one of the readily available vectors (see above and described herein), and confirmed, using a marker or signal (e.g., GFP emission).
  • a transiently transfected plant can be created by recombining a polynucleotide encoding a NVP with a DNA encoding a GFP -Hybrid fusion protein in a vector, and transfection said vector into a plant (e.g., tobacco) using different FECT vectors designed for targeted expression.
  • a polynucleotide encoding a NVP can be recombined with a pFECT vector for APO (apoplast localization) accumulation; a pFECT vector for CYTO (cytoplasm localization) accumulation; or pFECT with ersp vector for ER (endoplasm reticulum localization) accumulation.
  • APO apoplast localization
  • CYTO cytoplasm localization
  • ER endoplasm reticulum localization
  • An exemplary transient plant transformation strategy is agroinfection using a plant viral vector due to its high efficiency, ease, and low cost.
  • a tobacco mosaic virus overexpression system can be used to transiently transform plants with NVP. See TRBO, Lindbo JA, Plant Physiology, 2007, V145: 1232-1240, the disclosure of which is incorporated herein by reference in its entirety.
  • the TRBO DNA vector has a T-DNA region for agroinfection, which contains a CaMV 35S promoter that drives expression of the tobacco mosaic virus RNA without the gene encoding the viral coating protein. Moreover, this system uses the “disarmed” virus genome, therefore viral plant to plant transmission can be effectively prevented.
  • the FECT expression system additionally needs to co-express Pl 9, a RNA silencing suppressor protein from tomato bushy stunt virus, to prevent the post-transcriptional gene silencing (PTGS) of the introduced T-DNA (the TRBO expression system does not need co-expression of P19).
  • Pl9 a RNA silencing suppressor protein from tomato bushy stunt virus
  • the NVP ORF can be cloned into the Pac I and Avr II restriction sites of a FECT expression vector (pFECT) to create an NVP expression vector for the FECT transient plant expression system (pFECT - NVP).
  • pFECT FECT expression vector
  • some embodiments may have a FECT vector expressing the RNA silencing suppressor protein Pl 9 (pFECT -Pl 9) generated for co-transformation.
  • a vector can be recombined for use in a TRBO transient plant expression system, for example, by performing a routine PCR procedure and adding a Not I restriction site to the 3 ’-end of the NVP ORF described above, and then cloning the NVP ORF into Pac I and Not I restriction sites of the TRBO expression vector (pTRBO-NVP).
  • an Agrobacterium tumefaciens strain for example, commercially available GV3101 cells, can be used for the transient expression of a NVP ORF in a plant tissue (e.g., tobacco leaves) using one or more transient expression systems, for example, the FECT and TRBO expression systems.
  • An exemplary illustration of such a transient transfection protocol includes the following: an overnight culture of GV3101 can be used to inoculate 200 mL Luria-Bertani (LB) medium; the cells can be allowed to grow to log phase with OD600 between 0.5 and 0.8; the cells can then be pelleted by centrifiigation at 5000 rpm for 10 minutes at 4°C; cells can then be washed once with 10 mL prechilled TE buffer (Tris-HCl 10 mM, EDTA ImM, pH8.0), and then resuspended into 20 mL LB medium; GV3101 cell resuspension can then be aliquoted in 250 pL fractions into 1.5 mL microtubes; aliquots can then be snap-frozen in liquid nitrogen and stored at -80°C freezer for fiiture transformation.
  • LB Luria-Bertani
  • the pFECT-NVP and pTRBO-NVP vectors can then transformed into the competent GV3101 cells using a freeze-thaw method as follows: the stored competent GV3101 cells are thawed on ice and mixed with 1 to 5 pg pure DNA (pFECT-NVP or pTRBO-NVP vector). The cell-DNA mixture is kept on ice for 5 minutes, transferred to - 80°C for 5 minutes, and incubated in a 37°C water bath for 5 minutes. The freeze-thaw treated cells are then diluted into 1 mL LB medium and shaken on a rocking table for 2 to 4 hours at room temperature.
  • a 200 pL aliquot of the cell-DNA mixture is then spread onto LB agar plates with the appropriate antibiotics (10 pg/mL rifampicin, 25 pg/mL gentamycin, and 50 pg/mL kanamycin can be used for both pFECT-NVP transformation and pTRBO-NVP transformation) and incubated at 28°C for two days. Resulting transformed colonies are then picked and cultured in 6 mL aliquots of LB medium with the appropriate antibiotics for transformed DNA analysis and making glycerol stocks of the transformed GV3101 cells.
  • the appropriate antibiotics 10 pg/mL rifampicin, 25 pg/mL gentamycin, and 50 pg/mL kanamycin can be used for both pFECT-NVP transformation and pTRBO-NVP transformation
  • the transient transformation of plant tissues can be performed using leaf injection with a 3-mL syringe without needle.
  • the transformed GV3101 cells are streaked onto an LB plate with the appropriate antibiotics (as described above) and incubated at 28°C for two days.
  • a colony of transformed GV3101 cells are inoculated to 5 ml of LB-MESA medium (LB media supplemented with 10 mM MES, and 20 pM aceto syr ingone) and the same antibiotics described above, and grown overnight at 28°C.
  • the cells of the overnight culture are collected by centriftigation at 5000 rpm for 10 minutes and resuspended in the induction medium (10 mM MES, 10 mM MgCh, 100 pM acetosyringone) at a final OD600 of 1.0.
  • the cells are then incubated in the induction medium for 2 hours to overnight at room temperature and are then ready for transient transformation of tobacco leaves.
  • the treated cells can be infiltrated into the underside of attached leaves of Nicotiana benthamiana plants by injection, using a 3-mL syringe without a needle attached.
  • NVP ORF can also be integrated into plant genome using stable plant transformation technology, and therefore NVPs can be stably expressed in plants and protect the transformed plants from generation to generation.
  • the NVP expression vector can be circular or linear.
  • the NVP ORF, the NVP expression cassette, and/or the vector with polynucleotide encoding an NVP for stable plant transformation should be carefiilly designed for optimal expression in plants based on what is known to those having ordinary skill in the art, and/or by using predictive vector design tools such as Gene Designer 2.0 (Atum Bio); VectorBuilder (Cyagen); SnapGene® viewer; GeneArtTM Plasmid Construction Service (Thermo -Fisher Scientific); and/or other commercially available plasmid design services. See Tolmachov, Designing plasmid vectors. Methods Mol Biol. 2009; 542:117-29.
  • the expression of NVP is usually controlled by a promoter that promotes transcription in some, or all the cells of the transgenic plant.
  • the constructed NVP expression vector can be transfected into plant cells or tissues using many transfection technologies.
  • Agroinfection is a very popular way to transform a plant using an Agrobacterium tumefaciens strain or an Agrobacterium rhizogenes strain.
  • Particle bombardment also called Gene Gun, or Biolistics
  • Other less common transfection methods include tissue electroporation, silicon carbide whiskers, direct injection of DNA, etc.
  • RNA level and protein level A stably transformed plant can be evaluated at all of these levels and a transiently transformed plant is usually only evaluated at protein level.
  • the genomic DNA can be extracted from the stably transformed plant tissues for and analyzed using PCR or Southern blot.
  • the expression of the NVP in the stably transformed plant can be evaluated at the RNA level, for example, by analyzing total mRNA extracted from the transformed plant tissues using northern blot or RT-PCR.
  • the expression of the NVP in the transformed plant can also be evaluated in protein level directly. There are many ways to evaluate expression of NVP in a transformed plant.
  • total protein can be extracted from the transformed plant tissues for the direct evaluation of the expression of the NVP using a Bradford assay to evaluate the total protein level in the sample.
  • analytical HPLC chromatography technology Western blot technique, or iELISA assay can be adopted to qualitatively or quantitatively evaluate the NVP in the extracted total protein sample from the transformed plant tissues.
  • NVP expression can also be evaluated by using the extracted total protein sample from the transformed plant tissues in an microbe bioassay, for example, in some embodiments, the transformed plant tissue or the whole transformed plant itself can be used in microbe bioassays to evaluate NVP expression and its ability to provide protection for the plant.
  • a plant, plant tissue, plant cell, plant seed, or part thereof of the present disclosure can comprise one or more NVPs, or a polynucleotide encoding the same, said NVP comprising an amino acid sequence that is at least [0554] Confirming successful transformation
  • heterologous foreign DNA Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.
  • PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.
  • Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformed plant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, radiolabeled 32 P target DNA fragment to confirm the integration of introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).
  • RNA is isolated from specific tissues of transformed plant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by the polynucleotide encoding a NVP is then tested by hybridizing the filter to a radioactive probe derived from a NVP, by methods known in the art (Sambrook and Russell, 2001, supra).
  • a number of markers have been developed to determine the success of plant transformation, for example, resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like.
  • Other genes that encode a product involved in chloroplast metabolism may also be used as selectable markers.
  • genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone may find particular use.
  • Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310- 6314 (bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res.
  • genes disclosed herein are useful as markers to assess transformation of bacterial, yeast, or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell, propagule, embryo or progeny of the same are well known in the art. In one embodiment, the presence of the transgene is detected by testing for pathogenic microbe activity.
  • Fertile plants expressing a NVP and/or a polynucleotide encoding the same may be tested for pathogenic microbe activity, and the plants showing optimal activity selected for fiirther breeding. Methods are available in the art to assay for pathogenic microbe activity. Generally, the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology 78:290-293.
  • evaluating the success of a transient transfection procedure can be determined based on the expression of a reporter gene, for example, GFP.
  • GFP can be detected under UV light in tobacco leaves transformed with the FECT and/or TRBO vectors.
  • NVP expression can be quantitatively evaluated in a plant (e.g., tobacco).
  • a plant e.g., tobacco
  • An exemplary procedure that illustrates NVP quantification in a tobacco plant is as follows: 100 mg disks of transformed leaf tissue is collected by punching leaves with the large opening of a 1000 pL pipette tip. The collected leaf tissue is place into a 2 mL microtube with 5/32” diameter stainless steel grinding balls, and frozen in -80°C for 1 hour, and then homogenized using a Troemner-Talboys High Throughput Homogenizer.
  • TSP-SEI extraction solutions sodium phosphate solution 50 mM, 1:100 diluted protease inhibitor cocktail, EDTA ImM, DIECA lOmM, PVPP 8%, pH 7.0
  • the microtube is then left still at room temperature for 15 minutes and then centrifiiged at 16,000 g for 15 minutes at 4°C; 100 pL of the resulting supernatant is taken and loaded into pre-Sephadex G-50-packed column in 0.45 pm Millipore MultiScreen filter microtiter plate with empty receiving Costar microtiter plate on bottom.
  • the microtiter plates are then centrifiiged at 800 g for 2 minutes at 4°C.
  • the resulting filtrate solution herein called total soluble protein extract (TSP extract) of the tobacco leaves, is then ready for the quantitative analysis.
  • TSP extract total soluble protein extract
  • the total soluble protein concentration of the TSP extract can be estimated using Pierce Coomassie Plus protein assay.
  • BSA protein standards with known concentrations can be used to generate a protein quantification standard curve.
  • 2 pL of each TSP extract can be mixed into 200 pL of the chromogenic reagent (CPPA reagent) of the Coomassie Plus protein assay kits and incubated for 10 minutes.
  • the chromogenic reaction can then be evaluated by reading OD595 using a SpectroMax-M2 plate reader using SoftMax Pro as control software.
  • the concentrations of total soluble proteins can be about 0.788 ⁇ 0.20 pg/pL or about 0.533 ⁇ 0.03 pg/pL in the TSP extract from plants transformed via FECT and TRBO, respectively, and the results can be used to calculate the percentage of the expressed NVP in the TSP (%TSP) for the iELISA assay
  • an indirect ELISA (iELISA) assay can be used to quantitatively evaluate the NVP content in the tobacco leaves transiently transformed with the FECT and/or TRBO expression systems.
  • An illustrative example of using iELISA to quantify NVP is as follows: 5 pL of the leaf TSP extract is diluted with 95 pL of CB2 solution (Immunochemistry Technologies) in the well of an Immulon 2HD 96-well plate, with serial dilutions performed as necessary; leaf proteins obtained from extract samples are then allowed to coat the well walls for 3 hours in the dark, at room temperature, and the CB2 solution is then subsequently removed; each well is washed twice with 200 pL PBS (Gibco); 150 pL blocking solution (Block BSA in PBS with 5% non-fat dry milk) is added into each well and incubated for 1 hour, in the dark, at room temperature; after the removal of the blocking solution, a PBS wash of the wells, 100 pL of
  • the expressed NVP can be detected by iELISA at about 3.09 ⁇ 1.83 ng/pL in the leaf TSP extracts from the FECT transformed tobacco; and about 3.56 ⁇ 0.74 ng/pL in the leaf TSP extract from the TRBO transformed tobacco.
  • the expressed NVP can be about 0.40% total soluble protein (%TSP) for FECT transformed plants and about 0.67% TSP in TRBO transformed plants.
  • N or “% v/v” or “volume per volume” refers to the volume concentration of a solution (“v/v” stands for volume per volume).
  • v/v can be used when both components of a solution are liquids.
  • w/w or “% w/w” or “weight per weight” refers to the weight concentration of a solution, i.e., percent weight in weight (“w/w” stands for weight per weight).
  • w/w expresses the number of grams (g) of a constituent in 100 g of solution or mixture.
  • a mixture consisting of 30 g of ingredient X, and 70 g of water would be expressed as “ingredient X 30% w/w.”
  • Percent weight per weight (% w/w) is calculated as follows: (weight of solute (g)/ weight of solution (g)) x 100; or (mass of solute (g)/ mass of solution (g)) x 100.
  • /N or “% w/v” or “weight per volume” refers to the mass concentration of a solution, i.e., percent weight in volume (“w/v” stands for weight per volume).
  • w/v expresses the number of grams (g) of a constituent in 100 mL of solution. For example, if 1 g of ingredient X is used to make up a total volume of 100 mL, then a “1% w/v solution of ingredient X” has been made. Percent weight per volume (% w/v) is calculated as follows: (Mass of solute (g)/ Volume of solution (mL)) x 100.
  • NVPs or NVP-antimicrobial proteins described herein e.g., an NVP having an amino acid sequence as set forth in SEQ ID NOs: 4-19, or 66, or an agriculturally acceptable salt thereof can be used to create a mixture and/or composition, wherein said mixture and/or composition consists of at least one NVP.
  • the present disclosure comprises, consists essentially of, or consists of, a combination, a mixture, or a composition comprising, consisting essentially of, or consisting of, one or more NVPs, one or more NVP-antimicrobial proteins, and/or combinations thereof.
  • the invention contemplates a mixture of one or more NVPs, one or more NVP-antimicrobial proteins, and/or combinations thereof.
  • one or more NVPs, one or more NVP-antimicrobial proteins, and/or combinations thereof can be blended together in in varying proportions.
  • the invention contemplates a combination of one or more NVPs, one or more NVP-antimicrobial proteins, and/or combinations thereof.
  • one or more NVPs, one or more NVP-antimicrobial proteins, and/or combinations thereof can be provided as a combination, e.g., in the same container, or in different containers.
  • the invention contemplates a composition of one or more NVPs, one or more NVP-antimicrobial proteins, and/or combinations thereof.
  • one or more NVPs, one or more NVP-antimicrobial proteins, and/or combinations thereof can be provided as a composition fiirther comprising an excipient.
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NCR13 variant peptide (NVP) having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence having an N-
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence having an N-
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence having an N-
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence having an N-
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence having an N-
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence according to Formula (
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence according to Formula (
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence according to Formula (
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence according to Formula (
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having an amino sequence as set forth in any one of SEQ ID NOs: 4-19, or 66, or an agriculturally acceptable salt thereof.
  • the combination, mixture, or composition comprises, consists essentially of, or consists of, an NVP having an amino sequence as set forth in any one of SEQ ID NOs: 5, 7, 8, 9, 11, 13, 16, and 17, or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • VTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 4), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • MTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 7), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • FKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 8), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • PTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 10), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical,
  • TKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 12), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • YTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 13), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • NKTPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 14), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • EKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 17), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • KTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 18), or an agriculturally acceptable salt thereof.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP having antimicrobial activity against one or more microbe species, said NVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP, wherein said NVP homopolymer or heteropolymer of two or more NVPs, wherein the amino acid sequence of each NVP is the same or different.
  • a combination, mixture, or composition of the present disclosure can comprise, consist essentially of, or consist of, an NVP that is a fiised protein comprising two or more NVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each NVP may be the same or different.
  • the antimicrobial compositions described herein may be made by formulating either the NVP, NVP-antimicrobial protein, or agriculturally acceptable salt thereof, with the desired agriculturally-acceptable carrier.
  • the compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline and/or other buffer.
  • the formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application.
  • a composition can comprise, consist essentially of, or consist of, an NVP-antimicrobial protein and an excipient.
  • a composition of the present disclosure can comprise: an NVP, an NVP-antimicrobial protein, or an agriculturally acceptable salt thereof, and an excipient; wherein the NVP, NVP-antimicrobial protein, or agriculturally acceptable salt thereof is in an amount ranging from about 0.000001% w/w to about 99.99999% w/w of the total composition, or from about 0.01% to about 99.99%; from about 0.02% to about 99.98%; from about 0.03% to about 99.97%; from about 0.04% to about 99.96%; from about 0.05% to about 99.95; from about 0.06% to about 99.94%; from about 0.07% to about 99.93%; from about 0.08% to about 99.92%; from about 0.09% to about 99.91%; from about 1% to about 99%; from about 2% to about 98%; from about 3% to about 97%; from about 4% to about 96%; from about 5% to about 95%; from about 6% to about 95%; from about 6% to about
  • NVP, an NVP-antimicrobial protein, or an agriculturally acceptable salt thereof is in an amount of about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
  • compositions of the present disclosure comprising an NVP, an NVP-antimicrobial protein, or an agriculturally acceptable salt thereof, and an excipient, may be made available in a spray product as an aerosol-based application, including aerosolized foam applications. Pressurized cans are the typical vehicle for the formation of aerosols.
  • a liquefied-gas type propellant is used.
  • an effective amount of an NVP, an NVP-antimicrobial protein, or an agriculturally acceptable salt thereof can depend on the specific use pattern, the microbe against which control is most desired and the environment in which the NVP or NVP- antimicrobial protein will be used. Therefore, an effective amount of an NVP, an NVP- antimicrobial protein, or an agriculturally acceptable salt thereof is sufficient that control of a microbe is achieved.
  • a fabric treatment may contain an amount of an NVP, or an agriculturally acceptable salt thereof, ranging from about 0.005 wt% to about 99 wt%.
  • a fabric treatment may contain an amount of an NVP- antimicrobial protein, or an agriculturally acceptable salt thereof, ranging from about 0.005 wt% to about 99 wt%.
  • compositions or formulations comprising an NVP and an excipient, or comprising an NVP -antimicrobial protein and an excipient, for coating walls, floors and ceilings inside of buildings, and for coating a substrate or non-living material.
  • inventive compositions comprising an NVP, an NVP-antimicrobial protein, or an agriculturally acceptable salt thereof, and an excipient, can be prepared using known techniques for the purpose in mind. Preparations of compositions comprising an NVP-antimicrobial protein and an excipient, could be so formulated to also contain a binder to facilitate the binding of the compound to the surface or other substrate. Agents useftd for binding are known in the art and tend to be polymeric in form.
  • Typical binders are poly vinyl alcohol, modified starch, poly vinyl acrylate, polyacrylic, polyvinyl acetate co polymer, polyurethane, and modified vegetable oils.
  • Suitable binders can include latex dispersions derived from a wide variety of polymers and co-polymers and combinations thereof.
  • a surface-treatment composition may contain an amount of an NVP, or an agriculturally acceptable salt thereof, ranging from about 0.005 wt% to about 99 wt%.
  • a surface-treatment may contain an amount of an NVP- antimicrobial protein, or an agriculturally acceptable salt thereof, ranging from about 0.005 wt% to about 99 wt%.
  • an antimicrobial formulation according to the present disclosure may consist of an NVP, an NVP-antimicrobial protein, or an agriculturally acceptable salt thereof, and an excipient, diluent or carrier (e.g., such as water), a polymeric binder, and/or additional components such as a dispersing agent, a polymerizing agent, an emulsifying agent, a thickener, an alcohol, a fragrance, or any other inert excipients used in the preparation of sprayable compositions known in the art.
  • an excipient e.g., such as water
  • a polymeric binder e.g., such as water
  • additional components such as a dispersing agent, a polymerizing agent, an emulsifying agent, a thickener, an alcohol, a fragrance, or any other inert excipients used in the preparation of sprayable compositions known in the art.
  • a dispersant may contain an amount of an NVP- antimicrobial protein, or an agriculturally acceptable salt thereof, ranging from about 0.005 wt% to about 99 wt%.
  • a composition comprising an NVP, an NVP- antimicrobial protein, or an agriculturally acceptable salt thereof, and an excipient, can be prepared in a number of different forms or formulation types, such as suspensions or capsules suspensions. And a person skilled in the art can prepare the relevant composition based on the properties of the particular NVP or NVP-antimicrobial protein, its uses, and also its application type.
  • the NVP or NVP-antimicrobial protein used in the methods, embodiments, and other aspects of the present disclosure may be encapsulated in a suspension or capsule suspension formulation.
  • An encapsulated NVP or NVP-antimicrobial protein can provide improved wash- fastness, and also a longer period of activity.
  • the formulation can be organic based or aqueous based, preferably aqueous based.
  • a suspension may contain an amount of an NVP, or an agriculturally acceptable salt thereof, ranging from about 0.005 wt% to about 99 wt%.
  • microcapsules are formed by emulsifying or dispersing the core material in an immiscible continuous phase and then an interfacial polymerization reaction is caused to take place at the surface of the core particles.
  • concentration of the NVP or NVP-antimicrobial protein present in the microcapsules can vary from 0.1 to 60% by weight of the microcapsule.
  • a microencapsulation may contain an amount of an NVP-antimicrobial protein, or an agriculturally acceptable salt thereof, ranging from about 0.005 wt% to about 99 wt%.

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Abstract

La présente divulgation concerne des peptides variants NCR13 antimicrobiens non naturels modifiés (NVP), ou des sels acceptables en agriculture de ceux-ci, ainsi que leurs procédés de fabrication et d'utilisation. La présente divulgation concerne également de nouvelles compositions antimicrobiennes, des formulations et leurs procédés d'utilisation, qui sont utiles pour la lutte contre des microbes pathogènes.
PCT/US2023/068490 2022-06-17 2023-06-15 Peptides variants de ncr13 antimicrobiens WO2023245100A1 (fr)

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