WO2024166076A1 - Production recombinée de peptides antimicrobiens in planta - Google Patents

Production recombinée de peptides antimicrobiens in planta Download PDF

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WO2024166076A1
WO2024166076A1 PCT/IB2024/051292 IB2024051292W WO2024166076A1 WO 2024166076 A1 WO2024166076 A1 WO 2024166076A1 IB 2024051292 W IB2024051292 W IB 2024051292W WO 2024166076 A1 WO2024166076 A1 WO 2024166076A1
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plant
amps
seq
pam
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Magdy Mahmoud MAHFOUZ
Mohammed Shahid CHAUDHARY
Zahir ALI
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King Abdullah University Of Science And Technology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/17—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced ascorbate as one donor, and incorporation of one atom of oxygen (1.14.17)
    • C12Y114/17003—Peptidylglycine monooxygenase (1.14.17.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag

Definitions

  • This invention is generally in the field of antimicrobial peptide production in planta.
  • AMPs Antimicrobial peptides
  • HDPs host defense peptides
  • AMPs evolved as part of the immune systems in many species and kill bacterial cells (including drug-resistant strains) 1 by interacting with their membranes followed by multimodal mechanisms that can include membrane perturbation, inhibition of cell wall synthesis and inhibition of internal targets including synthesis of macromolecules, 2,3 acting more rapidly than classical antibiotics, 4 and limiting the evolution of drug resistance.
  • 5,6 AMPs can also exhibit potent activity against bacterial biofilms, independent of AMP activity, 7 and diverse immunomodulatory effects. 8
  • the pervasive collateral sensitivity of AMPs towards drugresistant bacterial strains, 9 and their marked functional synergism with current antibiotics 10 underscores their potential use as effective therapeutic drugs.
  • the development of clinically translated AMPs has only recently begun to accelerate.
  • AMPs are particularly challenging and costly to manufacture synthetically, slowing down their clinical translation.
  • Conventional AMP manufacturing relies on solid-phase peptide synthesis (SPPS) with a cost between $100 and $600 per gram, 2 although certain efficiencies can be gained by optimizing large-scale synthesis that help bring down costs.
  • SPPS solid-phase peptide synthesis
  • solid-phase peptide synthesis suffers from the prohibitive limitation of peptide length, which should be no more than 50 amino acids, 14 the presence of hydrophobic peptides that tend to aggregate in the solvents used for synthesis, 15 and the need to use hazardous chemicals and solvents throughout the peptide synthesis and purification procedures. 16
  • Synthetic biology offers the promise of sustainable, scalable, and cost-effective production of AMPs, based on genetically engineered organisms.
  • AMPs can be produced in bacterial or yeast cells and purified to homogeneity, the use of plants as a production host for complex biologies is deemed safer, demands less infrastructure, and has the potential for rapid scaling-up of production.
  • producing proteins in plants is estimated to cost 10- to 50-times less than E. coli fermentation.
  • 21 In planta production of peptides has proven difficult, presumably due to proteolysis by plant proteases.
  • various strategies have been deployed, such as the downregulation of genes encoding interfering plant proteases 22 or restricting AMP production to a specific organelle. 23 Despite these strategies, the typical yields from plant-produced peptides have generally been low. 24 ’ 25
  • compositions and methods for the controlled in planta production of amidated AMPs are disclosed.
  • the disclosed methods use a targeted combination of (a) stable and (b) transient expression modules in transgenic plants.
  • the bifunctional peptidylglycine a-amidating monooxygenase (PAM) enzyme preferably from rats (Rattus norvegicus) is used to introduce the mammalian C-terminal amidation pathway into plants, for example, N. benthamiana plants.
  • PAM monooxygenase
  • a first aspect relates to heterologous production of amidated antimicrobial peptide in plant expressed as mutated SUMO-fused domain.
  • the nucleic acid sequences are also comprised of cleavable linkers which can be cleaved orthogonally by the orthogonal protease SENP EUH protease, flexible linker sequences allowing independent movement of N and C terminal, and the presence of C terminal glycine residue which is required as a substrate for amidation.
  • the sequences are typically expressed transiently and are not integrated into the host cell chromosome.
  • AMPs preferably, cationic AMPs in plants
  • a purification tag such as the eight amino-acid Strep-tag II sequence is added to the AMPs for high-affinity binding to the engineered streptavidin Strep-Tactin.
  • the construct is designed to encode a fusion protein containing a purification tag, an optional epitope such as a hemagglutinin (HA) epitope, for example, human influenza hemagglutinin epitope, an optional linker, for example, GGSGGS (SE ID NO: 54); a cleavage sequence such as small ubiquitin-related modifier (bdSUMO) containing mutations at SUMO-interacting positions (bdSUMOEul) and the AMP sequence of interest, with a terminal glycine residue (hereinafter, AMP-fusion protein expression construct).
  • a hemagglutinin (HA) epitope for example, human influenza hemagglutinin epitope
  • an optional linker for example, GGSGGS (SE ID NO: 54
  • a cleavage sequence such as small ubiquitin-related modifier (bdSUMO) containing mutations at SUMO-interacting positions (bdSUMOEul) and the AMP sequence of interest, with
  • Exemplary AMP sequences with a terminal glycine residue include 1018-G (VRLIVAVRIWRRG) (SEQ ID NO:51), 1002-G (VQRWLIVWRIRKG) (SEQ ID NO:52), and 3002-G (ILVRWIRWRIQWG) (SEQ ID NO:53).
  • Epitope tagging is a method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques. The fusion protein can then be detected and/or purified using a monoclonal antibody specific for the epitope tag.
  • a vector containing an AMP-fusion protein expression construct and a vector containing a construct encoding PAM are transiently co-expressed in a plant.
  • a vector containing an AMP-fusion protein expression construct is transiently expressed in a plant stably expressing PAM.
  • the plant is engineered for cytosolic accumulation of the expression product i.e., the AMP fusion protein.
  • a second aspect includes transient expression of PAM1, PAM2 and PAM3 enzymes from Rattus norvegicus. These enzyme sequences are expressed transiently and not integrated into the host the cell chromosome.
  • a third aspect include the generation of PAM1 transgenic plants, wherein the enzyme sequence is integrated into the plant host cell chromosome using, preferably, by Agrobacterial- mediated delivery.
  • Transgenic plant cell which include one or more nucleic acid sequences containing the AMP-fusion protein construct and encoding an AMP-fusion protein and/or one or more nucleic acid sequences encoding PAM, are also provided.
  • a fourth aspect relates to the large-scale purification of SENP EuH protease enzyme from an E. coli host.
  • a fifth aspect relates a peptide purification method from plants employing high- performance liquid chromatography methods to obtain peptide as chloride salts, which are nontoxic compared to antimicrobial peptide produced as Tri-flouroacetic acid salts.
  • FIGs. 1A-1G shows establishment of a SynBio chassis for in planta expression of AMPs.
  • FIG. 1A is a schematic diagram of the AMP expression cassette for in planta expression, using the backbone of the pEAQ-HT vector.
  • Strep-II high affinity strep-tag II
  • HA human influenza hemagglutinin epitope
  • linker flexible GGSGGS (SEQ ID NO:54) linker
  • bdSUMO Eul small ubiquitin-related modifier (bdSUMO) from Brachypodium distachyon containing mutations at SUMO-in teracting positions
  • AMP1, AMP2 and AMP3 with a terminal glycine residue.
  • IB is a flowchart summarizing the plant-based production and purification of biologically active AMPs.
  • the individual plasmids are transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana; leaves are harvested at 6 days post infiltration (dpi); total protein is harvested and applied to Strep-Tactin Superflow resin.
  • His-tagged SENP EUH is removed using Ni-affinity chromatography and isolated AMPs are further purified by size-exclusion chromatography (SEC).
  • SEC size-exclusion chromatography
  • the pooled SEC fractions are applied to a reverse-phase high-performance liquid chromatograph (RP-HPLC) for final purification of AMPs.
  • RP-HPLC reverse-phase high-performance liquid chromatograph
  • FIG. ID is an immunoblot confirmation of purified SUMO-fused AMPs.
  • the separated proteins were transferred onto a polyvinylidene difluoride membrane and probed with a monoclonal anti-HA antibody for detection of bdSUMO Eul -AMPs ( ⁇ 15.5 kDa).
  • Total proteins extracted from non-infiltrated leaves served as negative control, NTC; HA-tagged protein was used as a positive control, PTC. Two independent blots were performed with similar results. The arrowhead indicates the expected size of protein.
  • FIG. IE is a gel shift assay for AMP release. Proteins were separated on a 18% Tricine-SDS gel to detect the release of the AMP peptide ( ⁇ 1.5-1.7 kDa) from the bdSUMO Eul domain ( ⁇ I4 kDa). The arrowheads indicate the uncleaved (top arrowhead) and cleaved proteins (bottom arrowhead) respectively.
  • FIG. IF is an RP-HPLC purification of AMPs. Pooled fractions from SEC were separated on a ZORBAX RX-C8 column using an acetonitrile gradient. Purified AMPs were separated on a 18% Tricine-SDS gel. Two independent Tricine-SDS -PAGE gels were performed with similar results.
  • FIG. 1G is a mass analysis of plant-purified AMPs using ESI-MS.
  • the y-axis shows the signal intensity, and the x-axis displays the m/z value of each peptide.
  • the AMP peak values were added to the mass chromatographs.
  • Black arrowheads indicate the expected size of peptides.
  • FIGs. 2A-2E show plant-based platform for production of amidated AMPs.
  • FIG. 2A is a schematic diagram of chimeric cassettes with different variants of bifunctional rat PAMs and the different domains: PHM (peptidylglycine a-hydroxylating monooxygenase domain); PAL (peptidyl-a-hydroxyglycine a-amidating lyase domain); A (region encoded by exon 16 separating the PHM and PHL domains); T (transmembrane domain); C (cytoplasmic domain). The HA epitope was added for immunodetection of PAMs.
  • FIG. 2B shows in planta transient expression of PAM enzymes. Each plasmid was individually co-infiltrated in N.
  • FIG. 2C shows in planta transient co-expression of AMPs and PAM enzymes. Constructs encoding AMPs and PAMs were transiently co-expressed in N.
  • FIG. 2D shows in planta amidation of AMPs in transgenic plants expressing PAM1.
  • Transgenic N. benthamiana lines (T4 generation) overexpressing a PAM1 variant were infiltrated with constructs encoding glycine-extended AMPs.
  • AMPs were isolated and subjected to separation in RP-HPLC using 9.4 x 250 mm ZORBAX RX-C8 with an acetonitrile gradient from 20 to 80% in 0.01 M HC1 and monitored at the 215 nm wavelength.
  • Purified peptides were eluted as a double peak, with the major peak belonging to amidated peptide with retention times of 10.2 min (AMP1), 9.7 min (AMP2), 10.2 min (AMP3) and the minor peak belonging to the non-amidated form with retention times 9.5 min (AMP1), 9.6 min (AMP2) and 9.7 min (AMP3).
  • FIG. 2E shows confirmation of AMP amidation via ESLMS. Mass analysis of purified AMPs isolated from the PAM transgenic plants showing major peak belonging to amidated AMPs along the minor non- amidated peak.
  • FIGs. 3A-3B show that plant-purified peptides display low toxicity in mammalian cells
  • FIG. 3A shows representative dose-response curves
  • FIGs. 4A-4F show experimental validation of antimicrobial activity of plant purified AMP1 against ESKAPE pathogens and their prevention of biofilm formation.
  • FIG. 4A-4C show that purified peptides exhibit a similar efficacy as synthetic peptides.
  • 10 6 colonyforming units (CFU)/ml of each ESKAPE E. coli PI-7, MRS A USA300, P. aeruginosa, K. pneumoniae, A. junii, E.
  • coli PI-7 in OD600 at a concentration of pp: 50 pg/ml, sp: 50 pg/ml, >90% of inhibition of MRSA USA300 (pp: 25 pg/ml, sp: 25 pg/ml), P. aeruginosa (pp: 25 pg, sp: 25 pg), K. pneumoniae (pp: 6.25 pg/ml, sp: 6.25 pg/ml), A. junii (pp: 50 pg/ml, pp: 12.5 pg/ml ), E.
  • FIG. 4D-4F show bactericidal activity of synthetic and purified peptide for the prevention of biofilm formation after 24 h of incubation in biofilm medium containing various concentrations of peptides. Results are expressed as biofilm mass, measured using crystal violet staining, in arbitrary units (au). Data are mean ⁇ SD of three independent experiments performed in duplicates.
  • the purified AMP1 abolish >90% of MRSA USA300 biofilms at 12.5 pg/ml, P.
  • FIGs. 5A-5F show that plant purified peptide AMP1 causes rapid membrane permeabilization and killing of MRSA USA300.
  • FIG. 5A-5F show that plant purified peptide AMP1 causes rapid membrane permeabilization and killing of MRSA USA300.
  • FIG. 5A shows antimicrobial activity (expressed as a minimal inhibitory concentration [MIC] of plant purified AMP1 and vancomycin, evaluated against 10 6 CFU/ml of MRSA USA300.
  • FIG. 5D shows percentage of PI- positive MRSA USA300 was calculated after addition of antimicrobial agents until their respective time points.
  • FIG. 5E shows scanning electron micrographs of MRSA USA300 treated with either PBS or 2x MIC of plant purified AMP1.
  • FIG. 5F shows mean cell width, as measured from SEM images by manually tracing the dimensions of individual cells. A standard two-tailed paired t test for analyzing the significance in the size of bacteria cells before (control) and after peptide treatment was applied. Data are means ⁇ SD from three independent experiments.
  • FIGs. 6A-6B show that purified peptides synergize with AZM by increasing the membrane permeability of carbapenem-resistant E. coli PI-7.
  • FIGs. 6A and 6B show time -kill curves (FIG. 6A) and prevention of biofilm formation assay (FIG. 6B) in E. coli PI-7.
  • Data are means ⁇ SD and represent the average of duplicates from 3 independent experiments.
  • FIG. 7A-7F Establishment of a SynBio chassis for in planta expression of AMPs.
  • FIG. 7A Cleaved peptide fractions were run on a 18% Tricine-SDS-PAGE with gel loading dye containing bromophenol blue or without bromophenol blue. Native low molecular- weight peptides migrated to the bottom of the gel and the 32.6-kDa protease band can be seen at the top of the gel. Protein extracts obtained from plants infiltrated with empty pEAQ-HT vector was used as negative control.
  • FIG. 7B Cleaved peptide fractions were run on a 18% Tricine-SDS-PAGE with gel loading dye containing bromophenol blue or without bromophenol blue. Native low molecular- weight peptides migrated to the bottom of the gel and the 32.6-kDa protease band can be seen at the top of the gel. Protein extracts obtained from plants infiltrated with empty pEAQ-HT vector was used
  • Cleaved AMP fractions were purified using size exclusion chromatography (SEC) in buffer containing 150 mM NaCl, 5% (v/v) CH3CN, 0.01 M HC1 and analyzed on 18% Tricine-SDS-PAGE gel. Two independent Tricine-SDS- PAGE have been performed with similar results.
  • FIG. 7C Pooled SEC fractions were run on a 9.4 x 250 mm ZORBAX RX-C8 column and monitored at the two wavelengths 215 nm and 280 nm.
  • FIG. 7D Pooled SEC fractions were run on a 9.4 x 250 mm ZORBAX RX-C8 column and monitored at the two wavelengths 215 nm and 280 nm.
  • FIG. 7E The immunoblot was stripped and reprobed with a monoclonal anti-GFP antibody to test HA- tagged SUMO-GFP accumulation. Two independent blots have been performed with similar results.
  • FIG. 7F Table showing concentration of proteins recovered at each step in the downstream process for in planta peptide purification. The black arrowheads indicate to the corresponding proteins and peptides.
  • FIG. 8A- 8B Wild type extract activity against ESKAPE pathogens and their characterization using ESI-MS.
  • FIG. 8A Protein extract obtained from wild type N. benthamiana were subjected to the same purification procedure and dissolved in the peptide buffer containing 0.025% (v/v) acetic acid and 0.1% [w/v] bovine serum albumin (BSA). The activity was determined by incubating with ESKAPE pathogens for 24 h followed by absorbance readings at OD600.
  • E. coli PI-7 100 pg/mL sodium azide
  • MRSA USA300 40 pM colistin
  • P. aeruginosa 40 pM colistin
  • FIG. 8B Mass analysis of wild type N. benthamiana extract using ESI- MS. The y-axis shows the signal intensity, and the x-axis displays the m/z value. Source data are provided as a Source Data file.
  • FIG. 10A-10F show experimental validation of antimicrobial activity of plant purified AMP2 against ESKAPE pathogens and their prevention of biofilm formation.
  • FIG. 10A- 10D Plant-produced and synthetic peptides have the same efficacy in bacterial growth inhibition.
  • CFU colony-forming units
  • coli PI-7 in OD600 at a concentration of pp: 50pg/mL, sp: 50 pg/mL, >90% of inhibition of MRSA USA300 (pp: 25 pg/mL, sp: 12.5 pg/mL), K. pneumoniae (pp: 25 pg/mL, sp: 6.25 pg/mL), A. junii (pp: 25 pg/mL, pp: 12.5 pg/mL), E. faecalis (pp: 50 pg/mL, sp: 50 pg/mL), P. aeruginosa (pp: 25 pg/mL, sp: 12.5 pg/mL).
  • FIG. 10E-10F Bactericidal activity of purified peptides against prevention of biofilms after 24 h of incubation in biofilm media containing various concentrations of peptides. Results are expressed as biofilm mass, measured using crystal violet staining, in arbitrary units (au). Values are medians of two independent experiments. *, significantly different (*P ⁇ 0.05, **P ⁇ 0.01, and ***P ⁇ 0.001) compared to control (0 pg/mL), as calculated using the two-tailed Mann-Whitney rank sum test.
  • Source data are provided as a Source Data file.
  • FIG. 11A-11D show the experimental validation of antimicrobial activity of plant purified peptide AMP3 against ESKAPE pathogens and their prevention of biofilm formation.
  • FIG.ll -11B Plant-produced peptides are effective against ESKAPE pathogens. For each concentration of peptide, 10 6 colony-forming units (CFU)/mL of each ESKAPE pathogens were treated with 100, 50, 25, 12.5, 6.25, 3.215, 1.56 pg/mL of peptides in cation- adjusted Mueller-Hinton broth for 24 h. Percentage inhibition was up to 30% reduction for carbapenem-resistant E.
  • CFU colony-forming units
  • FIG. 11C and 11D Bactericidal activity of purified peptides against prevention of biofilms after 24 h of incubation in biofilm medium containing various concentrations of peptides.
  • Results are expressed as biofilm mass, measured using crystal violet staining, in arbitrary units (au). Values are medians of two independent experiments. *, significantly different (*P ⁇ 0.05, **P ⁇ 0.01, and ***P ⁇ 0.001) compared to control (0 pg/mL), as calculated using the two- tailed Mann- Whitney rank sum test.
  • FIG. 12 shows gating strategy for flow cytometry-based analysis of PI accumulation in MRSA USA300 cells .
  • Cells were washed, suspended in 500 DL of 1 x PBS and analyzed on BD LSRFortessaTM Cell Analyzer. Cells were gated on forward and side scatter profiles. Positive and negative cell populations were gated based on staining the fluorescently-PI dye. At least 1000 events were analyzed. Control cells showed negligible or very low accumulation of PI stain.
  • FIG. 13 shows base-case techno-economic analysis for industrial scale production of AMPs in plants.
  • FIG. 14 shows base-case techno-economic analysis for industrial scale production of AMPs in plants. Downstream process flowsheet for N. benthamiana base case scenario in the SuperPro Designer model with a production capacity at 300 Kg/year.
  • the peptides are 1018-G (VRLIVAVRIWRRG) (SEQ ID NO:51), 1002-G (VQRWLIVWRIRKG) (SEQ ID NO:52), and 3002-G (ILVRWIRWRIQWG) (SEQ ID NO:53) designated as AMP1, AMP2 and AMP3 respectively, which differ from the parent sequences by having an additional Gly at the C- terminus.
  • the examples demonstrate successful production of both the non-amidated Gly precursors as well as the final PAM processed amidated AMP counterparts.
  • a titer for these amidated peptides of 1.4 mg per 20 g of transgenic plant tissue, was attained.
  • agroinfiltration refers to a method in plant biology to transfer genetic cassettes from Agrobacterium into a plant.
  • a suspension of Agrobacterium tumefaciens is injected into a plant leaf, where it transfers the desired gene to plant cells.
  • the benefit of agroinfiltration when compared to traditional plant transformation is speed and convenience.
  • cell refers to a membrane -bound biological unit capable of replication or division.
  • construct refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences. Genetic constructs used for transgene expression in a host organism include a series of cassettes including units with (in the 5 ’-3’ direction), a promoter sequence; a sequence encoding a gene of interest; and a termination sequence. The construct may also include selectable marker gene(s) and other regulatory elements for expression.
  • a “cultivar” refers to a cultivated variety.
  • derivative species, germplasm or variety refers to any plant species, germplasm or variety that is produced using a stated species, variety, cultivar, or germplasm, using standard procedures of sexual hybridization, recombinant DNA technology, tissue culture, mutagenesis, or a combination of any one or more said procedures.
  • expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • expression vector refers to a vector that includes one or more expression control sequences.
  • expression control sequence refers to a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
  • Control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and the like.
  • expression vector refers to a vector that includes one or more expression control sequences regardless of the origin of the sequence (prokaryote or eukaryote).
  • gene refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein.
  • gene also refers to a DNA sequence that encodes an RNA product.
  • gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5’ and 3’ ends.
  • germplasm refers to one or more phenotypic characteristics, or one or more genes encoding said one or more phenotypic characteristics, capable of being transmitted between generations.
  • genome as used herein, referring to a plant cell encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondria, or plastid) of the cell.
  • heterologous means from another host.
  • the other host can be the same or different species.
  • plant is used in its broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative crop or cereal, and fruit or vegetable plant. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant’s development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc.
  • plant cell refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
  • plant cell culture refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
  • plant material refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
  • plant organ refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
  • plant part or “part of a plant” can include, but is not limited to cuttings, cells, protoplasts, cell tissue cultures, callus (calli), cell clumps, embryos, stamens, pollen, anthers, pistils, ovules, flowers, seed, petals, leaves, stems, and roots.
  • plant tissue includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.
  • plant part refers to a plant structure, a plant organ, or a plant tissue.
  • promoter refers to a regulatory nucleic acid sequence, typically located upstream (5’) of a gene or protein coding sequence that, in conjunction with various elements, is responsible for regulating the expression of the gene or protein coding sequence.
  • progenitor refers to any of the species, varieties, cultivars, or germplasm, from which a plant is derived.
  • “Stable expression” as used herein relates to the introduction of genetic material into chromosomes of the targeted cell where it integrates and becomes a permanent component of the genetic material in that cell. Gene expression after stable introduction can permanently alter the characteristics of the cell and its progeny arising by replication leading to stable transformation.
  • stable refers to the introduction of gene(s) into the chromosome of the targeted cell where it integrates and becomes a permanent component of the genetic material in that cell. Gene expression after stable transformation/transfection can permanently alter the characteristics of the cell leading to stable transformation.
  • An episomal transformation is a variant of stable transformation in which the introduced gene is not incorporated in the host cell chromosomes but rather is replicated as an extrachromosomal element. This can lead to stable transformation of the characteristics of a cell.
  • Transiently refers to the introduction of a gene into a cell to express the nucleic acid, e.g., the cell expresses specific proteins, peptides or RNA, etc. The introduced gene is not integrated into the host cell genome and is accordingly eliminated from the cell over a period of time. Transient expression relates to the expression of a gene product during a period of transient transfection.
  • transgenic plant/cell refers to a plant/cell that contains recombinant genetic material which has been introduced into the plant/cell in question (or into progenitors of the plant) by human manipulation.
  • a plant that is grown from a plant cell into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually).
  • transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems etc.
  • transgene refers to an artificial gene, manipulated in the molecular biology lab that incorporate all appropriate elements critical for gene expression generally derived from a different species.
  • transformed refers to a host organism such as a bacterium or a plant into which a exogenous nucleic acid molecule has been introduced.
  • vector refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • the vectors can be expression vectors.
  • COMPOSITIONS Genetically modified constructs containing a gene encoding AMP of to be introduced into a plant, plant vectors including the constructs, as well as plant/plant parts genetically engineered using the disclosed constructs and vectors, alone or additionally, genetically engineered to express with the bifunctional PAM enzymes encompassing the peptidylglycine a- hydroxylating monooxygenase (PHM) domain, peptidyl-a-hydroxylglycine a-amidating lyase (PHL) domain, the transmembrane domain, and the cytosolic region, are disclosed.
  • the AMP is positively charged.
  • AMPs are typically small peptides, ranging from about 5 to 50 amino acids, but can be as large as over 100 amino acids. Most AMPs are positively charged (+2 to +9) due to their high proportions of arginine and lysine residues, although negatively charged AMPs do also exist. In a preferred embodiment, the AMP is positively charged.
  • Exemplary AMPs include, but are not limited to SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, FK13 (Human) (Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg)(SEQ ID NO:55), Guavanin 2, WLBU2, CONGA, DBS1, Mastoparan 4,1, cancrin, which has an amino acid sequence of GS AQPYKQLHKVVNWDPYG (SEQ ID NO:65), etc., reviewed in Huan, et al., Front.
  • WLBU2 is an engineered cationic AMP with promising antibacterial activity. It is composed of 24 amino acids including; 13 arginine, 8 valine and 3 tryptophan residues (RRWVRRVRRWVRRVVRVVRRWVRR) (SEQ ID NO: 68) (Salem, et al., Turk J Pharm Sci. 2022;19(l):l 10-116), Deslouches, et al., doi.org/10.1128/aac.49.8.3208-3216.2005).
  • Nucleic acid constructs which include expression cassettes designed to encode a fusion protein containing a purification tag, an optional epitope such as a hemagglutinin (HA) epitope, for example, human influenza hemagglutinin epitope, an optional linker, for example, GGSGGS (SE ID NO: 54) linker; a cleavage sequence such as small ubiquitin-related modifier (bdSUMO) containing mutations at SUMO-interacting positions (bdSUMOEul) and the AMP sequence of interest, with a terminal glycine residue (hereinafter, AMP-fusion protein expression construct), to be introduced into a plant cell are disclosed.
  • an optional epitope such as a hemagglutinin (HA) epitope, for example, human influenza hemagglutinin epitope
  • an optional linker for example, GGSGGS (SE ID NO: 54) linker
  • a cleavage sequence such as small ubiquitin-related modifier (b
  • linker used to separate moieties in a fusion protein can be used, and preferably include flexible peptides or polypeptides.
  • a “flexible linker” herein refers to a peptide or polypeptide containing two or more amino acid residues joined by peptide bond(s) that provides increased rotational freedom for two polypeptides linked thereby than the two linked polypeptides would have in the absence of the flexible linker.
  • Exemplary flexible peptides/polypeptides include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser- Gly-Ser (SEQ ID NO:57), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO:58), (Gly4-Ser)3 (SEQ ID NO:59), and (Gly4-Ser)4 (SEQ ID NO:60), GSGSGSGS (SEQ ID NO:61), SGSG (SEQ ID NO:62), CGGSGSGSG (SEQ ID NO:63) or GSGC (SEQ ID NO:64).
  • exemplary a purification tags include, but are not limited to c-myc, polyhistidine, or FlagTM (Kodak), polyhistidine affinity tag, also known as the His-tag or Hise, usually consists of six consecutive histidine residues, but can vary in length from two to ten histidine residues; glutathione S-transferase (GST); Maltose binding protein (MBP), calmodulin binding peptide (CBP); the intein-chitin binding domain (intein-CBD), the streptavidin tag, etc.
  • GST glutathione S-transferase
  • MBP Maltose binding protein
  • CBP calmodulin binding peptide
  • intein-CBD the intein-chitin binding domain
  • streptavidin tag etc.
  • the nucleic acid construct is operably linked to a promoter, in a suitable expression vector.
  • a nucleic acid sequence or polynucleotide is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • operably linked means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the expression vector can be any expression vector suitable for plant transformation, such as a plasmid or a plant viral vector, such as Tobacco mosaic virus.
  • plasmid plasmid
  • vector plasmid
  • cassette as used herein refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of double- stranded DNA.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage, or nucleotide sequences, in linear or circular form, of a single- or doublestranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a polynucleotide of interest into a cell.
  • promoters suitable for use in the constructs of this disclosure are functional in plants.
  • Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser & Fraley, Science 244:1293-99 (1989)).
  • promoters are selected from those of eukaryotic or synthetic origin that are known to yield high levels of expression in plant and algae cytosol.
  • promoters are selected from those of plant or prokaryotic origin that are known to yield high expression in plastids.
  • the promoters are inducible. Inducible plant promoters are known in the art.
  • the promoter is an egg cell-specific promoter.
  • promoters are publicly known. These include constitutive promoters, inducible promoters, tissue- and cell-specific promoters and developmentally -regulated promoters. Exemplary promoters and fusion promoters are described, e.g., in U.S. Pat. No. 6,717,034, which is herein incorporated by reference in its entirety.
  • Suitable constitutive promoters for nuclear-encoded expression include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Pat. No. 6,072,050; the core CAMV 35S promoter, (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.
  • Tissue-preferred promoters can be used to target a gene expression within a particular tissue such as seed, leaf or root tissue.
  • Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157- 168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331 - 1341 ; Van Camp et al (1996) Plant Physiol. 112(2):525-535; Canevascini et al.
  • seed-preferred promoters include both “seed- specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seedgerminating” promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10:108.
  • seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19Bl (maize 19 kDa zein); milps (myo-inositol- 1 -phosphate synthase); and celA (cellulose synthase).
  • Gama-zein is a preferred endosperm-specific promoter.
  • Glob-1 is a preferred embryo-specific promoter.
  • seed-specific promoters include, but are not limited to, bean P-phaseolin, napin P-conglycinin, soybean lectin, cruciferin, oleosin, the Lesquerella hydroxylase promoter, and the like.
  • seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. Additional seed specific promoters useful for practicing this invention are described in the Examples disclosed herein.
  • Eeaf-specific promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
  • Root-preferred promoters are known and may be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al.
  • MAS mannopine synthase
  • Plant Cell 3( 1 ):1 l'-22 full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean. See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid.
  • promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. Proc. Natl. Acad. Sci. USA 88: 10421-10425 (1991) and McNellis et al. Plant J. 14(2):247-257( 1998)) and tetracyclineinducible and tetracycline -repressible promoters (see, for example, Gatz et al. Mol. Gen. Genet. 227:229-237 (1991), and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference in their entirety.
  • a polyadenylation signal refers to any sequence that can result in polyadenylation of the mRNA in the nucleus prior to export of the mRNA to the cytosol, such as the 3’ region of nopaline synthase (Bevan, et al. Nucleic Acids Res. 1983, 11, 369-385).
  • Genetic constructs may encode a selectable marker to enable selection of transformation events. There are many methods that have been described for the selection of transformed plants [for review see (Miki et al., Journal of Biotechnology, 2004, 107, 193-232) and references incorporated within]. Selectable marker genes that have been used extensively in plants include the neomycin phosphotransferase gene nptll (U.S. Patent Nos. 5,034,322, U.S. 5,530,196), hygromycin resistance gene (U.S. Patent No. 5,668,298), the bar gene encoding resistance to phosphinothricin (U.S. Patent No.
  • 5,767,378 describes the use of mannose or xylose for the positive selection of transgenic plants. Methods for positive selection using sorbitol dehydrogenase to convert sorbitol to fructose for plant growth have also been described (WO 2010/102293). Screenable marker genes include the beta-glucuronidase gene (Jefferson et al., 1987, EMBO J. 6: 3901- 3907; U.S. Patent No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., 1995, Trends Biochem. Sci. 20: 448-455; Pan et al., 1996, Plant Physiol. 112: 893-900).
  • Transformation events can also be selected through visualization of fluorescent proteins such as the fluorescent proteins from the nonbioluminescent Anthozoa species which include DsRed, a red fluorescent protein from the Discosoma genus of coral (Matz et al. (1999), Nat Biotechnol 17: 969-73).
  • DsRed a red fluorescent protein from the Discosoma genus of coral
  • An improved version of the DsRed protein has been developed (Bevis and Glick (2002), Nat Biotech 20: 83-87) for reducing aggregation of the protein.
  • Visual selection can also be performed with the yellow fluorescent proteins (YFP) including the variant with accelerated maturation of the signal (Nagai, T. et al.
  • a transgenic plant includes, for example, a plant that comprises within its genome an exogenous polynucleotide introduced by a transformation step.
  • the exogenous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.
  • a transgenic plant can also comprise more than one heterologous polynucleotide within its genome. Each exogenous polynucleotide may confer a different trait to the transgenic plant.
  • a heterologous polynucleotide can include a sequence that originates from a foreign species, or, if from the same species, can be substantially modified from its native form.
  • Suitable plant families include but are not limited to, Alliaceae, Amaranthaceae, Amaryllidaceae, Apocynaceae, Asteraceae, Boraginaceae, Brassicaceae, Campanulaceae, Caryophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Gramineae, Hyacinthaceae, Labiatae, Leguminosae-Papilionoideae, Liliaceae, Linaceae, Malvaceae, Phytolaccaceae, Poaceae, Pinaceae, Rosaceae, Scrophulariaceae, Solanaceae, Tropaeolaceae, Umbelliferae and Violaceae.
  • Such plants include, but are not limited to. Allium cepa, Amaranthus caudatus, Amaranthus retroflexus, Antirrhinum majus, Arabidopsis thaliana, Arachis hypogaea, Artemisia sp., Avena sativa, Bellis perennis, Beta vulgaris, Brassica campestris, Brassica campestris ssp. Napus, Brassica campestris ssp.
  • Pekinensis Brassica juncea, Calendula officinalis, Capsella bursa-pastoris, Capsicum annuum, Catharanthus roseus, Chemanthus cheiri, Chenopodium album, Chenopodium, amaranticolor, Chenopodium foetidum, Chenopodium quinoa, Coriandrum sativum, Cucumis melo, Cucumis sativus, Glycine max, Gomphrena globosa, Gossypium hirsutum cv.
  • Fig. 1A An exemplary vector is shown in Fig. 1A, and it can be used to transiently express the genes of interest as exemplified herein.
  • the vector can include or exclude the HA epitope shown in Fig. 1.
  • a construct coding for the PAM can introduced into plant leaves callus, seed or embryonic tissue. Stably-transformed plants (events) are then recovered. Briefly, vectors containing the various PAM genes are introduced into Agrobacterium (Agrobacterium tumefaciens) strain GV3101 by electroporation. Stable Agrobacterium- mediated leaf disc transformation can be performed according to a previously described standard protocol. 89 Transgenic plants are propagated until the homozygous T4 generation and are screened using immunoblot for accumulation of the PAM protein.
  • transgenic plants are selected on a known substrate such as Murashige and Skoog (MS) (Sigma) medium containing 100 pg/ml kanamycin in a growth chamber with the temperature set to 28 °C and a 13-h light/11- h dark regime.
  • MS Murashige and Skoog
  • One- week-old seedlings can be acclimatized and transferred to soil in greenhouse with the temperature set to -28-30 °C for continued growth until maturity.
  • a vector containing an AMP-fusion protein expression construct and a vector containing a construct encoding PAM are transiently co-expressed in a plant.
  • a vector containing an AMP-fusion protein expression construct is transiently expressed in a plant stably expressing PAM, for example, PAM1, PAM2 or PAM3 from Rattus norvegicus.
  • the plant is engineered for cytosolic accumulation of the expression product i.e., the AMP fusion protein.
  • the plant transformation method does not employ a whole virus such as, Tobacco mosaic virus as the vector for introducing nucleic acid constructs into a plant.
  • a method for large-scale purification of SENP EuH protease enzyme from an E. coli hos the method of which is exemplified below under “Purification of SENPEuH protease” and incorporated herein by reference.
  • a peptide purification method from plants employing high-performance liquid chromatography methods to obtain peptide as chloride salts, which are non-toxic compared to antimicrobial peptide produced as Tri-flouroacetic acid salts the method of which is exemplified below under “Large-scale purification of peptides”, and incorporated herein by reference.
  • the method includes protein purification via reverse-phase high-performance liquid chromatography (RP-HPLC), which served as an additional desalting step, using acetonitrile as the organic modifier and HC1 as ion-pairing agent rather than traditional trifluoro-acetic acid that has inherent toxicity and would need to be exchanged for a biocompatible ion.
  • RP-HPLC reverse-phase high-performance liquid chromatography
  • SEC size exclusion chromatography
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation.
  • the disclosed methods do not include plastid transformation/ the constructions do not include additional targeting sequences for plasmid expression of periplasmic secretion of the expressed protein.
  • the constructs used herein do not include nucleic acid sequences encoding a periplasmic targeting signal and an antimicrobial peptide.
  • Periplasmic targeting signal peptide sequences generally derived from a protein that is secreted in a Gram negative bacterium (U.S. Patent No. 7,579005).
  • Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection, electroporation, Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al. WO US98/01268), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed.
  • a preferred method is an agrobacterium mediated transformation, exemplified in the Examples of this application, the method of which is incorporated herein.
  • the A. tumefaciens-mediated plant genetic transformation process requires the presence of two genetic components located on the bacterial Ti-plasmid.
  • the first essential component is the T-DNA, defined by conserved 25-base pair imperfect repeats at the ends of the T-region called border sequences.
  • the second is the virulence (vir) region, which is composed of at least seven major loci (virA, virB, virC, virD, virE, virF, and virG) encoding components of the bacterial protein machinery mediating T-DNA processing and transfer.
  • the VirA and VirG proteins are two-component regulators that activate the expression of other vir genes on the Ti-plasmid.
  • the VirB, VirC, VirD, VirE and perhaps VirF are involved in the processing, transfer, and integration of the T-DNA from A. tumefaciens into a plant cell (Hwang et al., 2017 doi.org/10.1199/tab.O186).
  • a suspension of Agrobacterium tumefaciens is injected into a plant leaf, where it transfers the desired gene to plant cells.
  • the first step of the protocol is to introduce a gene of interest to a strain of Agrobacterium. Subsequently the strain is grown in a liquid culture and the resulting bacteria are washed and suspended into a suitable buffer solution. This solution is then placed in a syringe (without a needle). The tip of the syringe is pressed against the underside of a leaf while simultaneously applying gentle counterpressure to the other side of the leaf. The Agrobacterium solution is then injected into the airspaces inside the leaf. Vacuum infiltration is another way to penetrate Agrobacterium deep into plant tissue.
  • leaf disks, leaves, or whole plants are submerged in a beaker containing the solution, and the beaker is placed in a vacuum chamber.
  • the vacuum is then applied, forcing air out of the stomata.
  • the pressure difference forces solution through the stomata and into the mesophyll.
  • the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene producing the desired level of desired polypeptide(s) in the desired tissue and cellular location.
  • the cells that have been transformed may be grown into plants in accordance with conventional techniques. See, for example, McCormick et al. Plant Cell Reports 5:81-84(1986). These plants may then be grown, and either pollinated with the same transformed variety or different varieties, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.
  • the engineered SUMO Eul module was previously shown to resist proteolytic cleavage by endogenous deSUMOylases in eukaryotic cell lysates, facilitating the isolation of protein complexes from eukaryotic extracts A
  • the sequences of AMP genes were codon-optimized to increase the translational efficiency in the production host Nicotiana benthamiana.
  • Each synthetic gB locks template was PCR amplified with primers that added Agel and Xhol restriction sites to the 5’ and 3’ ends of the PCR product, respectively, for subsequent cloning into the Agel/Xhol- digested Cowpea mosaic virus-based vector pEAQ-HT (Leaf Expression Systems, Norwich, UK).
  • oligonucleotides were purchased from Integrated DNA Technologies (IDT, Leuven, Belgium) and were HPLC-purified by the manufacturer. Sequences of the oligonucleotides are listed in the T able Below.
  • Plasmids encoding the rat variants of PAM enzymes were kindly provided by Prof. Betty Eipper, University of Connecticut Health Center, USA.
  • the coding sequence encoding the bifunctional PAM enzymes encompassing the peptidylglycine a-hydroxylating monooxygenase (PHM) domain, l-rz- hydroxylglycine a-amidating lyase (PHL) domain, the transmembrane domain, and the cytosolic region were amplified from plasmid DNA.
  • the PAM2 variant lacks exon 16 located adj acent to the sequence encoding the protease-sensitive region separating the PHM and PHL domain, whereas PAM3 variant lacks the sequence encoding trans- membrane domain.
  • Public database used for rat PAM enzyme sequence include UniProt (www.uniprot.org/uniprotkb/ A0A8I5ZMRl/entry).
  • the forward primer was preceded by the four nucleotides CACC.
  • the reverse primer contained unique restriction sites for Hindlll and Xbal to ligate the annealed HA primers with overhanging sticky ends complimentary to Hindll l/Xbal.
  • the subcloned vectors containing the PAM-HA construct were verified by Sanger sequencing using overlapping primers.
  • the inserts were recombined into the plant transformation vector pK2GW7 using Gateway cloning to drive the expression of PAM genes under the control of the constitutively active cauliflower mosaic virus (CaMV) 35 S promoter.
  • CaMV constitutively active cauliflower mosaic virus
  • the pK2GW7 binary vectors containing the various PAM genes generated above were introduced into Agrobacterium (Agrobacterium tumefaciens) strain GV3101 by electroporation. Stable Agrobacterium- mediated leaf disc transformation was performed according to a standard protocol 89 . Briefly, 2-week-old Nicotiana benthamiana leaf explants infiltrated with Agrobacterium containing PAM genes in MES buffer (10 mM 2-[N- morpholino] -ethanesulfonic acid, pH 5.6, 10 mM MgCL.
  • MES buffer 10 mM 2-[N- morpholino] -ethanesulfonic acid, pH 5.6, 10 mM MgCL.
  • kanamycin-resistant lines forming proper roots were acclimatized to the soil in greenhouse under plastic domes with the temperature set to -28-30 °C for continued growth until maturity.
  • Transgenic plants were propagated until the homozygous T4 generation and were screened using immunoblot for accumulation of the PAM protein.
  • a plasmid encoding the Brachypodium distachyon mutated protease His-TEV- SENP EUH was purchased from Addgene (plasmid number 149689) and transformed into E. coli BL21 (DE3) pLysS cells (New England Biolabs Inc., Hitchin, England).
  • SENP1 EUH protease was produced by growing bacteria into 2 L of Terrific broth (IBI Scientific) containing kanamycin. Cells were grown at 37 °C until reaching an GD600 of 0.5-0.7; protein production was induced by the addition of isopropyl-[3-D-thiogalactopyranoside (IPTG) at a final concentration of 0.3 mM.
  • the cells were grown at 18 °C for 19 h, harvested by centrifugation at 5,500 x g for 15 min at 4 °C, then resuspended in ice- chilled lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 4.5 mM MgCL. 5% [v/v] glycerol, 20 mM imidazole, 100 mM PMSF and complete EDTA-free protease inhibitor cocktail tablet/50 mL [Roche, UK]).
  • ice- chilled lysis buffer 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 4.5 mM MgCL. 5% [v/v] glycerol, 20 mM imidazole, 100 mM PMSF and complete EDTA-free protease inhibitor cocktail tablet/50 mL [Roche, UK]).
  • the cells were subjected to lysis using lysozyme (Sigma) at a con- centration of 1 mg/mL on ice for 1 h, followed by mechanical disruption using sonication (Qsonica Q700). Cell debris were then removed by centrifugation at 10,000 x g for 40 min at 4 °C and the decanted supernatant was passed through a Nalgene disposable bottle top filter with a 0.45-pm membrane (Thermo Fisher Scientific, USA).
  • the filtered supernatant was loaded onto a 5-mL HisTrapTM HP column (GE Healthcare Biosciences) pre-equilibrated with buffer A (50 mM Tris- HC1 pH 7.5, 500 mM NaCl, 20 mM imidazole, 5% [v/v] glycerol) using an AKTA pure instrument (UNICORN 6.3, GE Healthcare Biosciences).
  • buffer A 50 mM Tris- HC1 pH 7.5, 500 mM NaCl, 20 mM imidazole, 5% [v/v] glycerol
  • AKTA pure instrument UNICORN 6.3, GE Healthcare Biosciences
  • the fractions containing the SENP EUH protease was analyzed using SDS-PAGE, pooled, and dialyzed overnight in Snakeskin-pleated dialysis tubing (Thermo Fisher Scientific, USA) against dialysis buffer (25 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 10% [v/v] glycerol).
  • dialysis buffer 25 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 10% [v/v] glycerol.
  • the dialyzed sample was concentrated to 1 mL using centrifugal filters with a membrane NMWL of 10-kDa (Millipore, USA).
  • the concentrated protein was then loaded onto a HiLoad 16/600 Superdex 200 pg gel filtration column (GE Healthcare Biosciences) equilibrated with storage buffer (25 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 10% [v/v] glycerol). Fractions containing the protease were pooled, flash-frozen in liquid nitrogen and stored at -80 °C until use.
  • Leaves infiltrated with each AMP construct were harvested 6 days post-infiltration and ground in liquid nitrogen to a fine powder with pre-cooled mortars and pestles.
  • Total proteins were extracted from the leaf powder by the addition of 2-3 x (w/v) ice-cold extraction buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 3 mM DTT, 4% [w/v] polyvinylpolypyrrolidone [PVPP], 0.1% [v/v] Triton X- 100, 100 mM PMSF and Complete EDTA-free protease inhibitor cocktail tablet/30 mL [Roche, UK]), followed by mechanical disruption using sonication at 30% amplitude.
  • 2-3 ice-cold extraction buffer 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 3 mM DTT, 4% [w/v] polyvinylpolypyrrolidone
  • the phenol adsorbent polymer PVPP is highly insoluble in polar solvents, it was directly added to the ground leaf powder.
  • the slurry was completely squeezed through 2-3 layers of Miracloth, clarified by centrifugation at 10,000 x g for 1 h at 4 °C and filtered through a Nalgene disposable bottle top filter with a 0.45-pm membrane (Thermo Fisher Scientific, USA).
  • the filtered supernatant was applied to 5 mL of Strep-Tactin Superflow resin (Qiagen, Hilden, Germany) in gravity flow Econocolumns® (BioRad), incubated for 2 hr at 4 °C with gentle rotation, followed by resin washes with buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 3 mM DTT) to remove loosely bound proteins. After washing the resin, it was immediately resuspended in SUMO digestion buffer (45 mM Tris-HCl pH 7.5, 2 mM MgCh, 250 mM NaCl, 10 mM DTT, 0.1% [v/v] NP-40).
  • Recombinant AMPs were released under native form by overnight cleavage with 17 pg of purified SENP EUH protease in the presence of 1 M Urea at 4 °C under gentle rotation. Urea was added to the protease reaction buffer for precise cleavage of the peptide and to prevent any nonspecific activity. Cleaved AMPs were collected and loaded onto a 5- mL HisTrapTM HP column (GE Healthcare Biosciences) using buffer A (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM imidazole) via AKTA pure (GE Health- care Biosciences) to remove His-tagged SENP EUH protease.
  • buffer A 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM imidazole
  • the wave- lengths used to monitor the cleaved peptides were 215 nm (peptide bond absorbs light at 215 nm) and 280 nm since all three prototypical AMPs have an aromatic-side chain of tryptophans that absorbs light in the UV range of 250-290 nm, thereby providing a convenient means for peptide detection.
  • Flow- through fractions that were devoid of protease but contained the native AMPs were collected and immediately freeze-dried in a lyophilizer to concentrate the fractions.
  • the lyophilized extract was then resuspended in size-exclusion chromatography (SEC) buffer (5% [v/v] HPLC-grade CH3CN, 0.01 M HC1 and 150 mM NaCl) and centrifuged at 10,000 g maintained at room temperature for 10 min.
  • SEC size-exclusion chromatography
  • the cleared supernatant was injected in a 1.5-mL loop, loaded onto a SEC-buffer pre-equilibrated Superdex 30 Increase 3.2/300 (GE Healthcare Biosciences) with a flow rate of 0.01 mL/min, monitored at 215 nm/280 nm and eluted in SEC buffer.
  • ESI-TOF-MS of peptides Mass identification of peptides was carried using a MicroTOF-Q spec- trometer (Broker Daltonics, Inc, Germany). The machine was calibrated in positive ionization mode using 1 % (v/v) formic acid in acetonitrile/HPLC-grade water solution (CH3CN/H2O, 50/50, v/v). Dried peptide samples were dissolved in solvent containing 50% (v/v) CH3CN and 1% (v/v) formic acid and injected into the ESI source using a stainless-steel needle syringe at a flow rate of 10 pL/min.
  • Data were acquired by the TOF analyzer (Compass for otofSeries 1.7 Version 3.4, Bruker Daltonics GmbH) at a rate of 1 acquisition/sec from m/z 200 to m/z 2000.
  • the optimized voltage was set to +3 kV for the capillary and dry nitrogen gas heated to 150 °C was used for better nebulization.
  • Data were acquired and processed with the Compass DataAnalysis software (Bruker Compass DataAnalysis 4.2 SR2, Bruker Daltonics GmbH).
  • azithromycin (Cat. no. PZ0007), colistin sulfate (Cat. no. C4461), meropenem (Cat. no. PHR1772), ceftazidime (Cat. no. C3809), vancomycin sulphate (Cat. no. 861987), sulfamethoxazole (Cat. no. S7507), gentamicin (Cat. no. G1914), kanamycin (Cat. no. BP861), levofloxacin (Cat. no. 28266), ciprofloxacin (Cat. no. 17850) were purchased from Sigma.
  • the stock solutions of antibiotics were prepared in molecular biology grade 1 x phosphate-buffered saline (PBS) (Corning Inc., Corning NY, USA). In case of azithromycin, trace amounts of glacial acetic acid was added for complete solubility. Synthetic QCed peptides AMP1, AMP2 were kindly provided by Prof. Robert Hancock (University of British Columbia, Canada). Peptides were dissolved in endotoxin-free sterile water (Corning Inc., Corning NY, USA) containing 0.025% (v/v) acetic acid and 0.1% [w/v] bovine serum albumin (BSA) for in vitro experiments.
  • PBS molecular biology grade 1 x phosphate-buffered saline
  • the pathogenic strains used in this study were carbapenem-resistant Escherichia coli PI-7 (a New Delhi metallo-P-lactamase -positive strain previously isolated from municipal wastewater in Saudi Arabia), methicillin-resistant Staphylococcus aureus USA300, extended- spectrum P-lactamase-producing Klebsiella pneumoniae ATCC 700603, Acinetobacter Junii DSMZ 14968, Pseudomonas aeruginosa ATCC 9027, Enterobacter faecalis ATCC 29212.
  • Pathogenic Escherichia coli PI-7 was grown in UB broth containing 8 pg/rnU meropenem, methicillin-resistant Staphylococcus aureus USA300 was grown in tryptic soy broth (TSB; Difco, Detroit) containing 10 pg/mL chlor- amphenicol, while all remaining strains were grown in UB broth with-out any antibiotic added.
  • TLB tryptic soy broth
  • MIC Minimal inhibitory concentration
  • All plant expression constructs carried the sequence encoding a triple N-terminal hemagglutinin (HA)-epitope tag to analyze production abundance by immunoblot. Leaves were harvested post-infiltration and total protein was extracted from 100 mg of sample using extraction buffer (100 mM Tris-HCl pH 8, 5 mM EDTA, 150 mM NaCl, 10 mM DTT, 0.5% [v/v] Triton X-100 along with protease inhibitor cocktails consisting of 1 mM PMSF, 15 pg/mL leupeptin, 1 pg/mL aprotinin, 1 pg/mL pepstatin, 5 pg/mL antipain, 5 pg/mL chymostatin, 2 mM NaiVCh.
  • extraction buffer 100 mM Tris-HCl pH 8, 5 mM EDTA, 150 mM NaCl, 10 mM DTT, 0.5% [v/v] Triton X-100
  • a mid-logarithmic growth-phase culture was diluted to 1 x 10 8 CFU/mL in Ca-MHB and was exposed to antimicrobial agents for the estimated time as evaluated in time-kill kinetic assay for each respective agent.
  • Twenty microliters of propidium iodide (PI, Molecular Probes, Invitrogen) with a final concentration of 1 pg/mL were then added to the cells and incubated in the dark for 30 min.
  • the percent influx of PI stain was then analyzed using a BD LSRFortessaTM Cell Analyzer (BD FACS- Diva Software, Version 6.2, BD Biosciences, San Jose, CA, USA) and calculated using FlowJo 10.6.2 software (BD Biosciences).
  • HEK-293 cells Human embryonic kidney 293 (HEK-293) cells (Thermo Fisher Scientific, Cat. no. 51-0035) were cultured in 75 T flasks and incubated in a humidified incubator maintained at 37 °C with 5% (v/v) CO2 using DMEM/high-glucose medium supplemented with Glutamax, 10% (v/v) fetal bovine serum (FBS), and 1% (w/v) penicillin/streptomycin (GIBCO, Thermo Fisher Scientific, USA). The culture medium was replaced every 2 days until the cells reached 80% confluency. Cells were sub- cultured and seeded at a density of IxlO 4 cells per well in 96 well-plates.
  • each peptide 50 pg/mL was added to the cells. After 2 days of incubation, 2 mM of calcein AM and 4 mM ethidium homodimer- 1 (LIVE/DEAD® Viability/Cytotoxicity Kit, Life TechnologiesTM) was added to the wells and incubated for 40 min in the dark. Before imaging, the staining solution was removed, and fresh PBS was added. Stained cells were imaged under an inverted confocal microscope (Zeiss Microscope, Germany).
  • a CellTiter-Glo® luminescent 3D cell viability assay was used to determine proliferation of cells according to the amount of ATP produced as an indicator of cellular metabolic activity.
  • About IxlO 4 of cells were seeded per well of a 96-well plate. Then, 50 pg/mL of each peptide was added to the cells. After the incubation time, the kit was equilibrated at room temperature for approximately 30 min.
  • CellTiter-Glo® Reagent equal to the volume of cell culture medium present in each well was added. The contents were mixed for 5 min and then incubated for 30 min. After incubation, the luminescence was recorded using a plate reader (PHERAstar FS, Germany).
  • Immunostaining was performed after the incubation of each peptide with cells for 84 hr as described previously 91 . Briefly, cells were fixed with 4% (w/v) paraformaldehyde solution for 30 min and incubated in cold cytoskeleton buffer (3 mM MgCL, 300 mM sucrose and 0.5% [v/v] Triton X-100 in PBS) for 5 min for permeabilization. The permeabilized cells were incubated in blocking buffer solution (5% [v/v] FBS, 0.1% [v.v] Tween-20, and 0.02% [w/v] sodium azide in PBS) for 30 min at 37 °C.
  • blocking buffer solution 5% [v/v] FBS, 0.1% [v.v] Tween-20, and 0.02% [w/v] sodium azide in PBS
  • F-Actin, rhodamine -phalloidin (1:300) was added to the cells that were then incubated at room temperature in the dark for 1 h, followed by washing three times with IX PBS. Further, the cells were incubated in DAPI (1:2,000) in water for five min to counterstain the nucleus before the DAPI solution was removed by washing with IX PBS. The stained cells were observed and imaged using a laser scanning con- focal microscope (Leica Application Suite X, Leica Stellaris Confocal Microscope, Germany).
  • a mid-logarithmic growth-phase culture was diluted in BM2 medium (62 mM potassium phosphate buffer, pH 7, 7 mM (NH ⁇ SCL, 2 mM MgSCL, 10 pM FeSCh and 0.4% [w/v] glucose) to IxlO 8 CFU/mL and 90 pL of this suspension was seeded in polypropylene microtiter plates (Corning Inc., Corning NY, USA). Bacterial cells were then exposed to varying concentration of AMPs (100 pg/mL to 1.56 pg/mL) and grown overnight at 37 °C in a humidified atmosphere. As an untreated control, bacteria were exposed to BM2 medium without any peptide.
  • biofilms were fixed with 100% methanol for 15 min, washed with PBS and finally air-dried. Dried biofilms were stained with 1% (w/v) crystal violet (Sigma) for 30 min, washed with PBS, and solubilized in 95% (v/v) ethanol for 1 h.
  • the optical density at 595 nm was recorded using TECAN Infinite 200 PRO series (Tecan i-control 2; 2.0.10.0, Austria, a measure of biofilm mass.
  • Untreated bacterial cells were prepared in Ca-MHB and fixed overnight with modified Karnovsky’s fixative (2.5% [w/v] glutaraldehyde and 2% [w/v] paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.35) at 4 °C.
  • modified Karnovsky’s fixative (2.5% [w/v] glutaraldehyde and 2% [w/v] paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.35) at 4 °C.
  • peptide-treated cells suspended cells were filtered using a commercial 50-mL vacuum filter with a 0.22-pm pore-size membrane (Corning Inc., Corning NY, USA) and directly used for fixation.
  • specimens were post-fixed with 1.5% (w/v) potassium ferrocyanide, and 1% (w/v) osmium tetroxide prepared in 0.1 M sodium cacodylate buffer, dehydrated through a graded ethanol series, dried using a critical point dryer (CPD300, Leica, Germany) and sputter- coated with a 10-nm thick platinum layer. All specimens were imaged using a FEI Nova Nano 630 SEM (SmartSEM Version 6.09, Serial Number Merlin-61-95, Oregon, USA) equipped with an Everhart- Thornley detector (ETD) and through a lens detector (TLD) operating at 3 kV.
  • ETD Everhart- Thornley detector
  • TLD lens detector
  • inert protein tags juxta posed by cleavage sequences for the tag release were selected for use in the engineered peptide constructs. Cytosolic accumulation was selected to avoid adding a targeting signal before the desired AMPs.
  • flexible linker GGGSGGGS was added to preserve the functionality of the fused protein, by allowing independent movement of the N and C portions (Fig. 1A).
  • AMPs selected for this study harbor leucine and arginine residues at their N termini, which would normally make them more susceptible to protease degradation via the N-end rule pathway 48,49 .
  • AMPs can take on the characteristics of a signal peptide due to their high hydrophobicity and strong positive charge 50 , and are prone to degradation by the proteases of the secretory path- way between the endoplasmic reticulum (ER) and the Golgi apparatus 51 .
  • SUMO Eul an engineered version of the plant SUMO (Small Ubiquitin-like Modifier) domain termed SUMO Eul was added to the N terminus of the target amino acid sequences of the HDP to increase peptide stability and solubility in the plant cytosol while also ensuring exact cleavage site production without extra residues.
  • SUMO EU1 domain contains three amino acid changes that render it resistant to degradation except by its cognate SUMO-specific protease, SENP EUH53 .
  • this cleavage reaction leaves no residual amino acids 54-56 , thus allowing release of AMPs in their native form, concomitantly averting the elution of non-specific background binders 53 .
  • the protease was previously reported to be efficient in cleaving domains of proteins immobilized on cellulose beads in vitro or within the confined environment of cells in vivo 57 , thereby demonstrating the robustness and precise activity of protease.
  • a C-terminal glycine residue was added to all AMPs as a substrate for eventual PAM-mediated amidation (Fig. 1A).
  • pEAQ-HT carrying parts of genomic RNA2 from cowpea mosaic virus (Fig. 1A), which facilitates hypertranslation of heterologous constructs in plants, and allows the production of AMPs at high titer in the infected leaves.
  • Fig. 1A cowpea mosaic virus
  • the vector used here can be readily delivered into leaves using Agrobacterium, and it does not pose a risk of biocontamination to the environment since it is harboring a deconstructed virus backbone.
  • pEAQ-HT also harbors a P19 post-transcriptional gene silencing suppressor gene, further enhancing gene expression levels.
  • Fig. IB The purification protocol summarized in Fig. IB was utilized. Agrobacteria harboring the /WP-cx pressing constructs was infiltrated into the leaves of N benthamiana at an ODeoo of 0.5. Six days later, total extracts from the infiltrated leaves were probed for protein accumulation by SDS-PAGE (Fig. 1C) and immunoblotting (Fig. ID). During protein purification, polyvinylpolypyrrolidone was added to sequester phenolic contaminants and prevent unwanted proteolysis with a cocktail of protease inhibitors. Solubilized SUMO Eul - AMPs was purified by Strep-tag II affinity chromatography and eluted using 2.5 mM d- desthiobiotin.
  • SEC size exclusion chromatography
  • PAM enzymes stably accumulated and amidated transiently expressed glycine- extended AMPs in plants
  • the PAM cDNAs from the rat genome were then subcloned and expressed individually in N. benthamiana.
  • the encoded PAM enzymes had both PHL and PAM domains, and PAM transcripts often undergo alternative splicing resulting in either integral membrane-bound (PAM1/2) or soluble (PAM3) forms of the enzyme63.
  • the coding sequence of each PAM isoform was cloned into the binary vector pK2GW7 and transiently expressed individually in N. benthamiana leaves (Fig. 2A). Following confirmation of expression (Fig. 2B), the ability of each PAM isoform was assessed to amidate glycine- extended AMPs in planta via co-expression by immunoblotting from total protein extracts using anti-HA antibodies.
  • Recombinant proteins produced in E. coli are generally contaminated with endotoxin, which greatly limits their use as bacterially produced therapeutics.
  • AMPs themselves have an inherent risk of collateral toxicity due to their ability to disrupt mammalian cellular membranes, 8 which often needs to be carefully verified when preparing AMP-based therapeutics before clinical studies.
  • Plant-produced AMPs demonstrated robust killing of ESKAPE pathogens and prevented the formation of their biofilms
  • E. coli PI-7 Escherichia coli PI-7; MRSA USA300: Methicillin resistant Staphylococcus aureus
  • P. aeruginosa Pseudomonas aeruginosa
  • K. pneumoniae Klebsiella pneumoniae
  • A. junii Acinetobacter junii
  • E. faecalis Enterobacter faecalis
  • E. coli PI-7 Escherichia coli PI-7; MRSA USA300: Methicillin resistant Staphylococcus aureus USA300; P. aeruginosa: Pseudomonas aueruginosa; K. pneumoniae: Klebsiella pneumoniae; A. junii: Acinetobacter junii; E. faecalis: Enterobacterfaecalis
  • Extracts obtained from wild type N. benthamiana didn’t exhibit any inhibition in the growth of ESKAPE pathogens (data now shown).
  • purified AMP1 was slightly effective against E. coli PI-7 (50 pg/ml), a BSL-2 class pathogen and antibiotic-resistant strain, 70 against which colistin is the last resort antibiotic and drug of choice for treatment.
  • the three peptides were also efficacious at preventing K. pneumoniae (Fig. 4D, 4F and Fig. 9, 10D, 11 A, and 11B), A. junii (Fig. 4D and 4F, Fig. 9, 10D, 11A and 11B), E. faecalis (Fig. 4D, 4F and 9, 10D, 11 A and 1 IB), and P. aeruginosa (Fig. 4D and 4E, and Fig. 9, 10C, 11 A and 1 IB) biofilm formation, reflecting the widespread and robust antimicrobial activity of plant-produced peptides.
  • Plant-produced AMP1 permeabilized the bacterial membrane, and killed cells
  • Bacterial killing by synthetic AMP1 involves interaction with the bacterial outer membrane, followed by cytoplasmic membrane interaction/permeabilization. 72 To ascertain the mode of action of plant purified AMP1, its killing kinetics on the community-acquired multi-drug resistant clinical isolate MRSA USA300 strain in Ca-MHB (cation-adjusted Mueller-Hinton broth) was determined. Vancomycin (last resort antibiotic that is effective against MRSA USA300) was used as a control that kills bacteria independently of membrane lysis. At a concentration of 2 x MIC, the plant purified AMP1 completely killed an inoculum of 10 8 colony-forming units (CFUs) of bacterial cells within 30-60 min of treatment (Fig. 5A and 5B), as observed previously for the native peptide. In contrast, the control antibiotic vancomycin required >2.5 h for bacterial killing, as expected.
  • CFUs colony-forming units
  • AMP1 was >50% (53.1%) as compared to control (Fig. 5D and Fig. 14), suggesting membrane permeabilization while vancomycin, a cell-wall biosynthesis inhibitor, showed negligible PI accumulation (1.5%) (Fig. 5C and 5D).
  • SEM scanning electron microscopy
  • Colistin is usually a last resort antibiotic for carbapenem-resistant infections, 73 but its pharmacokinetics properties bring major risks for dose-dependent nephrotoxicity and uncertainties in optimal dosing. 74 To investigate whether plant-purified peptides could act synergistically with other antibiotics against which E. coli PI-7 has developed resistance, susceptible antibiotics were screened for, using the standard broth-dilution method. E.
  • coli PI-7 was highly resistant to gentamicin, kanamycin, ceftazidime, sulfamethoxazole, levofloxacin, ciprofloxacin (>350 pM), azithromycin (312.5 pM), but susceptible to colistin (20 pM) (Tabe 1).
  • E. coli PI-7 isolated from sewage water MIC- Mean inhibitory concentration expressed in micromolar concentration. Antibiotic resistance profile of E. coli PI-7 showing that the strain is resistant to antibiotics belonging to the fluoroquinolone class (MIC for levofloxacin and ciprofloxacin: >350 pM), macrolide (MIC for Azi- thromycin: 312.5 p M), cephalosporin (MIC for ceftazidime: >350 pM), aminoglycoside (MIC for gentamicin and kanamycin: >350 pM), sulfonamide (MIC for sulfamethoxazole: >350 pM) but highly susceptible to polymyxin (MIC for colistin: 20 pM).
  • Source data are provided as a Source Data file.
  • Table 4 Unit cost of electricity, labor and utilities referred to the Saudi Arabian.
  • the base case scenario assumes to produce 91 batches a year with each upstream processing batch yielding 9,520 kg N. benthamiana plant FW containing 9.52 kg AMP, assuming an expression level of 1 g AMP per Kg plant FW.
  • the upstream processing steps (37% of total cost, Fig. 12) include growing the plants, large-scale preparation of agrobacteria, vacuum-based infiltration, and post-infiltration incubation.
  • the downstream processing steps (67% of total cost, Fig. 13) involve harvesting leaves, homogenizing whole tissues, and extraction, retrieval, and chromatography-based purification of bulk AMPs.
  • the SuperPro Designer® 13.0 software computed the cost of goods sold (COGS) at $74/g for amidated AMP (Table 5). Table 5. Prices of reagents used in the production of peptides adapted from
  • the final cost encompasses all materials (both raw and consumables), as well as the production costs for the additional chromatography step and protease purification from the E. coli strain that can secrete the target enzyme in the base case scenario. Additionally, the cost of each reagent used has been added in Table 6, and the general COGS using different host chassis (E. coli 18 ’ 16 , mammalian cells 77 ) is summarized in Table 7.
  • Table 7 Table showing economic capital investment, operating expenditures (with and without depreciation) and calculated the cost of goods sold (COGS) for plantbased AMP production scenario.
  • AMPs constitute a promising alternative, since they possess potent antimicrobial and antibiofilm activity even against multi-drug resistant pathogens. 79 Despite decades of research and longstanding promise, no AMPs have been approved by the FDA, except cyclic lipopeptides and gramicidin S, although a few clinical trials have taken place or are underway.
  • N. Benthamiana plants overexpressing rat PAM1 were used to catalyze amidation in planta. These plants tolerated the stable integration of rat PAM1 and exhibited no obvious morphological defects.
  • PAM1 plants were phenotypically normal and retained the ability to produce PAM1 at least up to the T4 generation, although they did produce far fewer seeds for an unknown reason. This effect on the reproductive system should however not constitute a major limitation for biotechnological applications. While efficient peptide amidation has been achieved so far in transgenic rabbits (Oryctolagus cunicidus), 36 this approach requires a sizeable investment in centralized facilities for transgenesis, in contrast to plant transgenesis, which can be performed with minimal infrastructure. Besides, transgenic rabbits producing amidated peptides were reported to have precocious mammary development and reproductive problems. 86
  • plant-produced peptides exhibited marked synergism with azithromycin in curtailing the growth rate of carbapenem-resistant strain E. coli PI-7, potentially adding another antibiotic to clinical management for this strain for which colistin is the last resort drug.
  • the protocol disclosed herein yielded a substantial amount of pure amidated AMPs (> 90%), and this prompted computation of the scalability of this process for industrial-scale production of AMPs
  • the techno-economic analysis simulation estimated the total cost of goods sold (COGS) at $74/g for plant-based production of AMPs. This cost is quite competitive considering that chemical synthesis of the same peptide was priced at $95.29/mg (based on a price quote from a commercial company) and compared against the COGS of E. coli produced cationic peptides produced in batches which ranges from $44.5-$268.16/mg.

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Abstract

La présente invention concerne des compositions et des procédés pour la production maîtrisée in planta d'AMP amidés. Les procédés divulgués utilisent une combinaison ciblée de modules d'expression (a) stables/transitoires et (b) transitoires dans des plantes transgéniques. L'enzyme bifonctionnelle peptidylglycine a-amidante mono-oxygénase (PAM) est utilisée pour introduire la voie d'amidation C-terminale des mammifères dans les plantes et une construction conçue pour coder une protéine de fusion contenant une étiquette de purification, un lieur; une séquence de clivage telle qu'un petit modificateur lié à l'ubiquitine (bdSUMO) contenant des mutations à des positions d'interaction avec SUMO (bdSUMOEul) et la séquence AMP d'intérêt, avec un résidu glycine terminal. Cette stratégie permet d'accumuler des niveaux considérables d'AMPS dans les plantes transgéniques, par comparaison avec les plantes non transgéniques, ainsi qu'avec les procédés précédemment divulgués d'expression des AMP dans les plantes.
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