WO2024166076A1 - Recombinant production of antimicrobial peptides in planta - Google Patents

Abstract

Compositions and methods for the controlled in planta production of amidated AMPs are disclosed. The disclosed methods use a targeted combination of (a) stable/transient and (b) transient expression modules in transgenic plants. The bifunctional peptidylglycine a-amidating mono-oxygenase (PAM) enzyme is used to introduce the mammalian C-terminal amidation pathway into plants and a construct designed to encode a fusion protein containing a purification tag, a 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. This strategy results in accumulation of substantial levels of AMPS in transgenic plants, when compared to nontransgenic, as well as to previously disclosed methods of expression plants to express AMPs.

Description

RECOMBINANT PRODUCTION OF ANTIMICROBIAL PEPTIDES IN PLANTA CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application No. 63/484,445 filed February 10, 2023, the entire content of which is incorporated herein by reference for all purposes in its entirety.

FIELD OF THE INVENTION

This invention is generally in the field of antimicrobial peptide production in planta.

BACKGROUND OF THE INVENTION

Antimicrobial peptides (AMPs) and host defense peptides (HDPs) hold considerable promise for clinical applications. 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 antibiotics¹⁰ underscores their potential use as effective therapeutic drugs. However, despite having myriad applications in clinical medicine, 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. Moreover, 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.¹⁷ Although 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.²⁰ In fact, producing proteins in plants is estimated to cost 10- to 50-times less than E. coli fermentation.²¹ However, in planta production of peptides has proven difficult, presumably due to proteolysis by plant proteases. To circumvent this incompatibility, various strategies have been deployed, such as the downregulation of genes encoding interfering plant proteases²² 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

There is still a need for improved methods for expressing and purifying AMPs from plants.

It is an object of the present invention to provide compositions for improved expression of in AMPs in plants.

It is also an object of the present invention to provide methods for improved expression of AMPs in plants.

SUMMARY OF THE INVENTION

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. Thus, 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.

Constructs for effecting expression of AMPs, preferably, cationic AMPs in plants are disclosed. To ensure high-efficiency purification, 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). 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.

Improved methods for expression of AMPs, preferably, cationic AMPs in plants are disclosed. In one embodiment, a vector containing an AMP-fusion protein expression construct and a vector containing a construct encoding PAM are transiently co-expressed in a plant. In a more preferred embodiment, a vector containing an AMP-fusion protein expression construct is transiently expressed in a plant stably expressing PAM. In a preferred embodiment, the plant is engineered for cytosolic accumulation of the expression product i.e., the AMP fusion protein.

Thus, 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, transgenic plants/plant parts 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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. FIG. 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. Following protease cleavage, His-tagged SENP^EUH is removed using Ni-affinity chromatography and isolated AMPs are further purified by size-exclusion chromatography (SEC). Next, the pooled SEC fractions are applied to a reverse-phase high-performance liquid chromatograph (RP-HPLC) for final purification of AMPs. FIG. 1C shows analysis of the isolated proteins by SDS-PAGE. Strep-II affinity-captured SUMO-fused AMPs were separated by SDS-PAGE and gels were stained with Coomassie Brilliant blue. 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. benthamiana leaves along with a plasmid encoding P19 (OD600 = 0.2). Leaves were harvested 3 dpi, total proteins were extracted and analyzed by immunoblotting using a monoclonal anti-HA antibody. The predicted molecular masses were 120 kDa (PAM1), 105 kDa (PAM2) and 95 kDa (PAM3). The three black arrowheads indicate the bands corresponding to PAM1, PAM2, and PAM3 enzymes. Two independent blots were performed with similar results. FIG. 2C shows in planta transient co-expression of AMPs and PAM enzymes. Constructs encoding AMPs and PAMs were transiently co-expressed in N. benthamiana (1:1 ratio) and the extracts were immunoblotted using anti-HA antibody. Bottom arrowheads indicate the expected size of proteins and peptides, top arrowheads indicate possible in planta modified AMPs, while the asterisk indicates the nonspecific bands. Two independent blots were performed with similar results. 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 and FIG. 3B is a bar graph illustration of metabolic index of both peptide treated (300, 200, 100, 50, 25, 12.5, 6.25 pg/ml) and untreated cells as measured by estimating the cellular ATP levels using luciferase-based CellTiter-Glo® reagent. Cell viability was quantified as the ratio of ATP levels-dependent luminescence in treated / untreated cells expressed as percentage. Untreated: cells in a tissue culture plate treated with PBS; Negative control: cells treated with 1% [v/v] Triton-X 100 (± SD, n=3 independent biological replicates).

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. For each assay, 10⁶ colonyforming units (CFU)/ml of each ESKAPE (E. coli PI-7, MRS A USA300, P. aeruginosa, K. pneumoniae, A. junii, E. faecalis) pathogen were treated with 1=100 pg/ml, 2=50 pg/ml, 3=25 pg/ml, 4=12.5 pg/ml, 5=6.25 pg/ml, 6=3.215 pg/ml, 7=1.56 pg/ml of peptides in cation-adjusted Mueller-Hinton broth for 24 h. Up to >50% reduction of carbapenem resistant 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. faecalis (pp: 25 pg/ml, sp: 25 pg/ml); pp: purified peptide; sp: synthetic peptide. Data are mean ± SD of three independent experiments performed in duplicates. 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. aeruginosa at 25 pg/ml, A. junii at 50 pg/ml, K. pneumoniae at 6.25 pg/ml, E.faecalis at 50 pg/ml (P = 0.0022). *, 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. FIGs. 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 10⁶ CFU/ml of MRSA USA300. MIC for pp AMP1 was ~25 pg/ml in tryptic soy broth; MIC for vancomycin was 0.7 pM (± SD, n = 3 independent experiments). FIGs. 5B and 5C show killing kinetics (FIG. 5B) n = 3 independent experiments and influx of propidium iodide dye (FIG. 5C) as measured by flow cytometry. The controls used were as follows: x-axis, 0 = no antimicrobial activity; MRSA USA300 exposed to 70% ethanol is equivalent to 100% killing or influx of PI dye; and cell-wall biosynthesis inhibitor vancomycin (used at 2x MIC). 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. P = 0.0046, one-way ANOVA, Dunnett’s test and P = 0.0286, two-tailed Mann-Whitney rank sum test.

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 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. Abundance of Strep- II tag-captured SUMO-fused FK13, YI12, Guavanin 2, WLBU2, CONGA, DBS1, GFP, Mastoparan 4,1 as detected with a monoclonal anti-HA after transient infiltration in transgenic N. benthamiana expressing PAM1. 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. As a positive control, E. coli PI-7 (100 pg/mL sodium azide), MRSA USA300 (40 pM colistin), P. aeruginosa (40 pM colistin), K. pneumoniae (40 pM colistin), A. junii (16 pg/mL meropenem), E.faecalis (2% chlorhexidine prepared in 70% ethanol) were used. Data are means ± SEM of two independent experiments. 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. 9. Plant-purified peptides display low toxicity in mammalian cells. Bar graph illustration of metabolic index of both peptides treated (50 pg/mL) and untreated HEK293 cells as measured by estimating the cellular ATP levels using luciferase-based CellTiter- Glo® reagent. Cell viability was quantified as the ratio of ATP levels-dependent luminescence in treated / untreated cells expressed as percentage (± SD, n = 3 independent biological replicates).

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. For each concentration of peptide, 10⁶ colony-forming units (CFU)/mL of each ESKAPE pathogens were treated with 1=100 pg/mL, 2=50 pg/mL, 3=25 pg/mL, 4=12.5 pg/mL, 5=6.25 pg/mL, 6=3.215 pg/mL, 7=1.56 pg/mL of peptides in cation-adjusted Mueller-Hinton broth (Ca-MHB) for 24 h. Percentage inhibition was up to >40% on carbapenem-resistant E. 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). pp, plant-purified peptide; sp, synthetic peptide. Data are means ± SD of two independent experiments performed in duplicates. 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, 10⁶ 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. coli PI-7 in OD600, at a concentration of pp: 50 pg/mL, 90% of inhibition of MRSA USA300 (pp: 25 pg/mL), K. pneumoniae (pp: 25 pg/mL), A. Junii (pp: 25 pg/mL), E. faecalis (pp: 50 pg/mL), P. aeruginosa, (pp: 12.5 pg/mL). pp, plant-purified peptide. Data are means ± SD of two independent experiments performed in duplicates. 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 . Mid-logarithmic growth-phase culture of MRSA USA300 (OD 600 = 0.5 = 108 CFU cells/mL) without any treatment were interrogated with 25 DL of 1 mg/mL propidium iodide for 30 mins in dark. 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. Upstream process flowsheet for N. benthamiana base case scenario in the SuperPro Designer model with a production capacity at 300 Kg/year.

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.

DETAILED DESCRIPTION OF THE INVENTION

This work describes a unique approach for the controlled production of amidated AMPs by a targeted combination of transgenic and transient expression modules in N. benthamiana. PAM enzymes from rats (Rattus norvegicus) are used to introduce the mammalian C-terminal amidation pathway into N. benthamiana plants. For transient production of AMPs, the amino acid sequences of three synthetic HDPs (host defense peptides) that have demonstrate potent antimicrobial, antibiofilm and immunomodulatory activity profiles against drug-resistant pathogens in vitro and in vivo, are selected as prototypes.^7,41,42 Specifically, 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. Moreover, a titer for these amidated peptides of 1.4 mg per 20 g of transgenic plant tissue, was attained. Importantly, these purified peptides are biologically active against ESKAPE pathogens, with low collateral toxicity towards mammalian cells. Overall, the data in this application highlight the exceptional flexibility of plant-based production platforms for potential large-scale production of amidated AMPs.

I. DEFINITITIONS

The term “agroinfiltration” as used herein refers to a method in plant biology to transfer genetic cassettes from Agrobacterium into a plant. In the method 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.

The term "cell" refers to a membrane -bound biological unit capable of replication or division.

The term “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.

As used herein, a “cultivar” refers to a cultivated variety.

As used herein, the term “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.

The term “expression” as used herein 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.

The term “expression vector” refers to a vector that includes one or more expression control sequences.

The term “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, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. In eukaryotes the term “expression vector” refers to a vector that includes one or more expression control sequences regardless of the origin of the sequence (prokaryote or eukaryote).

The term “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. The term “gene” also refers to a DNA sequence that encodes an RNA product. The term 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.

As used herein, “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.

The term “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.

As used herein the term “heterologous” means from another host. The other host can be the same or different species.

The term “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.

The term “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.

The term “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.

The term “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.

The term “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.

As used herein, “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. The term “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. The term “plant part” as used herein refers to a plant structure, a plant organ, or a plant tissue.

The term “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.

As used herein, the term “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.

The term "stable" as used herein 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" as used herein 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.

The term “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. Thus, 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). It is understood that the term 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.

A “transgene” as used herein 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.

The terms “transformed,” “transgenic,” “transfected” and “recombinant” refer to a host organism such as a bacterium or a plant into which a exogenous nucleic acid molecule has been introduced.

The term “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.

Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/- 10%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 5%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 2%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the description and does not pose a limitation on the scope of the description unless otherwise claimed.

All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the embodiments unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

II. 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. In a preferred embodiment, 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. Microbiol., Volume 11 - 2020 I https://doi.org/10.3389/fmicb.2020.582779; FPLTWLKWWKWKK-C0NH2) (SEQ ID NO: 66); YI12 (YLRLIRYMAKMI-C0NH2 (SEQ ID NO: 67)(Das, et al., Nat Biomed Eng 5, 613-623 (2021)); 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).

A. Genetically modified Constructs encoding AMP

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. Any known 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).

Other exemplary a purification tags (an amino acid sequence that allows the polypeptides to be captured onto an affinity matrix), include, but are not limited to c-myc, polyhistidine, or Flag™ (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.

B. Expression vectors

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. For example, 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; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “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. The terms “plasmid”, “vector” and “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

The 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)). In one embodiment, promoters are selected from those of eukaryotic or synthetic origin that are known to yield high levels of expression in plant and algae cytosol. In another embodiment, promoters are selected from those of plant or prokaryotic origin that are known to yield high expression in plastids. In certain embodiments the promoters are inducible. Inducible plant promoters are known in the art. In one embodiment, the promoter is an egg cell-specific promoter.

Many plant 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. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); and ALS promoter (U.S. Pat. No. 5,659,026). Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142.

"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. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129- 1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.

"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. Such 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. For dicots, 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. For monocots, 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. (1991) 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. Depending upon the objective, 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. Other chemical-regulated 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.

Transcription Termination Sequences

At the extreme 3’ end of the transcript of the transgene, a polyadenylation signal can be engineered. 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).

Selectable Markers

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. 5,276,268), the expression of aminoglycoside 3”- adenyltransferase (aadA) to confer spectinomycin resistance (U.S. Patent No. 5,073,675), the use of inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase (U.S. Patent No. 4,535,060) and methods for producing glyphosate tolerant plants (U.S. Patent No. 5,463,175; U.S. Patent No. 7,045,684). Methods of plant selection that do not use antibiotics or herbicides as a selective agent have been previously described and include expression of glucosamine-6-phosphate deaminase to inactive glucosamine in plant selection medium (U.S. Pat. No. 6,444,878) and a positive/negative system that utilizes D-amino acids (Erikson et al., Nat Biotechnol, 2004, 22, 455-8). European Patent Publication No. EP 0 530 129 Al describes a positive selection system which enables the transformed plants to outgrow the non-transformed lines by expressing a transgene encoding an enzyme that activates an inactive compound added to the growth media. 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). 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. (2002), Nat Biotech 20: 87-90), the blue fluorescent protein, the cyan fluorescent protein, and the green fluorescent protein (Sheen et al. (1995), Plant J 8: 777-84; Davis and Vierstra (1998), Plant Molecular Biology 36: 521-528). A summary of fluorescent proteins can be found in Tzfira et al. (2005), Plant Molecular Biology 57: 503-516) and Verkhusha, et al. (2004), Nat Biotech 22: 289-296) whose references are incorporated in entirety. Improved versions of many of the fluorescent proteins have been made for various applications. Use of the improved versions of these proteins or the use of combinations of these proteins for selection of transformants will be obvious to those skilled in the art. It is also practical to simply analyze progeny from transformation events for the presence of the PHB thereby avoiding the use of any selectable marker.

C. Modified Plants/Plant parts Recombinant/transgenic plant and plant parts are disclosed in which the disclosed constructs have been introduced. 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.

Plant Types

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. Siv'on, Gypsophila elegans, Helianthus annuus, Hyacinthus, Hyoscyamus niger, Lactuca sativa, Lathyrus odoratus, Linum usitatissimum, Lobelia erinus, Lupinus mutabilis, Lycopersicon esculentum, Lycopersicon pimpinellifolium, Melilotus albus, Momordica balsamina, Myosotis sylvalica, Narcissus pseudonarcissus, Nicandra physalodes, Nicotiana benthamiana, Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana rustica, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana edwardsonii, Ocimum basilicum, Petunia hybrida, Phaseolus vulgaris, Phytolacca Americana, Pisum sativum, Raphanus sativus, Ricinus communis, Rosa sericea, Salvia splendens, Senecio vulgaris, Solarium lycopersicum, Solatium melongena, Solanum nigrum, Solanum tuberosum, Solanum pimpinellifolium, Spinacia oleracea, Stellaria media, Sweet Wormwood, Trifolium pratense, Trifolium repens, Tropaeolum majus, Tulipa, Vicia faba, Vida villosa and Viola arvensis. Other plants that may be infected include Zea maize, Hordeum vulgare, Triticum aeslivum, Oryza sativa and Oryza glaberrima.

III. METHODS OF MAKING AND USING

The disclosed constructions and vectors are introduced into a plant of choice using methods known in the art, discussed briefly below, and exemplified in the Examples of the present application. 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. In each generation, 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. 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.

In some forms, a vector containing an AMP-fusion protein expression construct and a vector containing a construct encoding PAM are transiently co-expressed in a plant. In some forms, 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. In a preferred embodiment, the plant is engineered for cytosolic accumulation of the expression product i.e., the AMP fusion protein.

In some forms, 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. Also disclosed is 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.

Also disclosed is 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. The use of low molecular- weight filters to separate the released peptide from uncleaved fusion protein was avoided, since peptides tend to adsorb to filter matrices by ionic and hydrophobic interactions. Instead, size exclusion chromatography (SEC) using organic modifiers was utilized, for two reasons: i) SEC facilitates desalting in contrast to affinity-based chromatography that includes salts that could result in nonspecific matrix interactions with the hydrophobic, cationic peptides, and ii) SEC columns allow buffer exchange into the highly volatile mobile phase buffer for subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) analysis. All three AMP peptides eluted at the earliest stage between 1- and 2-ml at a flow rate of 0.01 ml/min (Extended Data Fig. lb); all eluted fractions covering the peaks were collected for purification via RP-HPLC, which served as an additional desalting step.61 RP-HPLC was performed 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.

A. Methods of Plant Transformation

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. In a preferred embodiments, 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. Thus, 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. Gamborg and Phillips (Springer- Verlag, Berlin); and McCabe et al. Biotechnology 6:923-926 (1988)). 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). This system has been extensively exploited in agarobacterium mediated plant transformations. One method used floral dip. In this method, transformation of female gametes is accomplished by simply dipping plant inflorescences for a few seconds into a 5% sucrose solution containing 0.01-0.05% (vol/vol) of the surfactant Silwet L-77. The optimal growth stage for transformation by floral dip was when plants contained numerous unopened floral buds. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants, (described in detail in Clough et al., The Plant Journal 16(6):735-743 1998). The floral dip method is exemplified in the Examples section of this disclosure. Another method uses agroinfiltration. In the method 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. In this procedure, 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. When the vacuum is released, the pressure difference forces solution through the stomata and into the mesophyll.

B. Methods for Reproducing Transgenic Plants

Following transformation by any one of the methods described above, 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 present invention will be further understood by reference to the following nonlimiting examples.

EXAMPLES

Methods

Construction of synthetic glycine-extended AMPs for in planta expression

All synthetic antimicrobial gene cassettes comprising sequences encoding the streptavidin-tag II, 3x HA epitope, mutated bdSUMO^Eul module fused to the N terminus of each AMP and the terminal glycine residues were ordered as gBlocks gene fragments (shown below, IDT, Leuven, Belgium; gBlocks sequences containing sequences encoding:

Strep-tag II, underlined; followed by,

HA epitope in italics; followed by,

Flexible linker: in bold font, followed by, mutated SUMO domain, italics and underlined: respective AMP sequences), followed by

AMP sequence or GFP. gBLOCK AMP1

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGA7TACGC7TATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G77GC.47C.4GAC7GG7GG7AGGAGGTGGGCCAGGAGGCTAGCCTTTGCTTTTAGGAGGGGGTAACTCGA

GGAT (SEQ ID NO:1). gBLOCK AMP2

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGA7TACGC7TATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G7TGCATCAGACTGGTGG7GTACAGAGATGGTTGATAGTATGGAGAATAAGAAAGGGATAATTTTTTTC

TCGAGGAT (SEQ ID NOG). gBLOCK AMP3

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGA7TACGC7TATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G77GC.47C.4GAC7GG7GG7ATTTTGGTTAGGTGGATTAGGTGGAGGATTCAGTGGGGATAATTTTTTTCT

CGAGGAT (SEQ ID NOG). gBlock GFP

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGA7TACGC7TATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G7TGCATCAGACTGGTGGTATGGCTAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTT

GAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCTACATA

CGGAAAGCTTACACTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCAC

TACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAG

AGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGAC

GCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAA

AGAAGATGGAAACATTCTCGGACACAAATTAGAGTACAACTATAACTCACACAATGTATACATCACGG

CAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGT

TCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACC

ATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTG

AGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAACTCGAGGAT (SEQ ID NO:4). gBlock CONGA

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGAT7ACGCT7ATCCC7A

CGACGTGCCTGA7TATGCA7ACCCA7ATGATGTCCCCGAC7ATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G77GCA7CAGAC7GG7GG7AGGAGGTGGGCCAGGAGGCTAGCCTTTGCTTTTAGGAGGGGGTAACTCGA

GGAT (SEQ ID NO:5). gBlock DBS1

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGAT7ACGCT7ATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G77GCA7CAGAC7GG7GG7AGGAGAGGTTGGGCTAGGAGACTTTTTTTCGCTTATGGTAGGAGGGGGTA

ACTCGAGGAT (SEQ ID NO:6). gBlock FK13

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAGTACCCA7ACGATGTTCCAGAT7ACGCT7ATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G7TGCATCAGACTGGTGG7TTTCCCTTGACATGGTTGAAGTGGTGGAAGTGGAAGAAGGGGTAACTCGA GGAT (SEQ ID NO:7). gBlock Guavanin 2

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGA7TACGC7TATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G77GCA7CAGAC7GG7GG7AGACAATATATGAGACAAATAGAGCAGGCCTTGAGATATGGTTATAGAAT

TTCAAGAAGAGGGTAACTCGAGGAT (SEQ ID NO:8). gBlock Mastoparan R1

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGA7TACGC7TATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G77GC.47C.4G.4C7GG7GG7AAGATTTTGAAGAGACTAGCAGCAAAGATTAAGAAGATTTTGGGGTAACT

CGAGGAT (SEQ ID NO:9). gBlock Mastoparan R4

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGAT7ACGCT7ATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G7TGCATCAGACTGGTGGTATAAACCTAAAAAAGCTAGCAGCAAGGATAAAGAAGAAGATAGGGTAAC

TCGAGGAT (SEQ ID NO: 10). gBlock WLBU2

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGAT7ACGCT7ATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA

GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT

G77GC.47C.4G.4C7GG7GG7AGGAGGTGGGTTAGAAGGGTCCGACGCGTATGGCGCCGAGTGGTCAGAG

TCGTACGACGCTGGGTGCGACGCGGGTAACTCGAGGATGAT (SEQ ID NO:11). gBlock YI12

ATCACCGGTATGTGGAGCCACCCGCAGTTCGAAAAG7ACCCA7ACGATG7TCCAGAT7ACGCT7ATCCC7A

CGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCGGTGGGGGTGGGAGCGGTGGGGG

TGGGAGCCATATTAATTTGAAGGTTAAGGGACAAGATGGGAATGAGGTATTTTTCCGTATTAAACGGTCTACA

CAGTTGAAGAAGCTTATGAACGCCTACTGTGATAGACAGTCTGTTGACATGAAGGCAATAGCATTTCTCTTTAA GGGACGTAGATTGAGAGCTGAGAGAACGCCAGATGAACTTGAAATGGAGGACGGGGATGAGATTGACGCTAT G7TGCATCAGACTGGTGG7TATTTGAGATTGATAAGATATATGGCAAAGATGATAGGGTAACTCGAGGA T (SEQ ID NO: 12).

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). The constructs were verified by Sanger sequencing using a forward primer complementary to the vector backbone and a reverse primer complementary to the 3’ end sequence of the AMP gene cassette. All 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.

Cloning of the rat PAM gene

Plasmids encoding the rat variants of PAM enzymes (PAM 1, 2 & 3) were kindly provided by Prof. Betty Eipper, University of Connecticut Health Center, USA. Using PCR, 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). To facilitate directional cloning into the inter- mediate vector pENTR™ D-TOPO® (Invitrogen), 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. Next, 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.

Generation of transgenic N. benthamiana plants overexpressing PAM

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 protocol⁸⁹. 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. 100 pM acetosyringone) were grown onto solid MS media (4.4 g/L Murashige and Skoog basal salts [MS], 30 g/L sucrose) in a growth chamber with the temperature set to 28 °C. After 2 days, the explants bearing the integrated transgene were selected and regenerated onto media (4.4 g/L MS salts, 1 mg/L 6- benzylaminopurine ,

0.1 mg/L 1 -naphthaleneacetic acid, 30 g/L sucrose, 50 mg/L kanamycin, 200 mg/L timentin, pH 5.8) in a growth chamber with the temperature set to 25 °C and a 13-h light/11-h dark regime. Following growth hormones-mediated shoots induction which take at least 3-4 weeks, the shoots were excised and transferred for rooting onto media (2.2 g/L MS salts, 50 mg/L kanamycin). All the reagents were purchased from Sigma-Aldrich. Finally, kanamycin- resistant lines forming proper roots (2-3 weeks) 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.

Purification of SENP^EuH protease

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]). 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 HisTrap™ 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). The column was extensively washed with ten column volumes (CVs) of buffer A and the bound protease was eluted in ten CV fractions against buffer B (50 mM Tris pH 7.5, 500 mM NaCl, 500 mM imidazole, 5% [v/v] glycerol). 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). 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.

Large-scale purification of peptides

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. Since 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 HisTrap™ 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. 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. 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. The peak fractions were analyzed on 18% Tricine-SDS-PAGE gels, flash-frozen in liquid nitrogen, and stored at -80 °C until further processing. Peptide fractions were further desalted and separated using reverse -phase chromatography. The semipreparative column used was 9.4 x250 mm ZORBAX RX-C8 with particle size of 5 pm (Agilent Technologies, USA), fractions containing peptides were loaded onto the column using the 1-mL loop present in the 1260 Infinity HPLC system (OpenLAB CDS ChemStation C.01.07 SR2, Agilent Technologies, USA) and eluted using linear gradient from 5% (v/v) CH3CN/0.01 M HC1 to 80% (v/v) CH3CN/0.01 M HC1 at a flow rate of 1 mL/min. Fractions absorbing at 215 nm/280 nm were then collected, subjected to freeze- drying and stored at - 20 °C until use.

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).

Antibiotics and synthetic antimicrobial peptides

For in vitro studies, 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.

Bacterial strains and media

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.

Minimal inhibitory concentration (MIC) assay The MIC values were determined using broth microdilutions in accordance to the Clinical Laboratory Standards Institute (CLSI) guidelines using cation-adjusted Mueller- Hinton broth (Ca-MHB) with minor modifications⁹⁰. Briefly, bacteria were grown overnight in the appropriate media at 37 °C with shaking. The overnight culture was washed with PBS and centrifuged at 3220 xg at room temperature for 10 minutes with a final resuspension in PBS to an OD600 = 0.50 approximating 10⁸ CFU/mL. Bacterial stocks were diluted to an inoculum of 10⁶ CFU/mL in Ca-MHB. Ninety microliters of this sus- pension were added to each well of 96-well round bottom plates (Costar) along with 10 pL of diluted antibiotics/peptides at varying concentrations. The 96-well plates were wrapped in paraffin and placed in a shaking incubator at 37 °C. The OD600 readings were taken after 24 h using a TECAN Infinite 200 PRO series (Tecan i-control 2; 2.0.10.0, Austria, GmbH). The MIC was considered as the lowest con- centration of peptide that completely inhibited the visible growth of bacteria after 24 h of incubation of the plates at 37 °C. Data are presented as two independent experiments performed in duplicates.

Immunoblot analysis

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. 2 mM NaF, 10 pM MG132). Low molecular-weight pep- tides (<20 kDa) were separated on 18% Tricine-SDS-PAGE gels. Proteins > 20 kDa were resolved on 4-20% Tris-HEPES gels (Thermo Fisher Scientific, USA). The separated proteins were transferred to a poly- vinylidene difluoride membrane with a pore size of 0.45 pm (Amer- sham Hybond-P; GE Healthcare Life Sciences). The membranes were blocked with 5% (w/v) BSA (Sigma), incubated overnight with primary antibodies rat anti-HA (1: 1000, Sigma, clone name: 3F10), anti-GFP (1:1500, ab6556, polyclonal) and subsequently with respective HRP- conjugated secondary antibodies goat anti-rat IgG (1:4000, Sigma), goat anti-rabbit IgG (1:2000, ab205718). Immunoblotting bands were visualized using enhanced chemiluminescence reagent (ECL kit; Amersham Pharmacia Biotech) and blot images were acquired using a ImageQuant LAS 4000 (Version 1.0, GE Healthcare Biosciences). In case of SDS-PAGE, images were acquired using a ChemiDoc MP system (Image lab Version 6.0.1, BioRad). Membrane permeabilization assay

A mid-logarithmic growth-phase culture was diluted to 1 x 10⁸ 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 LSRFortessa™ 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).

Cell cultured and live/dead staining

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 IxlO⁴ cells per well in 96 well-plates. Then, 50 pg/mL of each peptide (maximum reported MIC) 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 Technologies™) 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).

Proliferation assay

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 IxlO⁴ 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).

Cytoskeleton staining

Immunostaining was performed after the incubation of each peptide with cells for 84 hr as described previously⁹¹. 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. Then, 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).

Prevention of biofilm formation

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 IxlO⁸ 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. After 24 h of incubation, planktonic bacterial growth was aspirated out, 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.

Scanning electron microscopy (SEM)

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. For 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. There- after, 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.

Techno-economic analysis for industrial scale production of AMPs

A techno-economic analysis was performed to ensure the feasibility of the developed module for industrial-scale production to establish the requirements, constraints, cost drivers, and capital cost estimation required for a large-scale AMP manufacturing facility. Plant-based AMP production and purification was modelled on a previously published base case scenario75 using the SuperPro Designer® 13.0 software (https://www.intelligen.com; accessed from December 2nd to 15th, 2022) considering the upstream processing batch yields 9,520 kg N. benthamiana plant FW containing 9.52 kg AMP, with an expression level of 1 g AMP per kg plant FW. The model proposed vacuum-based infiltration of N. benthamiana, wherein plants were infiltrated transiently using vacuum in batches. Seed germination efficiency was considered to be >95%, with an estimated cost of

$9.50/g seed. The overall COGS was calculated considering all materials (raw and consumables) including the production costs (utilities, facility-dependent costs, waste disposal and labor) divided by the product output. Electricity, labor, hot and cold-water charges were estimated as per the Saudi Arabia local standards value. All currency is listed in USD.

Statistical analysis

Where applicable, data are shown as means ± SD with error bars. Two-sample comparisons were made using a Mann-Whitney rank sum test. For all tests, P < 0.05 was considered significant.

Results

Design of constructs for in planta production of AMPs

To date, few successful examples have reported using plants as a platform for large- scale production of AMPs^43-45, perhaps reflecting an inherent incompatibility between the plant host and the heterologous peptides to be produced⁴⁶. The present studies leveraged synthetic biology to unlock the potential of N. benthamiana as a cost-effective chassis for large-scale production of hydrophobic-rich, cationic- charged AMPs. To ensure high- efficiency purification, the eight amino-acid Strep-tag II sequence were added to the AMPs for high-affinity binding to the engineered streptavidin Strep-Tactin⁴⁷ (Fig. 1A). An elastinlike polypeptide (ELP) tag was previously employed for purification of AMPs in plants⁴⁶. However, even though the ELP tag is inert, a tag or residual sequences in the final peptide backbone may alter the structural conformation of the final AMPs and reduce their overall efficacy. Therefore, 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. Lastly, the 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).

The prototypic 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. Moreover, AMPs can take on the characteristics of a signal peptide due to their high hydrophobicity and strong positive charge⁵⁰, and are prone to degradation by the proteases of the secretory path- way between the endoplasmic reticulum (ER) and the Golgi apparatus⁵¹. Here, 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. While eukaryotes possess endogenous SUMO proteases that can cleave SUMO- tagged proteins in vivo⁵², the SUMO^EU1 domain contains three amino acid changes that render it resistant to degradation except by its cognate SUMO-specific protease, SENP^EUH53. Importantly, 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 binders⁵³. Moreover, 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 vivo⁵⁷, 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).

All elements were introduced into the plant vector 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.⁵⁸ The use of whole-viruses to produce AMPs was previously described in plants⁵⁹ but suffers from several drawbacks in terms of transgene size that can be cloned, and concerns associated with biocontainment.⁶⁰ 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. Notably, pEAQ-HT also harbors a P19 post-transcriptional gene silencing suppressor gene, further enhancing gene expression levels.

Transient expression and purification of AMPs

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. All three recombinant SUMO-fused peptides were successfully detected in the leaf extracts (Fig. 1C and ID). The introduced SUMO^Eul domain facilitated the release of native AMPs from the rest of the produced protein via proteolytic cleavage with SENP^EUH, as indicated by the mass shift after electrophoresis (Figure IE). The gel was immersed directly in an aqueous Coomassie brilliant blue G-250 solution containing 40% methanol and 4% formaldehyde to help retain smaller peptides. To ascertain that the bands were not an artefact of bromophenol blue in the sample loading buffer, protein separation was performed with or without bromophenol blue in the loading dye and obtained the same results (Fig. 7A). Histidine-tagged protease was removed from the solution by Ni-affinity chromatography.

The use of low molecular- weight filters to separate the released peptide from uncleaved fusion protein was avoided, since peptides tend to adsorb to filter matrices by ionic and hydrophobic interactions. Instead, size exclusion chromatography (SEC) using organic modifiers was utilized, for two reasons: i) SEC facilitates desalting in contrast to affinitybased chromatography that includes salts that could result in nonspecific matrix interactions with the hydrophobic, cationic pep- tides, and ii) SEC columns allow buffer exchange into the highly vola- tile mobile phase buffer for subsequent reverse-phase high- performance liquid chromatography (RP-HPLC) analysis. All three AMP peptides eluted at the earliest stage between 1- and 2-mL at a flow rate of 0.01 mL/min (Fig. 7B); all eluted fractions covering the peaks were collected for purification via RP-HPLC, which served as an additional desalting step⁶¹. RP-HPLC was carried out 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 ion⁶². The three experimental peptides eluted from the column at around 80% acetonitrile between 9-12 min (Fig. 7C). Analysis of the fractions on 18% Tricine-SDS-PAG gels revealed single peptide bands for each peptide after staining with Coomassie brilliant blue G-250 solution containing 40% methanol and 4% formaldehyde (Fig. 1 IF), highlighting the high purity of the peptides. Peptide masses were confirmed by ESI- MS based on their expected m/z ratios (1018-G: 1593.01 m/z)', (1002-G: 1709.0361 m/z)-, (3002-G: 1781.0361 m/z) (Fig. 1G). Such peaks were not present in the extract obtained from wild type N. benthamiana (data not shown).

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. Specific signals of the expected molecular weights of 15 kDa (for AMPs) and 110 kDa (for PAM1) were observed in extracts from co-infiltrated leaves (Fig. 2C). A pattern of retarded bands was also noticed, migrating closely with the AMPs signal in extracts fromPAMl and AMPs co-infiltrated leaves, no such retarded bands pattern were observed in extracts from leaves infiltrated with the AMP constructs alone (Fig. 2C). The appearance of extra bands suggests the post-translational modification of AMPs mediated by transiently expressed PAM1 enzyme.

Therefore stable N. benthamiana transgenic lines expressing PAM1 as a source of in planta amidation were generated. Importantly, the resulting transgenic PAM1 lines exhibited no obvious morphological or developmental changes relative to nontransgenic N. benthamiana, aside from a considerable decrease in seed yield. Subsequent studies tested whether these transgenic lines were able to amidate glycine-extended AMPs produced from a transiently infiltrated construct. Following Agrobacterium-mediated infiltration of the AMP constructs, the three AMPs were cleaved by SUMO protease and purified as above. RP- HPLC analysis detected one major peak and one minor peak at 215 nm for each AMP after separation on a C8 column (Fig. 2D). The peaks were consistent with previous work on the in vitro amidation of a precursor peptide produced in E. coli.^M The percentage of amidation, was estimated by RP-HPLC, to be more than 80% of total AMP abundance. The amidation of the peptides was confirmed by electrospray ionization time-of-flight mass spectrometry (ESI- TOF-MS) analysis, as evidenced by the expected mA. ratios (Fig. 2E) (AMP1-1536.93, AMP2- 1653.05, AMP3-1725.12).

Using the established protocol herein, other AMPs were also successfully purified as SUMO fusions: FK13, YI12, Guavanin 2, WLBU2, CONGA, DBS1, Mastoparan 4,1, and even the green fluorescent protein (GFP) (Fig. 7D and 7E). Interestingly, the amount of peptide produced in the PAM1 transgenic plants was quite substantial (1.4 mg per 20 g of leaf biomass) relative to that obtained from nontransgenic N. benthamiana plants (0.39 mg per 20 g of leaf biomass). It could be possible that C-terminal amidation shields peptides from proteases in transgenic plants, as previously reported for peptides in human serum.⁶⁵ The overall yields of individual downstream processing step are also summarized (Fig. 7F). Taken together, these results demonstrate that this approach is suitable for plant-based production of AMPs with a defined terminal C-amide residue.

In planta purified peptides exhibited low toxicity towards mammalian cells

Recombinant proteins produced in E. coli, are generally contaminated with endotoxin, which greatly limits their use as bacterially produced therapeutics.⁶⁶ Minor traces of endotoxin impair cellular proliferation⁶⁷ and can induce programmed cell death.⁶⁸ While plants contain very low levels of endotoxin,⁶⁹ Agrobacterium in in the disclosed system could result in endotoxin contamination, since the bacteria can survive for weeks in the plant intercellular space. 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. We, therefore, tested the biocompatibility of each plant-produced purified AMP on in vitro cultured human embryonic kidney 293 (HEK293) cells. At a concentration of 50 pg/ml, each peptide exhibited no adverse effects on HEK293 cells, as shown by a live/dead cytotoxicity assay (data not shown). Adenosine triphosphate quantification assay further demonstrated no differences in ATP production between all groups, indicating that cell proliferation of treated cells is comparable to the control (FIG. 8). Further immunostaining of F-actin filament using fluorescently labeled phalloidin revealed no changes in the morphological characteristics of treated cells thereby asserting the low cytotoxic effect of each peptide on mammalian cells (data not shown). However, mild cytotoxicity was observed at a higher concentration of 100 pg/ml tested for each purified peptide, as shown by the sensitive ATP quantifying assay (Fig. 3B). The estimated ICso value of each peptide were 126.4, 147.9 and 140 pg/ml for AMP1, AMP2 and AMP3 respectively (Fig. 3A). Based on the data, the plant-produced peptides according to the methods disclosed herein have extremely low (50 pg/ml) to mild cytotoxicity at higher concentrations (100 pg/ml).

Plant-produced AMPs demonstrated robust killing of ESKAPE pathogens and prevented the formation of their biofilms The activity of purified plant-derived peptides, in parallel with synthetically generated peptides, was assessed against a panel of multi-drug resistant pathogens belonging to the group of clinically important ESKAPE (E. coli PI-7, MRSA USA300, P. aeruginosa, K. pneumoniae, A. junii, E. faecalis.) bacterial pathogens using a standard killing assay in cation-adjusted Mueller-Hinton broth medium. All three plant-purified AMPs inhibited the growth of ESKAPE pathogens at concentrations between 6.25 pg/ml and 50 pg/ml (Fig. 4A- 4C, Fig. 9, Fig. 4A, 4B and Tables 2, 3).

Table 2. Antimicrobial activity of plant purified AMP1 peptide.

E. coli PI-7: Escherichia coli PI-7; MRSA USA300: Methicillin resistant Staphylococcus aureus

USA300; P. aeruginosa: Pseudomonas aeruginosa; K. pneumoniae: Klebsiella pneumoniae; A. junii: Acinetobacter junii; E. faecalis: Enterobacter faecalis

Table 3. Antimicrobial activity of plant purified AMP2 peptide.

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). Notably, 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. In addition, the disclosed plant-purified peptides were highly effective against the community- acquired, BSL-2 class pathogen MRSA USA300 strain, which poses a threat to public health⁷¹ (MICs for AMP1, -2 and -3 = 25 pg/ml). The activity of all peptides was also tested against robust bacterial biofilm formation, which represents a notoriously drug-resistant state of microbes that is responsible for 65% of all infections, using crystal violet staining 24 h after treatment with the peptides. Complete inhibition (>90%) of the Gram-positive bacterium MRSA USA300 biofilm was achieved with 25 pg/ml of AMP1 (Fig. 4D and 4E and Fig. 11 A), 12.5 pg/mL of AMP2 (Fig. 5B and Fig. 9), 25 pg/mL of AMP3 (FIG. 10C) and >50% inhibition of E. coli PI-7 at 50 pg/mL of AMP1 (Fig. 4D, 4Eand Fig. 11 A). 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 10⁸ 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.

Further studies then looked for evidence for membrane permeabilization using flow cytometric analysis of influx of propidium iodide (PI, a normally impermeant dye) into bacterial cells at the time points mentioned above. The rate of PI incorporation due to plant- purified

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). To verify the observed peptide- induced permeabilization of the plasma membrane, scanning electron microscopy (SEM) imaging was employed before and following treatment. Before the addition of the peptide or antibiotic, the cytoplasmic membrane of all cells was intact, but exposure to the peptide disrupted the bacterial membrane, as seen by the emergence of damage on the surface of several cells as well as shrinking of the cell size overall (Fig. 5E and 5F) which is in agreement with a previous study demonstrating similar effects using the synthetic peptide. These data indicate that plant-purified AMP1 displays similar activity as its syn- thetic counterpart by interacting with the bacterial membrane, leading to permeabilization and cell death.

Purified peptide synergized with azithromycin to exert bactericidal activity against multi-drug resistant pathogen

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).

Table 1. Antibiotic resistance profile of New Delhi metallo-P-lactamase-positive strain

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.

Subsequent studies checked whether azithromycin could synergize with plant-purified AMP1, even in standard Ca-MHB, in which azithromycin alone has little or no activity. Indeed, significant synergy was observed at sub-MIC and pharmacologically attainable doses of both azithromycin + AMP1 (P = 0.0286; reduction by four orders of magnitude in CFUs/ml) against E. coli PI-7 (Fig. 6A). This result was confirmed with the marked prevention of biofilm formation when treated in combination (P = 0.046) compared to individual agents (Fig. 6B). Fluorescent microscopy showed a toroidal nucleoid morphology in azithromycin + AMP1 treated cells compared to either agent alone (Fig. C). This is probably because AMP1 markedly increased the membrane permeability, thus allowing for azithromycin to enter the cell more effectively and inhibit ribosomal protein synthesis.

Techno-economic analysis for industrial-scale production of AMPs

The platform established herein yielded a substantial amount of amidated AMPs; thus, a techno-economic analysis was performed to assess the feasibility of the developed module for industrial-scale production. Furthermore, a techno-economic analysis will establish the requirements, constraints, cost drivers, and capital cost estimation required to establish a large-scale AMP manufacturing facility. To this end, simulation-based techno-economic analysis was performed to predict the final cost of AMPs when produced on a large-scale. For the analysis, the previously published base case scenario,⁷⁵ was used, including the cost and installation of equipment, working capital, and the start-up cost, except for the cost of electricity, steam water, and labor charges which were estimated as per the local standard value (Table 4).

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

Sigma-Aldrich and GE Healthcare, Europe, GmbH.

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. coli¹⁸’¹⁶, mammalian cells⁷⁷) is summarized in Table 7.

Table 6. Table showing economic capital investment, operating expenditures

(with and without depreciation) and calculated the cost of goods sold (COGS) for plant- based AMP production scenario.

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.

Discussion

The rapid emergence of drug-resistant bacteria, especially among members of the ESKAPE panel, is predicted to threaten up to 10 million lives each year by 2050, ⁷⁸ underscoring the need for the judicious use of new strategies to develop antibiotics. 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.⁸⁰ The most significant obstacles impeding the clinical translation of peptides have been attributed to their poor serum stability, low efficacy due to shorter half-lives, and associated cost burden in manufacturing these expensive peptides.² To address these limitations, a rapid, plant-based approach was designed to produce amidated AMPs that are as efficacious as their synthetic counterparts and with the potential for scaled-up production.

Attempts to increase the production of cationic AMPs in plants have been met with lower yields than in E. coli cultures. In a previously reported transgenic N. benthamiana system, designed AMPs fused to the carrier protein P-glucuronidase (GUS) were not detectable by SDS-PAGE.⁴³ In another study, stable transformation of rice (Oryza sativa) with a cecropin A construct that restricted peptide production to seed endosperm with no negative effect on seed physiology, but the yield was relatively low (0.5-6 pg/g seed tissue weight).⁸¹ In a completely different approach using the deconstructed virus system, magnICON, AMP protegrin-1 was sequestered to the apoplast of N. tabacum leaves; however the yield was not reported in this case.⁸² The low yield of these studies may reflect the incompatibility of the plant host chassis in the production of AMPs, although other factors may contribute as well, such as the presence of a cryptic splicing site in designed peptides leading to unfavorable RNA processing,⁸³ transcript formation and stability,⁸⁴ or aggregation of peptides due to their amphipathic nature.⁸⁵ These bottlenecks have been overcome in one study using a whole virus strategy, demonstrating a yield for cationic SP-1 peptide of 0.5 mg per 20 g of plant tissue using tobacco mosaic virus system, where the designed AMP was fused to the viral coat protein.⁵⁹

Here, 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. In addition, 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

Production levels of 1.4 mg of peptide per 20 g of infiltrated leaf biomass were attained using the methods disclosed herein. As a standard for comparison, 1 L of well- aerated E. coli yields 10-100 mg of AMPs.⁸⁷ Although not easily comparable, the amount of peptide reported in the study herein may represent an equivalent yield to an E. coli culture. Recoverable yield of cationic AMPs have rarely been reported in plants thus far, as opposed to anionic AMPs that can accumulate to 4-113 mg per 200 g of plant tissue.⁴⁶ This discrepancy in charge-associated production has been attributed to the positive charge of peptides creating electrostatic attractions with the strong negatively charged plant membrane. Moreover, a meta-analysis study on all plant proteins in databases revealed that native AMPs belonging to the plant kingdom are less cationic than those from other taxa.⁴⁶

The data shows that both the addition of the SUMO domain and the post-translational amidation catalyzed by PAM1 resulted in the stable accumulation of cationic AMPs in plants. The use of the SUMO domain has further beneficial effects for improved protein accumulation, presumably by increasing protein stability and solubility, thus facilitating AMP purification without adding high concentrations of detergents. Compared to the disclosed expression module for AMP production, which can be applied to many plant species, plastid engineering is sophisticated and suffers from technical hurdles limiting its application. Also, compared to plastid-expressed AMPs associated growth defects, the engineered AMPs constructs expression did not inhibit plant growth⁸⁸. More importantly, this study provides a simple production strategy to obtain amidated AMPs, and this platform is amenable to other expression strategies, which will facilitate scale-up production of clinical grade AMPs.

Previous studies have demonstrated that the synthetic AMPs used in the studies herein are active against both Gram-positive and Gram-negative bacteria. The plant-produced AMPs, produced according to the methods herein were similarly active against several ESKAPE pathogens, indicating that their biological activity is preserved. Moreover, the plant-produced AMPs described here caused membrane permeabilization that likely contributed to the killing of the bacterial pathogens. Antibiofilm assays showed that plant produced peptides were highly potent in preventing biofilm formation against MRSA USA300,.⁴² Since biofilms represent a highly-drug resistant growth state of bacteria, this observation warrants further clinical validation for the management of biofilm-associated infections. In addition, 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.¹⁸ Furthermore, protein production in mammalian cells generally represents an expensive proposition with the associated cost usually priced at > $1450/g, and to produce similar titer with plant-based system costs < $100/g, representing an overall > 50% reduction in cost.⁷⁵ Hence, even though multiple chromatography steps can be involved, plant-based AMPs production still requires significantly less capital investment and lower cost of goods compared to E. coli fermentation and mammalian cultures. Thus, taking into consideration the techno-economic analysis, the disclosed manufacturing platform provides a sustainable approach towards the production of peptides by incorporating the green chemistry route and avoiding the use of hazardous materials. Moreover, this platform is amenable to peptides of varying lengths, which may be challenging to produce via SPPS.

References

1 de Breij, et al. Sci Transl Med 10, doi:10.1126/scitranslmed.aan4044 (2018).

2 Hancock, et al.. Nat Biotechnol 24, 1551-1557, doi:10.1038/nbtl267 (2006).

3 Zasloff, Biochim Biophys Acta 1788, 1693-1694, doi:10.1016/j.bbamem.2009.04.011 (2009).

4 Fantner, et al. Nat Nanotechnol 5, 280-285, doi: 10.1038/nnano.2010.29 (2010).

5 Yu, et al. Proc Biol Sci 285, doi: 10.1098/rspb.2017.2687 (2018).

6 Kintses, et al. Nat Microbiol 4, 447-458, doi:10.1038/s41564-018-0313-5 (2019).

7 Nijnik, et al. J Immunol 184, 2539-2550, doi:10.4049/jimmunol.0901813 (2010).

8 Hancock, et al. Nat Rev Immunol 16, 321-334, doi:10.1038/nri.2016.29 (2016).

9 Lazar, et al. Nat Microbiol 3, 718-731, doi:10.1038/s41564-018-0164-0 (2018).

10 Lin, et al. EBioMedicine 2, 690-698, doi:10.1016/j.ebiom.2015.05.021 (2015).

11 Sawyer, et al. Peptide Drug Discovery Raison d’Etre: Engineering Mindset, Design Rules and Screening Tools. Vol. 1417 1-25 (ACS Symposium Series; American Chemical Society, 2022).

12 Robertson, Nat Biotechnol 21, 470-471, doi:10.1038/nbt0503-470 (2003).

13 Muttenthaler, et al. Nat Rev Drug Discov 20, 309-325, doi:10.1038/s41573-020- 00135-8 (2021).

14 Raibaut, et al. Top Curr Chem 363, 103-154, doi:10.1007/128_2014_609 (2015). 15 Ajikumar, et al. J Pept Sci 7 , 641-649, doi:10.1002/psc.355 (2001).

16 Isidro-Llobet, et al. J Org Chem 84, 4615-4628, doi:10.1021/acs.joc.8b03001 (2019).

17 Khalil, et al. Nat Rev Genet 11, 367-379, doi:10.1038/nrg2775 (2010).

18 Gaglione, et al. N Biotechnol 51, 39-48, doi:10.1016/j.nbt.2019.02.004 (2019).

19 Cao, et al. ACS Synth Biol 7, 896-902, doi:10.1021/acssynbio.7b00396 (2018).

20 Ma, et al. Nat Rev Genet 4, 794-805, doi: 10.1038/nrgl l77 (2003).

21 Kusnadi, et al. Biotechnol Bioeng 56, 473-484, doi:10.1002/(SICI)1097- 0290(19971205)56:5<473::AID-BIT1>3.0.CO;2-F (1997).

22 Robert, et al. Plant Biotechnol J 13, 1169-1179, doi:10.1111/pbi.l2452 (2015).

23 Yang, et al. J Am Chem Soc 139, 5351-5358, doi:10.1021/jacs.6bl2637 (2017).

24 Lico, et al. Plant Cell Rep 31, 439-451, doi:10.1007/s00299-011-1215-7 (2012).

25 Viana, et al. Biopolymers 98, 416-427, doi:10.1002/bip.22089 (2012).

26 Deslouches, et al. Antimicrob Agents Chemother 49, 3208-3216, doi: 10.1128/AAC.49.8.3208-3216.2005 (2005).

27 Sieprawska-Lupa, et al. Antimicrob Agents Chemother 48, 4673-4679, doi: 10.1128/AAC.48.12.4673-4679.2004 (2004).

28 Di, et al. Sci Adv 6, eaay6817, doi: 10.1126/sciadv.aay6817 (2020).

29 Starr, et al. Biochim Biophys Acta Biomembr 1859, 2319-2326, doi: 10.1016/j.bbamem.2017.09.008 (2017).

30 Hakansson, et al. Front Cell Infect Microbiol 9, 174, doi: 10.3389/fcimb.2019.00174 (2019).

31 Deslouches, J Med Microbiol 65, 554-565, doi:10.1099/jmm.0.000258 (2016).

32 Deslouches, et al. J Antimicrob Chemother 60, 669-672, doi:10.1093/jac/dkm253 (2007).

33 Kong, et al. D Nat Biomed Eng 4, 560-571, doi:10.1038/s41551-020-0556-3 (2020).

34 Starr, et al. Proc Natl Acad Sci U SA 117, 8437-8448, doi:10.1073/pnas.1918427117 (2020).

35 Erik Strandberg, et al. Pure Appl. Chem 79, 717-2007 (2007).

36 Eipper, et al. Anna Rev Physiol 50, 333-344,

37 Lee, et al. Biochem J 334 ( Pt 1), 99-105, doi:10.1042/bj3340099 (1998).

38 Mor, et al. J Biol Chem 269, 31635-31641 (1994).

39 Moore, et al. Pept Res 7, 265-269 (1994).

40 Eipper, et al. Annu Rev Neurosci 15, 57-85, doi: 10.1146/annurev.ne.15.030192.000421 (1992).

41 Mansour, et al. J Pept Sci 21, 323-329, doi:10.1002/psc.2708 (2015).

42 Haney, et al. Sci Rep 8, 1871, doi:10.1038/s41598-018-19669-4 (2018).

43 Okamoto, et al. Plant Cell Physiol 39, 57-63, doi: 10.1093/oxfordjournals.pcp.a029289 (1998).

44 Lee, et al. Plant Biotechnol J 9, 100-115, doi:10.1111/j.l467-7652.2010.00538.x (2011).

45 Company, et al. PLoS One 9, el09990, doi:10.1371/journal.pone.0109990 (2014).

46 Ghidey, et al. N Biotechnol 56, 63-70, doi:10.1016/j.nbt.2019.12.001 (2020).

47 Schmidt, et al. Nat Protoc 2, 1528-1535, doi:10.1038/nprot.2007.209 (2007).

48 Varshavsky, et al. Genes Cells 2, 13-28, doi:10.1046/j.1365-2443.1997.1020301.x (1997).

49 Mogk, et al. Trends Cell Biol 17, 165-172, doi: 10.1016/j.tcb.2007.02.001 (2007).

50 Faye, et al. Vaccine 23, 1770-1778, doi:10.1016/j.vaccine.2004.11.003 (2005).

51 Doran, et al. Trends Biotechnol 24, 426-432, doi:10.1016/j.tibtech.2006.06.012 (2006).

52 Peroutka, et al. Protein Sci 17, 1586-1595, doi: 10.1110/ps.035576.108 (2008). 53 Rodriguez, et al. J Cell Biol 218, 2006-2020, doi:10.1083/jcb.201812091 (2019).

54 Malakhov, et al. J Struct Funct Genomics 5, 75-86, doi:10.1023/B:JSFG.0000029237.70316.52 (2004).

55 Butt, et al. Protein Expr Purif 43, 1-9, doi:10.1016/j.pep.2005.03.016 (2005).

56 Islam, et al. Plant Biotechnol J 17 , 1094-1105, doi:10.1111/pbi.13040 (2019).

57 Kuo, et al. Methods Mol Biol 1177, 71-80, doi:10.1007/978-l-4939-1034-2_6 (2014).

58 Sainsbury, et al. Plant Biotechnol J 7, 682-693, doi: 10.1111/j.1467- 7652.2009.00434.x (2009).

59 Zeitler, B. et al. et al. Plant Mol Biol 81, 259-272, doi:10.1007/sl 1103-012-9996-9 (2013).

60 Scholthof, et al. Annu Rev Phytopathol 34, 299-323, doi: 10.1146/annurev.phyto.34.1.299 (1996).

61 Bommarius, et al. Peptides 31, 1957-1965, doi:10.1016/j.peptides.2010.08.008 (2010).

62 Cornish, et al. Am J Physiol 277, E779-783, doi:10.1152/ajpendo.l999.277.5.E779 (1999).

63 Eipper, et al. J Biol Chem 267, 4008-4015 (1992).

64 Ray, et al. Biotechnology (N Y) 11, 64-70, doi:10.1038/nbt0193-64 (1993).

65 Kaufmann, et al. Sci Rep 11, 15791, doi:10.1038/s41598-021-95305-y (2021).

66 Schwarz, et al. PLoS One 9, M 13840, doi:10.1371/journal.pone.0113840 (2014).

67 Nomura, et al. Regen Ther 'l, 45-51, doi: 10.1016/j.reth.2017.08.004 (2017).

68 Munshi, et al.. J Immunol 168, 5860-5866, doi:10.4049/jimmunol.l68.11.5860 (2002).

69 Magnusdottir, et al. Trends Biotechnol 31, 572-580, doi:10.1016/j.tibtech.2013.06.002 (2013).

70 Mantilla-Calderon, et al. Antimicrob Agents Chemother 60, 5223-5231, doi:10.1128/AAC.00236-16 (2016).

71 DeLeo, et al. Lancet 375, 1557-1568, doi:10.1016/S0140-6736(09)61999-l (2010).

72 Jiale, et al. AMB Express 11, 49, doi:10.1186/sl3568-021-01208-6 (2021).

73 Biswas, et al. Expert Rev Anti Infect Ther 10, 917-934, doi:10.1586/eri.l2.78 (2012).

74 Spapen, et al. Ann Intensive Care 1, 14, doi:10.1186/2110-5820-l-14 (2011).

75 Nandi, et al. MAbs 8, 1456-1466, doi:10.1080/19420862.2016.1227901 (2016).

76 Ferreira, et al. Biotechnol Biofuels 11, 81, doi:10.1186/sl3068-018-1077-0 (2018).

77 Ecker, et al. MAbs 7, 9-14, doi:10.4161/19420862.2015.989042 (2015).

78 Price, et al. Eur J Hosp Pharm 23, 245-247, doi:10.1136/ejhpharm-2016-001013 (2016).

79 Lin, et al. Int J Antimicrob Agents 52, 667-672, doi: 10.1016/j.ijantimicag.2018.04.019 (2018).

80 Magana, et al. Lancet Infect Dis 20, e216-e230, doi: 10.1016/S 1473-3099(20)30327-3 (2020).

81 Bundo, et al. BMC PlantBiol 14, 102, doi:10.1186/1471-2229-14-102 (2014).

82 Patino-Rodriguez, et al. Plant Cell Tiss Org 115, 99-106, doi:10.1007/sl 1240-013- 0344-9 (2013).

83 Simpson, et al. Plant Mol Biol 32, 1-41, doi:10.1007/BF00039375 (1996).

84 Fujiki, et al. Virology 381, 136-142, doi:10.1016/j.virol.2008.08.022 (2008).

85 Cary, et al. Plant Sci 154, 171-181, doi: 10.1016/s0168-9452(00)00189-8 (2000).

86 McKee, et al. Nat Biotechnol 16, 647-651, doi:10.1038/nbt0798-647 (1998).

87 Li, Protein Expr Purif 80, 260-267, doi:10.1016/j.pep.2011.08.001 (2011).

88 Hoelscher, et al. Nat Commun 13, 5856, doi:10.1038/s41467-022-33516-l (2022). 89 A simple and general method for transferring genes into plants. Science 227, 1229- 1231, doi:10.1126/science.227.4691.1229 (1985).

90 Sakoulas, et al. J Mol Med (Berl) 92, 139-149, doi:10.1007/s00109-013-l 100-7 (2014). 91 Alshehri, et al. Biomacromolecules 22, 2094-2106, doi:10.1021/acs.biomac.lc00205

(2021).

Claims

We claim:

1. A method of producing an antimicrobial peptide (AMP) in a plant comprising introducing into the plant or part thereof, an AMP- fusion protein expression construct comprising nucleic acid sequences encoding: (a) a purification tag (b) an optional linker, (c) a cleavage sequence, and (d) a sequence coding for an AMP with a terminal glycine residue.

2. The method of claim 1, wherein the construct further comprises a nucleic acid sequence encoding an epitope tag.

3. The method of claim 2, wherein the epitope tag is Human influenza hemagglutinin or c-myc.

4. The method of any one of claims 1-3, wherein the purification tag is selected from the group consisting of His-tag, glutathione S-transferase (GST); Maltose binding protein (MBP), calmodulin binding peptide (CBP); the intein-chitin binding domain (intein-CBD), and the streptavidin tag.

5. The method of any one of claims 1-3, wherein the purification tag comprises a nucleic acid sequence encoding Strep-tag II (5'-TGGAGCCACCCGCAGTTCGAAAAG-3') (SEQ ID NO: 66).

6. The method of any one of claims 1-5, wherein the AMP sequence of interest, with a terminal glycine residue is selected from the group consisting of VRLIVAVRIWRRG) (SEQ ID NO:51), VQRWLIVWRIRKG SEQ ID NO:52), and ILVRWIRWRIQWG (SEQ ID NO:53), FK13, Guavanin 2, WLBU2, CONGA, DBS1, Mastoparan 4,1, or functional variants thereof, having more than 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs:51-53.

7. The method of any one of claims 1-6, wherein the linker is GGSGGS (SE ID NO: 54).

8. The method of any one of claims 1-7, wherein the cleavage sequence is small ubiquitin-related modifier (bdSUMO) containing mutations at SUMO-interacting positions (bdSUMOEul).

9. The method of any one of claims 1-8, wherein the plant to co-expresses a bifunctional peptidylglycine a-amidating mono-oxygenase (PAM) enzyme.

10. The method of any one of claims 1-9, wherein the plant stably expresses a bifunctional peptidylglycine a-amidating monooxygenase (PAM) enzyme. Attorney Ref: KAUST 2023-036-02 PCT

11. The method of claim 10, further comprising engineering the plant to transiently express PAM by contacting the plant with a construct comprising nucleic acid encoding PAM.

12. The method of any one of claims 1-11 wherein the PAM is a human PAM or a PAM from Rattus norvegicus.

13. The method of any one of claims 1-12, wherein the AMP-fusion protein expression construct is introduced into the plant by agrobacterium mediated transformation.

14. The method of any of claims 1-13, wherein the AMP-fusion protein is secreted into the plant cytosol.

15. The method of any one of claims 1-13, wherein the plant is selected from the group consisting of 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. Naptts, Brassica campestris ssp. Pekinensis, Brassica juncea, Calendula officinalis, Capsella bursa-pastoris, Capsicum annuum, Catharanthus roseus, Chemanthus cheiri, Chenopodium album, Chenopodium amara/uicolor, Chenopodium foetidum, Chenopodium quinoa, Conundrum sativum, Cucumis melo, Cucumis sativus, Glycine max, Gomphrena globosa, Gossypium hirsutum cv. Siv'on, Gypsophila elegans, Helianthus annuus, Hyacinthus, Hyoscyamus niger, Lactuca sativa, Lathyrus odoratus, Linum usitatissimum, Lobelia erinus, Lupinus mutahilis, Lycopersicon esculentum, Lycopersicon pimpine Hi folium, Melilotus albus, Momordica balsamina, Myosotis sylvatica, Narcissus pseudonarcissus, Nicandra physalod.es, Nicotiana benthamiana, Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana rustica, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana edwardsonii, Ocimum basilicum, Petunia hybrida, Phaseolus vulgaris, Phytolacca Americana, Pisum sativum, Raphanus sativus, Ricinus communis, Rosa sericea, Salvia splendens, Senecio vulgaris, Solanum lycopersicum, Solanum melungena, Solanum nigrum, Solanum tuberosum, Solanum pimpinellifalium, Spinacia. oleracea, Stellaria media, Sweet Wormwood, Trifolium pratense, Trifolium, repens, Tropaeolum majus, Tulipa, Viciafaba, Vida villosa. and Viola arvensis. Other plants that may be infected include Zea maize, Hordeum vulgare, Triticum aestivum, Oryza sativa and Oryza. glaberrima.

16. The method of any one of claims 1-14, wherein the plant is N. benthamiana. Attorney Ref: KAUST 2023-036-02 PCT

17. The method of any one of claims 1-16, comprising purifying AMP from plant leaves, by a method comprising extracting protein from the leaves of the plant using a buffer,

18. A transgenic plant produced according to the method of any one of claims 1- 17.

19. A transgenic plant/plant part comprising one or more exogenous nucleic acid constructs comprising one or more genes encoding a fusion protein comprising a purification tag, an optional epitope, an optional linker; a cleavage sequence and the AMP sequence of interest, comprising a terminal glycine residue.

20. The transgenic plant/plant part of claim 18, wherein the AMP sequence of interest, with a terminal glycine residue is selected from the group consisting of VRLIVAVRIWRRG) (SEQ ID NO:51), VQRWLIVWRIRKG SEQ ID NO:52), and ILVRWIRWRIQWG (SEQ ID NO:53), FK13, Guavanin 2, WLBU2, CONGA, DBS1, Mastoparan 4,1, or functional variants thereof, having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs:51-53.

21. The transgenic plant/plant part of claim 18 or 19, wherein the linker is GGSGGS (SE ID NO: 54).

22. The transgenic plant/plant part of claim 18-20, wherein the cleavage sequence is small ubiquitin-related modifier (bdSUMO) containing mutations at SUMO-interacting positions (bdSUMOEul).

23. The transgenic plant/plant part of claim 18-21 , wherein the plant part is selected from the group consisting of plant cuttings, cells, protoplasts, cell tissue cultures, callus (calli), cell clumps, embryos, stamens, pollen, anthers, pistils, ovules, flowers, seed, petals, leaves, stems, and roots.