FILING DATA

[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2015903984, filed on 30 September 2015, entitled "A method" and Australian Provisional Patent Application No. 2015904036, filed on 5 October 2015, entitled "A method", the entire contents of which, are incorporated herein by reference. This specification refers to a Sequence Listing. The "ST25.txt" file is in ANSI format. The file is hereby incorporated by reference in its entirety into the specification.

BACKGROUND

FIELD

[0002] The present disclosure relates generally to the production of cyclic peptides in plants.

DESCRIPTION OF RELATED ART

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] Proteases are abundant throughout nature and are essential for a wide range of cellular processes. They typically serve to hydrolyze polypeptide chains, resulting in either degradation of the target sequence or maturation to a biologically active form. Less frequently, proteases can act as ligases to link distinct polypeptides, producing new or alternately spliced variants. This unusual function has been reported for processes such as the maturation of the lectin, Concanavalin A (Sheldon et al. (1996) Biochem. J. 320:865- 870), peptide presentation by major histocompatibility complex class I molecules (Hanada et al. (2004) Nature 427:252-256) and anchoring of bacterial proteins to the cell wall (Mazmanian et al. (1999) Science (80) 285:760-763). This enzymatic transpeptidation has also been implicated in the backbone-cyclization of ribosomally synthesized cyclic peptides (Barber et al. (2013) J. Biol. Chem. 288: 12500-12510; Nguyen et al. (2014) Nat. Chem. Biol. 10:132-13%; Luo et al. (2014) Chem. Biol. 27: 1610-1617; Lee et al. (2009) J. Am. Chem. Soc. 131:2122-2124).

[0006] Gene-encoded cyclic peptides have been identified in a range of organisms including plants, fungi, bacteria and animals (Arnison et al. (2013) Nat Prod Rep 30: 108- 160). In plants, they are divided into four classes: cyclotides (e.g. the prototypical cyclotide kalata B l [kB l]) [Gillon et al. (2008) Plant J. 53:505-515; Saska et al. (2007) J. Biol. Chem. 282:29721-29728]; PawS-derived trypsin inhibitors (e.g. sunflower trypsin inhibitor (SFTI)) [Mylne et al. (2011) Nat. Chem. Biol. 7:257-9]; knottin trypsin inhibitors (e.g. Momordica cochinchinensis trypsin inhibitor (MCoTI-II)) [Mylne et al. (2012) Plant Cell 24:2765-78]; and orbitides (e.g. segetalins) [Barber et al. (2013) supra]. Of these, although the cyclotides are the best studied, their recombinant production in planta has hithertofore faced significant technical challenges.

[0007] Cyclotides were first identified in the African plant Oldenlandia affinis and exhibit insecticidal, nematocidal and molluscicidal activity against agriculturally relevant pests (Jennings et al. (2001) Proc. Natl. Acad. Sci. U. S. A. 98: 10614-10619; Plan et al. (2008) J. Agric. Food Chem. 56:5237-5241; Colgrave et al. (2008) Biochemistry 47:5581-5589; Colgrave et al. (2009) Acta Trop. 109: 163-6). Other reported functions include neurotensin antagonism (Witherup et al. (1994) . Nat. Prod 57: 1619-1625), anti-HIV activity (Gustafson et al. (2000) . Nat. Prod 63:176-178), anti-microbial activity (Tarn et al. (1999) Proc. Natl. Acad. Sci. U. S. A. 96:8913-8918), cytotoxic activity (Lindholm et al. (2002) Mol. Cancer Ther. i:365-369), uterotonic activity (Gran (1973) Acta pharmacol. toxicol. 33:400-408), and hemolytic (Tarn et al. (1999) supra) and anti-fouling properties (Goransson et al. (2004) J. Nat. Prod. (57: 1287-1290). Cyclotides are characterized by a cystine knot motif that, together with backbone cyclization, confers exceptional stability. This has generated much interest in the cyclotide framework as a pharmaceutical scaffold; a potential heightened by the successful grafting of bioactive sequences into both Mobius and trypsin inhibitor cyclotides (Poth et al. (2013) Biopolymers 00:480-91). Backbone cyclization can also increase the stability and oral activity of bioactive linear peptides, suggesting that this modification will find broad application (Clark et al. (2005) Proc. Natl. Acad. Sci. United States Am. 102: 13767- 13772; Clark et al. (2010) Angew. Chem. Int. Ed. Engl. 49:6545-8; Chan et al. (2013) Chembiochem 14:617-24). However, expression of cyclotides in transgenic plants that are not native cyclotide producers is poor, impeding the transfer of agriculturally relevant bioactivities to other plants (Gillon et al. (2008) supra; Conlan et al. (2012) J. Biol. Chem. 287:28037-46). Elucidating the mechanism of enzymatic cyclization intrinsic to cyclotide biosynthesis is, therefore, important not only for the realization of the pharmaceutical and agricultural potential of cyclotides, but also for increasing the cyclization efficiency of unrelated, bioactive peptides.

[0008] Cyclotides are produced from precursor molecules in which the cyclotide sequence is typically flanked by N- and C-terminal propeptides. The first processing event is the removal of the N-terminal propeptide, producing a linear precursor that remains linked to the C-terminal prodomain (Gillon et al. (2008) supra). The final maturation step involves enzymatic cleavage of this C-terminal region and subsequent ligation of the free C- and N- termini. However, only four native cyclases have been identified to date (Barber et al. (2013) supra; Nguyen et al. (2014) supra; Luo et al. (2014) supra; Lee et al. (2009) supra; Gillon et al. (2008) supra). The best characterized of these is the serine protease PatG, which is responsible for maturation of the bacterial cyanobactins (Lee et al. (2009) supra). In plants, the serine protease PCY1 reportedly facilitates cyclization of the segetalins, cyclic peptides from the Caryophyllaceae (Barber et al. (2013) supra). In the other three classes of plant-derived cyclic peptides, strong Asx sequence conservation at the PI residue of the C-terminal cleavage site suggested involvement of a group of cysteine proteases known as vacuolar processing enzymes (VPEs) or asparaginyl endopeptidases (AEPs) in this process (Gillon et al. (2008) supra).

[0009] An AEP termed butelase- 1 was isolated from the cyclotide producing plant Clitoria ternatea and shown to cyclize a modified precursor of kB 1 from O. qffinis, confirming the ability of this group of enzymes to mediate cyclization in vitro (Nguyen et al. (2014) supra) provided that the appropriate recognition squences are added at the end of the polypeptide precursor to be cyclized. Only one AEP with any cyclising ability has been produced recombinantly, and this was highly inefficient, producing mainly hydrolyzed substrate (Bernath-Levin et al. (2015) Chemistry & Biology 22: 1-12). No AEPs have been effectively utilized in planta for the production of cyclic peptides. There is a need to develop methodology to efficiently generate cyclic peptides in plants.

SUMMARY

[0010] The present disclosure teaches a method for producing a cyclic peptide in a plant. The term "cyclic peptide" includes but is not limited to a cyclotide. The cyclic peptide may be naturally cyclical or may be a naturally linear peptide rendered cyclic in the plant. In an embodiment, the cyclic peptide is extracted from the plant for use in a variety of applications including pharmacological such as the treatment of cancer, cardiovascular disease, infectious disease, immune diseases and pain. In another embodiment, the cyclic peptide protects the plant or parts of the plant from pathogen infection or infestation.

[0011] Accordingly, enabled herein is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny, the method comprising co- expressing recombinant nucleic acid encoding an asparaginyl endopeptidase (AEP) vacuolar processing enzyme with peptide cyclization activity and encoding a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide. Reference to "recombinant nucleic acid" includes a single polynucleotide or multiple polynucleotides. The recombinant nucleic acid may be integrated into the chromosome or maintained as an extra chromosomal element. The nucleic acid may be part of one or more microbial eukaryotic or viral vectors. Reference to "cyclic peptide" includes a "cyclotide". In an embodiment, the recombinant nucleic acid encoding the polypeptide precursor is introduced into cells of a plant which comprise a stably introduced recombinant nucleic acid encoding the AEP wherein a plant is regenerated expressing the AEP. Hence, transient expression of nucleic acid encoding the polypeptide precursor is contemplated herein. Alternatively, a nucleic acid encoding an AEP is introduced into a plant or seed cell encoding a polypeptide precursor.

[0012] Taught herein is an aspect wherein the recombinant nucleic acid encoding each of the AEP and polypeptide precursor is expressed in two respective nucleic acid constructs. Alternatively, the recombinant nucleic acid encoding each of the AEP and the polypeptide precursor is expressed in a single nucleic acid construct. An example of the latter is a multi-gene expression vehicle (MGEV) consisting of a polynucleotide comprising from two or more transcription segments, each segment encoding a functional protein, each segment being joined to the next in a linear sequence by a linker transcription segment encoding a linker peptide, the transcription segments all being in the same reading frame operably linked to a single promoter and terminator wherein at least one segment encodes an AEP and at least one other segment encodes the polypeptide precursor. Alternatively, the nucleic acid construct is a viral expression vector such as but not limited to a MGEV construct within a viral vector. As indicated above, in an embodiment, a plant is stably transformed with and expresses one of an AEP or precursor polypeptide and a nucleic acid encoding and expressing the other of an AEP or polypeptide is then introduced to a cell of the plant. The transformed plant cell is then regenerated into a plant. Viral vectors or microbial vectors or shotgun transformation can be used to introduce the second nucleic acid.

[0013] The nucleic acid may be operably linked to an inducible or constitutive promoter. In an embodiment, the promoter is a tissue- specific promoter. Examples include a tissue- specific promoter specific for tissue selected from the list consisting of a leaf, stem, flower, seed and root. In an embodiment, the promoter is a seed-specific promoter.

[0014] The linear polypeptide precursor comprises a C-terminal AEP processing site. Generally, but not exclusively, the C-terminal processing site is an amino acid sequence defined as comprising P3 to PI prior to the actual cleavage site and comprising Ρ 1' to P3' after the cleavage site towards the C-terminal end. In an embodiment, P3 to PI and PI to P3 have the amino acid sequence:

X ₂ X ₃ X ₄ X ₅ X ₆ X ₇

wherein X is an amino acid residue and:

X ₂ is optional or is any amino acid;

X ₃ is optional or is any amino acid;

X ₄ is N or D;

X ₅ is G, F, S or A;

X ₆ is L, A or I; and X ₇ is optional or any amino acid.

[0015] In an embodiment, X ₂ through X ₇ comprise the amino acid sequence:

X ₂ X ₃ NGLX ₇

wherein X ₂ , X ₃ and X ₇ are as defined above.

[0016] The N-terminal end of the linear polypeptide precursor may contain no specific AEP processing site or may contain a processing site defined by any one of PI through P3 wherein PI to P3 is defined by:

X ₉ X ₁₀ X ₁₁

wherein X is an amino acid residue:

X ₉ is G;

X10 is optional or any amino acid or L, A, F or I or a hydrophobic amino acid;

X ₁₁ is optional and any amino acid.

[0017] In an embodiment, X ₉ throughX ₁₁ comprise the amino acid sequence:

GLX ₁₁

whereinX ₁₁ is defined as above.

[0018] In an embodiment, the AEP processing site comprises N- and C-terminal end sequences comprising the sequence:

GLX ₁₁ [X _n ] X ₂ X ₃ NGLX ₇

whereinX ₁₁ , X ₂ , X ₃ , and X ₇ are as defined above and [X _n ] is absent (n=0) or any amino acid residue in a sequence of from 1 to 2000 amino acids.

[0019] In an embodiment, the C-terminal processing site comprises P4 to P1 and P1' to P4' wherein PI to P4 and ΡΓ to P4' comprise XiX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ wherein X ₂ to X ₇ are as defined above and X ₁ is optional or any amino acid and X ₈ is optional or any amino acid.

[0020] The AEP may be from any source such as but not limited to from the genus Oldenlandia. One such species is Oldenlandia affinis. Examples include OaAEPl _b (SEQ ID NO:2), OaAEPl (SEQ ID NO:4), OaAEP2 (SEQ ID NO:6) and OaAEP3 (SEQ ID NO:4) or a variant, derivative or hybrid form thereof which retains asparaginyl endopeptidase and cyclization activity. In an embodiment, the AEP has an amino acid sequence having at least 80% similarity to any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8 after optimal alignment. In an embodiment, the AEP has an amino acid sequence having at least 80% similarity to any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8 after optimal alignment. In an embodiment, the AEP has an amino acid sequence having at least 80% similarity to any one of SEQ ID NO:6 after optimal alignment. In an embodiment, the AEP is defined by an amino acid sequence selected from the list consisting of SEQ ID NOs:2, 4, 6 and 8 or SEQ ID NOs:2, 4 and 8 or SEQ ID NO:6.

[0021] In an embodiment, the cyclic peptide is extracted from the plant. It may have any one of a number of activities such as pharmacological useful for the treatment of cancer, cardiovascular disease, infectious disease, immune diseases and pain. The cyclic peptide may also comprise a functional portion fused or embedded in a backbone framework of a cyclotide. The cyclic peptide may also be generated to protect the plant from pathogen infection or infestation such as against a fungus, bacterium, nematode, schistosome, mollusc, helminth, virus or protozoan organism.

[0022] The method is generally practiced in a plant which can be mass produced to extract the peptide. Examples of plants include a plant selected from the listing consisting of N. benthamiana, tobacco, canola, potato, bush bean, corn, soybean, wheat, alfalfa, barley, castor bean, clover, cotton, flax, oat, oilseed rape, rice, rye, ryegrass, safflower, sorghum, sugarbeet, sunflower, tomato, lettuce, celery, broccoli, cauliflower, cucurbits, onions and an ornamental flowering plant. In an embodiment, the plant is selected from the list consisting of N. benthamiana, tobacco, canola, potato and bush bean. Arabidopsis is also a useful test model.

[0023] Further enabled herein is a genetically modified plant or its genetically modified progeny or seed of the plant or from its progeny having cells which comprise a recombinant nucleic acid encoding an asparaginyl endopeptidase (AEP) vacuolar processing enzyme with peptide cyclization activity and encoding a linear polypeptide precursor of a cyclic peptide wherein the cells produce the cyclic peptide. It may comprise two respective nucleic acid constructs encoding the AEP and polypeptide precursor or a single nucleic acid construct such as but not limited to a multi-gene expression vehicle consisting of a polynucleotide comprising one or more functional proteins, joined to each other in a linear sequence by a linker peptide, the polynucleotide being encoded in a single reading frame operably linked to a single promoter and terminator wherein at least one protein is an AEP and another protein is a polypeptide precursor for the cyclic peptide. As indicated above, a plant cell which stably expresses one or other of an AEP or precursor polypeptide can be employed as a host to receive a nucleic acid encoding the other of the AEP or precursor polypeptide.

[0024] Alternatively, the nucleic acid construct is a viral expression vector. For example, the viral expression vector may contain a MGEV encoding an AEP and a precursor polypeptide joined by a linker polypeptide which is cleaved to generated two separate proteins. Alternatively, a viral expression vector encoding a one or other of an AEP or precursor polypeptide may be introduced into a plant stably expressing the other of the AEP or precursor polypeptide. The plant may produce the cyclic peptide in all tissues or in specific tissues such as but not limited to a leaf, stem, flower, root or seed.

[0025] In an embodiment, the AEP is derived from Oldenlandia such as but not limited to OaAEPl _b , OaAEPl, OaAEP2, OaAEP3 or a variant, derivative or hybrid form thereof which retains asparaginyl endopeptidase and cyclization activity. In an embodiment, the AEP is OaAEPlb, OaAEPl or OaAEP3. In an embodiment, the AEP is OaAEP2. In another embodiment, the AEP is derived from a genus of plants which produce cyclotides. In yet another embodiment, the AEP is from a cyclotide-producing plant. Examples include a species of Petunia, Momordica and Viola. In a further embodiment, the AEPs are from Clitoria ternatea such as but not limited to CtAEP2 (SEQ ID NO:26) and CtAEP6 (previously known as CtAEP5) [SEQ ID NO:28].

[0026] As indicated above, the cyclic peptide is extracted from the plant and exhibits an activity such as a therapeutic activity useful for the treatment of cancer, obesity, cardiovascular disease, infectious disease, immune diseases and pain. The cyclic peptide may also comprise a functional peptide fused or embedded in a backbone framework of a cyclotide. Alternatively, the cyclic peptide protects the plant or seed from pathogen infection or infestation. In an embodiment, the pathogen is a fungus, bacterium, nematode, helminth, schistosome, mollusc, virus or protozoan organism.

[0027] The genetically modified plant includes a plant selected from the list consisting of tobacco, N. benthamiana canola, potato, bush bean, corn, soybean, wheat, alfalfa, barley, castor bean, clover, cotton, flax, oat, oilseed rape, rice, rye, rye grass, safflower, sorghum, sugarbeet, sunflower, tomato, lettuce, celery, broccoli, cauliflower, cucurbits, chickpea, sugarcane, banana, onions and an ornamental flowering plant as well as Arabidopsis.

[0028] Enabled herein is a system for generating and extracting a cyclic peptide from a plant, the system comprising maintaining a genetically modified plant which comprises cells expressing a recombinant nucleic acid encoding an asparaginyl endopeptidase (AEP) vacuolar processing enzyme with peptide cyclization activity wherein the system requires introducing a nucleic acid molecule which is capable of expressing a linear polypeptide precursor of the cyclic peptide into cells of the plant, regenerating a plant and growing the plant or its progeny under conditions sufficient for cells of the plant to produce the cyclic peptide and then extracting the cyclic peptide. The cyclic peptide may be in isolated form or contained in an extract or isolated plant or seed tissue.

[0029] Further taught herein is a system for generating and extracting a cyclic peptide from a plant, the system comprising maintaining a genetically modified plant which comprises cells expressing a recombinant nucleic acid encoding a precursor polypeptide wherein the system requires introducing a nucleic acid molecule which is capable of expressing an asparaginyl endopeptidase (AEP) into cells of the plant and growing the plant or its progeny under conditions sufficient for cells of the plant to produce the cyclic peptide and then extracting the cyclic peptide.

[0030] Such a system is a useful method for generating cyclic peptides in plants. The system can be scaled up for mass production of harvestable plant material which is then subject to processing to extract the cyclic peptide.

[0031] In another embodiment, the system comprises introducing the precursor of the cyclic peptide for stable transformation or having a stably transformed plant expressing the AEP and then introducing the precursor or a viral vector for transient expression and harvesting. A viral vector may comprise a MGEV which encodes both an AEP and a precursor polypeptide.

[0032] A summary of sequence identifiers used throughout the subject specification is provided in Table 1.

Table 1

Summary of sequence identifiers

BRIEF DESCRIPTION OF THE FIGURES

[0033] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0034] Figure 1A is a schematic representation of the Oakl gene. The precursor protein encoded by the Oakl gene is proteolytically processed to produce mature kB l. The domains shown in order are: ER signal peptide (ER SP), N-terminal propeptide (NTPP), N-terminal repeat (NTR), cyclotide domain (kalata B l), C-terminal propeptide (CTPP). Dashed lines indicate the N- and C-terminal processing sites and a bold asterisk denotes the rOaAEPl _b cleavage site. The C-terminal Pl/Pl' - P3/P3' sites are indicated. ΡΓ-Ρ3" denote the N-terminal residues that replace the Ρ -Ρ3' residues upon release of the C- terminal propeptide. Figure IB is a representation of the amino acid sequence encoded by the Oakl gene (SEQ ID NO: 14). The domains shown in order are the ER signal peptide (italics), the NTPP (grey), the NTR (underline), the cyclotide domain and the CTPP (bold).

[0035] Figure 2A is a representation of the amino acid sequence encoded by the OaAEPl _b gene isolated from O. qffinis genomic DNA (SEQ ID NO:2). Predicted ER signal sequence shown in grey; N-terminal propeptide shown in italics; the putative signal peptidase cleavage site is indicated by an open triangle and autocatalytic processing sites are indicated by filled triangles. The mature OaAEP-1 cyclase domain is underlined. Cys216 and His 175, presumed to be important for catalytic activity, are shown in bold and labeled with an asterisk. Figure 2B is a representation of the amino acid sequence encoded by the Cter M precursor gene from Clitoria ternatea (SEQ ID NO:56). The ER signal peptide is double underlined; the Cter M cyclotide domain is shown in bold; the spacer region is shown in italics; the albumin- 1 chain a domain is underlined and the C-terminal tail is shown in grey.

[0036] Figure 3A is a diagrammatic representation of the OaAEPl gene construct comprising the ER signal peptide (ER SP) and the OaAEPl _b coding sequence. Figure 3B is a diagrammatic representation of a kalata B 1 gene construct. In order to cyclize other peptides, the ER SP, NTPP, NTR and CTPP are retained and the kalata B l peptide domain is replaced by an exogenous peptide domain. Figure 3C is a diagrammatic representation of a kalata B 1 gene construct where the CTPP has been replaced with codons for the amino acids histidine and valine (the recognition sequence for butelase 1). Additionally, the kalata B 1 peptide domain can be replaced with another peptide for cyclization.

[0037] Figures 4A and 4B are diagrammatic representations of constructs encoding OaAEPl _b and a peptide domain linked by the MGEV linker EEKKND. Figure 4A shows the construct with OaAEPl _b ER signal followed by the OaAEPl _b gene linked to the kalata B 1 NTPP and NTR followed by the peptide domain. Figure 4B shows the construct with the kalata B l ER signal, NTPP and NTR followed by the peptide domain linked to OaAEPlb.

[0038] Figures 5A and 5B are diagrammatic representations of example constructs encoding OaAEPl _b and multiple peptide domains. OaAEPl _b is preceded by its ER signal peptide and the peptide domains are flanked by the kalata B l NTR and CTPP. The OaAEPl _b domain and the peptide domains may be linked via a MGEV linker (EEKKN) (Figure 5A) or joined directly (Figure 5B).

[0039] Figures 6A and 6B are diagrammatic representations of example constructs with multiple peptide domains. Figure 6A shows the construct with the kalata B l ER signal, NTPP and NTR, the first peptide domain and kalata B l CTPP linked via a MGEV linker (EEKKN) to a second peptide domain followed by a second kalata B 1 CTPP. Figure 6B shows the construct with the two peptide domains linked directly flanked at the N-terminal end by the kalata B l ER signal, NTPP and NTR and at the C-terminal by the kalata B l CTPP.

[0040] Figure 7A is a diagrammatic representation of the Oak2a(kB2-kB3) construct (SEQ ID NO:52) with the kalata ER signal, NTPP and NTR, kalata B2 cyclotide domain, CTPP, NTR, kalata B3domain and CTPP. Figure 7B is a diagrammatic representation of the Oak4 construct (SEQ ID NO:54) with the kalata ER signal, NTPP and NTR, kalata B2 cyclotide domain, CTPP, NTR, kalata B2domain, CTPP, NTR, kalata B2 domain and CTPP.

[0041] Figures 8A and 8B are a diagrammatic representations of a double stack construct suitable for stable plant transformation. The construct comprises the Oakl and OaAEPl (Figure 8A) or the Oakl-HV and CtAEPl (Figure 8B) plant transcription units that have been cloned into the multiple cloning site of the pBIN19 binary vector (Bevan (1984) Nucleic Acids Res 2:8711-8721).

[0042] Figure 9 is a diagrammatic representation of example constructs for the expression of linear peptide precursors. The construct comprises the Oakl flanking sequences with the kalata B 1 peptide domain being replaced by the sequence of the peptide to be cyclized. The peptide sequences include sunflower trypsin inhibitor 1 (SFTI-1) [SEQ ID NO:31] and Momordica cochinchinensis trypsin inhibitor II (V03R) (MCoTI-II) [SEQ ID NO:36], a bracelet type cyclotide, cycloviolacin 013 (Vo013) [SEQ ID NO:32], an anti-angiogenesis peptide grafted into kalata B l (cpr-3) [SEQ ID NO:33] and a non-cyclic peptide - conotoxin (Vcl.l) [SEQ ID NOs:34 and 35].

[0043] Figure 10 is a diagrammatic representation of constructs for testing the minimal precursor peptide requirements when OaAEPl _b is linked to Oakl via a MGEV linker. The constructs are pHEX 231 (SEQ ID NO:74), pHEX257 (SEQ ID NO:76), pHEX269 (SEQ ID NO:78) and pHEX270 (SEQ ID NO: 80).

[0044] Figure 11 is a diagrammatic representation of constructs for testing the minimal precursor peptide requirements when Oak 1 is linked to OaAEPl _b via a MGEV linker. The constructs are pHEX 232 (SEQ ID NO:82), pHEX286 (SEQ ID NO:84), and pHEX269 (SEQ ID NO:86).

[0045] Figure 12 is a diagrammatic representation of constructs for testing the minimal precursor peptide requirements of the Cter M precursor. The constructs are Cter M truncation 1 (no albumin- 1 a-chain) [SEQ ID NO:88] Cter M truncation 2 (ER signal peptide, cyclotide domain and spacer region) [SEQ ID NO: 90] and Cter M truncation 3 (ER signal peptide and cyclotide domain only) [SEQ ID NO:92] .

[0046] Figure 13 shows MALDI-TOF traces indicating the presence or absence of cyclic Oakl-MOG3 and Oakl-MOG3D when transiently expressed in N. benthamiana with or without co-expression of OaAEP1 _b ,.

[0047] Figure 14 is a graphical representation of the activity of rOa AEPl _b (~5 μg mL ^-1 total protein) and rhuLEG (1.1 μg mL ^-1 total protein) over time against the fluorogenic substrate Z-AAN-MCA (100 μΜ). Activity is tracked at 1 minute intervals for 90 minutes using excitation and emission wavelengths of 320 and 420 nm respectively. A single representative experiment is shown. RFU, relative fluorescence units.

[0048] Figure 15 is a graphical representation of rOaAEPl _b activity against the IQF peptide Abz-STRNGLPS-Y(3N0 ₂ ) [SEQ ID NO:49] in the presence of protease inhibitors. rOaAEPl _b (4.4 μg mL ^-1 total protein) was allowed to cleave the IQF peptide (11 μΜ) for 90 minutes. Enzyme activity against the IQF peptide in the presence of either the Ac- YVAD-CHO or Ac-STRN-CHO inhibitors is reported relative to a no inhibitor control at the 90 minutes time point.

[0049] Figures 16A and 16B are graphical representations of substrate specificity of plant and human AEPs for wt (SEQ ID NO:49) and L31A (SEQ ID NO:50) IQF peptide substrates. Initial velocity ofrOa AEPlb (-3.5 μg mL ^-1 total protein) (16A) and rhuLEG (1.1 μg mL ^-1 total protein) (16B) against 50 μΜ IQF peptide substrates is shown. The average of two independent experiments are shown and the error bars report the range. DETAILED DESCRIPTION

[0050] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps.

[0051] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cyclic peptide" includes a single cyclic peptide, as well as two or more cyclic peptides; reference to "an AEP" includes a single AEP, as well as two or more AEPs; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the present invention. All such aspects are enabled within the width of the present invention. Any variants and derivatives contemplated herein are encompassed by "forms" of the invention. The term "aa" is used herein to denote "amino acid"; "na" means "nucleic acid".

[0052] The present specification teaches a method of producing a cyclic peptide in a plant or seed or progeny thereof or seed of the progeny. The term "cyclic peptide" encompasses a "cyclotide". The method comprises the co-expression in a plant cell of a plant, a recombinant nucleic acid in a vector encoding: (i) an asparaginyl endopeptidase (AEP) vacuolar processing enzyme with peptide cyclization activity; and (ii) a linear polypeptide precursor of the cyclic peptide including appropriate enzyme recognition sequences. The AEP catalyzes the processing of the polypeptide precursor to facilitate excision and circularization of the cyclic peptide. Reference to a "linear polypeptide precursor" or the term "polypeptide" includes a "peptide". A "peptide" includes a "polypeptide". No size limitation or definition is to be implied by use of the terms "protein", "polypeptide" or "peptide". [0053] Hence, a plant is engineered to express single or multiple vectors encoding (i) an AEP; and (ii) a polypeptide precursor of the cyclic peptide. The term "plant" includes progeny and seed from the parent or progeny plant.

[0054] Alternatively, the method comprises maintaining a plant or cells of a plant which are genetically modified to stably produce a heterologous AEP or a precursor polypeptide and then a nucleic acid encoding and capable of expressing the other of the AEP or precursor polypeptide is introduced to enable production of a cyclic peptide.

[0055] The linear polypeptide precursor comprises a C-terminal AEP processing site. Generally, but not exclusively, the C-terminal processing site is an amino acid sequence defined as comprising P3 to PI prior to the actual cleavage site and comprising ΡΓ to P3' after the cleavage site towards the C-terminal end. In an embodiment, P3 to PI and PI to P3 have the amino acid sequence:

X ₂ X ₃ X ₄ X ₅ X ₆ X ₇

wherein X is an amino acid residue and:

X ₂ is optional or is any amino acid;

X ₃ is optional or is any amino acid;

X ₄ is N or D;

X ₅ is G, F, S or A;

X ₆ is L, A or I; and

X ₇ is optional or any amino acid.

[0056] In an embodiment, X ₂ through X ₇ comprise the amino acid sequence:

X ₂ X ₃ NGLX ₇

wherein X ₂ , X ₃ and X ₇ are as defined above.

[0057] The N-terminal end of the linear polypeptide precursor may contain no specific AEP processing site or may contain a processing site defined by any one of PI through P3 wherein PI to P3 is defined by: X ₉ X ₁₀ X ₁₁

wherein X is an amino acid residue:

X ₉ is G;

X10 is optional or any amino acid or L, A, F or I or an hydrophobic amino acid residue;

X ₁₁ is optional and any amino acid. [0058] In an embodiment, X ₉ through X ₁₁ comprise the amino acid sequence: GLX ₁₁

wherein X ₁₁ is defined as above.

[0059] In an embodiment, the AEP processing site comprises N- and C-terminal end sequences comprising the sequence:

GLX ₁₁ [X„] X ₂ X ₃ NGLX ₇

wherein X ₁₁ , X ₂ , X ₃ , and X ₇ are as defined above and [X _n ] is absent (n=0) or any amino acid residue in a sequence of from 1 to 2000 amino acids.

[0060] In an embodiment, the C-terminal processing site comprises P4 to PI and ΡΓ to P4' wherein PI to P4 and ΡΓ to P4' comprise X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ wherein X ₂ to X ₇ are as defined above and Xi is optional or any amino acid and X ₈ is optional or any amino acid.

[0061] A "vector" refers to a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host plant cell. The polynucleotide to be delivered comprises a coding sequence of an AEP and/or the polypeptide precursor. The term includes vectors that function primarily for insertion of a DNA or RNA into a plant cell and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. [0062] A vector includes a multi-gene expression vehicle (MGEV) as described in International Patent Application No. PCT/AU2007/000712. A MGEV consists of a polynucleotide comprising from 2 to 8 segments, each segment encoding a functional protein, each segment being joined to the next in a linear sequence by a linker segment encoding a linker peptide, the segments all being in the same reading frame operably linked to a single promoter and terminator. In accordance with the present invention, at least one segment encodes the AEP and at least one other encodes the polypeptide precursor. A vector also includes a viral expression vector.

[0063] In an embodiment, taught herein is a recombinant nucleic acid encoding each of the AEP and polypeptide precursor expressed in two respective nucleic acid constructs. Alternatively, the recombinant nucleic acid encoding each of the AEP and the polypeptide precursor is expressed in a single nucleic acid construct. These are expressed in a plant cell or seed of a plant or progeny thereof or seed of the progeny. An example is a multi- gene expression vehicle consisting of a polynucleotide comprising from two or more segments, each segment encoding a functional protein, each segment being joined to the next in a linear sequence by a linker transcription segment encoding a linker peptide, the transcription segments all being in the same reading frame operably linked to a single promoter and terminator wherein at least one segment encodes an AEP and at least one other segment encodes the polypeptide precursor.

[0064] Further taught herein is a system for generating and extracting a cyclic peptide from a plant, the system comprising maintaining a genetically modified plant which comprises cells expressing a recombinant nucleic acid encoding a precursor polypeptide wherein the system requires introducing a nucleic acid molecule which is capable of expressing an asparaginyl endopeptidase (AEP) into cells of the plant and growing the plant or its progeny under conditions sufficient for cells of the plant to produce the cyclic peptide and then extracting the cyclic peptide.

[0065] A "plant cell" refers to the structural and physiological unit of plants, consisting of a protoplast and the cell wall.

[0066] A "protoplast" is an isolated cell without cell walls, having the potency for regeneration into cell culture, tissue or whole plant.

[0067] A "host" cell encompasses a plant cell. A "host" also includes a whole plant.

[0068] The terms "nucleic acid", "polynucleotide" and "nucleotide" sequences are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage of a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.

[0069] A "gene" refers to a polynucleotide containing at least one open reading frame that encodes an AEP or polypeptide precursor and which is capable of being transcribed and translated.

[0070] As used herein, "expression" refers to the process by which a polynucleotide transcription unit is transcribed into mRNA and/or the process by which the transcribed mRNA (also referred to as "transcript") is subsequently being translated into an AEP or polypeptide precursor. The transcripts and the encoded polypeptides are collectedly referred to as a "gene product".

[0071] In the context of a linear polypeptide precursor a "linear sequence" is an order of amino acids in the polypeptide in an N- to C-terminal direction in which the amino acid residues that neighbour each other in the sequence are contiguous in the primary structure of the polypeptide. [0072] A "plant" includes N. benthamiana, tobacco, canola, potato, bush bean, corn, soybean, wheat, alfalfa, barley, castor bean, clover, cotton, flex, oat, oilseed rape, rice, rye, ryegrass, safflower, sorghum, sugarbeet, sunflower, tomato, lettuce, celery, broccoli, cauliflower, cucurbits, chickpea, sugarcane, banana, onions and an ornamental flowering plant as well as Arabidopsis. These plants may also be "genetically modified" to express an AEP and/or a polypeptide precursor or peptides to be conjugated. Hence, a plant may be generated which stably expresses a nucleic acid sequence encoding one of an AEP or linear polypeptide precursor. Such a plant is then used to introduce a recombinant nucleic acid sequence encoding the other of the linear polypeptide precursor or AEP, respectively. Arabidopsis is a useful test model.

[0073] A "pathogen" includes a plant pathogen selected from a fungus, bacterium, nematode, helminth, schistosome, mollusc, virus and a protozoan organism.

[0074] Enabled herein is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny, the method comprising co-expressing a vector comprising recombinant nucleic acid encoding an asparaginyl endopeptidase (AEP) vacuolar processing enzyme with peptide cyclization activity and encoding a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide.

[0075] Taught herein is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny, the method comprising introducing a nucleic acid molecule capable of expressing one or other of an AEP or precursor protein into a plant cell which has been genetically modified to stably produce the other of an AEP or precursor polypeptide thereby enabling the co-expression of an AEP and the precursor polypeptide for a time and under conditions sufficient to generate the cyclic peptide.

[0076] The present invention extends to any AEP with peptide cyclization activity. Encompassed, herein, is an AEP such as, but not limited to, OaAEPl _b (SEQ ID NO:2), OaAEPl (SEQ ID NO:4), OaAEP2 (SEQ ID NO:6) and OaAEP3 (SEQ IDNO:8) from Oldenlandia affinis. Other AEPs include an AEP having at least 80% amino acid similarity to SEQ ID NO:2 (OaAEPl _b ), SEQ ID NO:4 (OaAEPl), SEQ ID NO:6 (OaAEP2) or SEQ ID NO:8 (OaAEP3) after optimal alignment and which retains asparaginyl endopeptidase and peptide cyclization activity. Enabled herein is an AEP having at least 80% amino acid similarity to SEQ ID NO:2 (OaAEPl _b ), SEQ ID NO:4 (OaAEPl) or SEQ ID NO:8 (OaAEP3) after optimal alignment and which retains asparaginyl endopeptidase and peptide cyclization activity. Taught herein is an AEP having at least 80% amino acid similarity to SEQ ID NO:6 (OaAEP2) after optimal alignment. AEPs may also come from cyclotide producing plants such as but not limited to Petunia spp (e.g. Petunia hybrida), Momordica spp (e.g. Momordica cochinchinesis) and Viola spp. In a further embodiment, the AEPs are from Clitoria ternatea such as but not limited to CtAEP2 (SEQ ID NO:26) and CtAEP6 (previously known as CtAEP5) [SEQ ID NO:28].

[0077] Reference to "at least 80%" includes 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.

[0078] The term "similarity" as used herein includes exact identity between compared sequences at the amino acid level. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, amino acid sequence comparisons are made at the level of identity rather than similarity.

[0079] Terms used to describe sequence relationships between two or more polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" includes from at least 10 contiguous amino acid residues (e.g. from 10 to 100 amino acids). A "comparison window" refers to a conceptual segment of typically 10 contiguous amino acid residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (BLASTP 2.2.32+, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (1994-1998) In: Current Protocols in Molecular Biology, John Wiley & Sons Inc.

[0080] The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on an amino acid- by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, IIe, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the BLASTP 2.2.32+ computer program. Similar comments apply in relation to sequence similarity.

[0081] In an embodiment, taught herein is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny the method comprising co-expressing recombinant nucleic acid encoding an AEP vacuolar processing enzyme with peptide cyclization activity having an amino acid sequence with at least 80% similarity to SEQ ID NO:2 after optimal alignment and encoding a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide.

[0082] In a related embodiment, enabled herein is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny the method comprising co-expressing recombinant nucleic acid encoding an AEP vacuolar processing enzyme with peptide cyclization activity having an amino acid sequence with at least 80% similarity to SEQ ID NO:4 after optimal alignment and encoding a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide.

[0083] In a related embodiment, disclosed herein is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny the method comprising co-expressing recombinant nucleic acid encoding an AEP vacuolar processing enzyme with peptide cyclization activity having an amino acid sequence with at least 80% similarity to SEQ ID NO:6 after optimal alignment and encoding a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide.

[0084] In a related embodiment, taught herein is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny the method comprising co-expressing recombinant nucleic acid encoding an AEP vacuolar processing enzyme with peptide cyclization activity having an amino acid sequence with at least 80% similarity to SEQ ID NO:8 after optimal alignment and encoding a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide.

[0085] As indicated above, reference to "at least 80%" means 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. [0086] Cells of plant or plant seed may be engineered to stably co -express both an AEP and a precursor polypeptide or the plant, seeds or cells of the plant or seeds may stably express one or other of an AEP or precursor polypeptide which are then used to introduce genetic material encoding and expressing the other of the AEP or precursor polypeptide. The genetic material may be in any form such as a viral, microbial or eukaryotic vector and may or may not involve a MGEV.

[0087] In an embodiment, the AEP is selected based on it being identified as having cyclizing ability. Hence, a method is taught herein for identifying an AEP with cyclizing ability, the method comprising co-incubating an AEP to be tested with fluorescent peptide substrates and using the pattern of enzyme activity against these substrates to identify a cyclizing AEP. The substrates in this method comprise the generic AEP substrate Z-AAN- MCA (where Z is carboxybenzyl; MCA is 7-amido-4-methylcoumarin) and 8-residue internally-quenched fluorescent (IQF) peptides. Enzymatic cleavage of each substrate is indicated by an elevation of fluorescence intensity over time due to spatial separation of a fluorescence donor/quencher pair. In an embodiment activity against an IQF peptide in the absence of activity against the generic Z-AAN-MCA substrate is indicative of cyclization ability. In an embodiment, the IQF peptides tested are Abz-STRNGLPS-Y(3N0 ₂ ) and Abz-STRNGAPS-Y(3N0 ₂ ) [where Abz is o-aminobenzoic acid and (Y[3NC¾) is 3- nitro tyro sine] and activity against the first peptide in the absence of activity against the second peptide is indicative of cyclization ability. In an embodiment, the fluorescence intensity is monitored over time at excitation/emission wavelengths 320/420nm (IQF peptides) or 360/460 nm (generic AEP substrate). Such AEPs include those having at least 80% similarity to SEQ ID NOs:2, 4, 6 and/or 8. AEPs include those having at least 80% similarity to SEQ ID NOs:2, 4 and/or 8. AEPs include those having at least 80% similarity to SEQ ID NO: 6.

[0088] The nucleic acid encoding the AEP and the polypeptide precursor may be present in separate nucleic acid vectors or be part of a single vector such as a multi-gene expression vehicle. In either event, the nucleic acid is operably linked to a promoter in the vector which enables expression of the nucleic acid to produce the AEP and a linear form of the polypeptide precursor which is then processed into the cyclic peptide. In another embodiment, a plant is maintained which comprises cells which are genetically modified to produce the AEP and cells of the plant are then subsequently transformed with any given nucleic acid encoding a polypeptide precursor. This includes the transient expression of nucleic acid encoding the polypeptide precursor or peptides in a plant which stably expresses an AEP.

[0089] Taught herein is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny the method comprising co-expressing recombinant nucleic acid encoding an AEP with peptide cyclization activity and encoding a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide, wherein the recombinant nucleic acid encoding each of the AEP and the polypeptide precursor is expressed in a single nucleic acid construct and wherein the single construct is a multi-gene expression vehicle (MGEV) consisting of a polynucleotide comprising from at least two transcription segments, each transcription segment encoding a functional protein, each transcription segment being joined to the next in a linear sequence by a linker segment encoding a linker peptide, the transcription segments all being in the same reading frame operably linked to a single promoter wherein at least one transcription segment encodes the AEP and at least one other encodes the polypeptide precursor.

[0090] In an embodiment, the AEP comprises an amino acid sequence having at least 80% similarity to any one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and/or SEQ ID NO:8. In an embodiment, the AEP comprises an amino acid sequence having at least 80% similarity to any one or more of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:8. In an embodiment, the AEP comprises an amino acid sequence having at least 80% similarity to any one or more of SEQ ID NO: 6. Again, reference to "at least 80%" means 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. The at least two transcription segments means from 2 to 8.

[0091] In another embodiment, enabled here in is a method for producing a cyclic peptide in a plant or seed of a plant or progeny thereof or seed of the progeny the method comprising co-expressing recombinant nucleic acid encoding an AEP with peptide cyclization activity and encoding a linear polypeptide precursor of the cyclic peptide for a time and under conditions sufficient to generate the cyclic peptide, wherein the recombinant nucleic acid encoding each of the AEP and polypeptide precursor is expressed in two respective nucleic acid constructs.

[0092] The AEP comprises an amino acid sequence having at least 80% similarity to SEQ As above, reference to "at least 80%" means 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

[0093] Techniques and agents for introducing and selecting for the presence of heterologous DNA or RNA in plant cells and/or tissue are well-known. Genetic markers allowing for the selection of heterologous DNA or RNA in plant cells are well-known, e.g. genes carrying resistance to an antibiotic such as kanamycin, hygromycin, gentamicin, or bleomycin. The marker allows for selection of successfully transformed plant cells growing in the medium containing the appropriate antibiotic because they will carry the corresponding resistance gene.

[0094] Techniques for genetically engineering plant cells and/or tissue with an expression cassette comprising an inducible promoter or chimeric promoter fused to a heterologous coding sequence or transcription unit and a transcription termination sequence are to be introduced into the plant cell or tissue by Agrobacterium-mediated transformation, electroporation, microinjection, particle bombardment or other techniques known to the art.

[0095] A nucleic acid construct carrying a plant-expressible AEP gene or a transcription unit encoding a polypeptide precursor can be inserted into the genome of a plant by any suitable method. Such methods may involve, for example, the use of liposomes, electroporation, diffusion, particle bombardment, microinjection, gene gun, chemicals that increase free DNA uptake, e.g. calcium phosphate coprecipitation, viral vectors, and other techniques practiced in the art. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, such as those disclosed by Herrera- Estrella et al. (1983) EMBO J 2:987-995; Bevan et al. (1983) Nucleic Acids Res ii(2J:369-385; Klee et al. (1985) Bio/Technology 3:637-642 and EPO publication 120,516 (Schilperoort et al, European Patent Publication 120, 516). In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells.

[0096] The choice of vector in which the nucleic acid encoding the AEP or polypeptide precursor is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g. replication, protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant nucleic acid molecules.

[0097] Typical expression vectors capable of expressing a recombinant nucleic acid sequence in plant cells and capable of directing stable integration within the host plant cell include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens.

[0098] A transgenic plant, also referred to as a genetically modified plant, can be produced by any standard means known to the art, including but not limited to Agrobacterium tumefaciens-mediated DNA transfer, generally with a disarmed T-DNA vector, electroporation, direct DNA transfer, and particle bombardment. Techniques are well- known to the art for the introduction of DNA into monocots as well as dicots, as are the techniques for culturing such plant tissues and regenerating those tissues. Examples of transgenic plants include tobacco, canola, potato, bush bean, corn, soybean, wheat, alfalfa, barley, castor bean, clover, cotton, flax, oat, oilseed rape, rice, rye, ryegrass, safflower, sorghum, sugarbeet, sunflower, tomato, lettuce, celery, broccoli, cauliflower, cucurbits, onions and an ornamental flowering plant. [0099] Because not all plants are natural hosts for Agrobacterium, alternative methods such as transformation of protoplasts may be employed to introduce the subject vectors into the host cells. For certain monocots, transformation of the plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. See, for example, Potrykus et al (1985) Mol. Gen. Genet, 199: 169 111, Fromm et al. (1986) Nature, 319:191-193 (1986) and Callis et al. (1987) Genes and Development, 1: 183-1200. Applicability of these techniques to different plant species may depend upon the feasibility to regenerate that particular plant species from protoplasts. A variety of methods for the regeneration of cereals from protoplasts are known in the art.

[0100] In addition to protoplast transformation, particle bombardment is an alternative and convenient technique for delivering the invention vectors into a plant host cell. Specifically, the plant cells may be bombarded with microparticles coated with a plurality of the subject vectors. Bombardment with DNA-coated microprojectiles has been successfully used to produce stable transformants in both plants and animals (see, for example, Sanford et al. (1993) Methods in Enzymology, 217:483 509). Microparticles suitable for introducing vectors into a plant cell are typically made of metal, preferably tungsten or gold. These microparticles are available, for example, from BioRad (e.g., Bio- Rad's PDS-1000/He). Those skilled in the art will know that the particle bombardment protocol can be optimized for any plant by varying parameters such as He pressure, quantity of coated particles, distance between the macrocarrier and the stopping screen and flying distance from the stopping screen to the target.

[0101] Vectors can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al. (1983) Methods in Enzymology, 101 :433. Other techniques for introducing nucleic acids into a plant cell include: (a) Hand inoculations. Hand inoculations are performed using a neutral pH, low molarity phosphate buffer, with the addition of celite or carborundum (usually about 1%). One to four drops of the preparation is put onto the upper surface of a leaf and gently rubbed, (b) Mechanized inoculations of plant beds. Plant bed inoculations are performed by spraying (gas -propelled) the vector solution into a tractor-driven mower while cutting the leaves. Alternatively, the plant bed is mowed and the vector solution sprayed immediately onto the cut leaves, (c) High pressure spray of single leaves. Single plant inoculations can also be performed by spraying the leaves with a narrow, directed spray (50 psi, 15 to 30cm from the leaf) containing approximately 1% carborundum in the buffered vector solution, (d) Vacuum infiltration. Inoculations may be accomplished by subjecting a host organism to a substantially vacuum pressure environment in order to facilitate infection.

[0102] Once introduced into a suitable host cell, expression of the transgene can be determined using any assay known in the art. For example, the presence of transcribed sense or anti- sense strands of the transgene can be detected and/or quantified by conventional hybridization assays (e.g. Northern blot analysis), amplification procedures (e.g. RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based technologies (see e.g. U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934). The transgene encodes an AEP or polypeptide precursor or peptides to be conjugated.

[0103] Expression of the polynucleotide can also be determined by examining the protein product. A variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunoflourescent assays, mass spectrometry and PAGE- SDS.

[0104] In general, determining the protein level involves (a) providing a biological sample containing polypeptides; and (b) measuring the amount of any immuno specific binding that occurs between an antibody reactive to the transgene product and a component in the sample, in which the amount of immuno specific binding indicates the level of expressed proteins. Antibodies that specifically recognize and bind to the protein products of the transgene are required for immunoassays. These may be purchased from commercial vendors or generated and screened using methods well known in the art. The sample of test proteins can be prepared by homogenizing the plant cell transformants or their progenies made therefrom or seeds therefrom, and optionally solubilizing the test protein using detergents, preferably non-reducing detergents such as triton and digitonin. The binding reaction in which the test proteins are allowed to interact with the detecting antibodies may be performed in solution, or on a solid tissue sample, for example, using tissue sections or solid support that has been immobilized with the test proteins. The formation of the complex can be detected by a number of techniques known in the art. For example, the antibodies may be supplied with a label and unreacted antibodies may be removed from the complex; the amount of remaining label thereby indicating the amount of complex formed. Results obtained using any such assay on a sample from a plant transformant or a progeny thereof is compared with those from a non-transformed source as a control.

[0105] The plant host cells of this invention are grown under favorable conditions to effect transcription of the polynucleotide. The host cells may also be employed to generate transgenic plants comprising the recombinant DNA or RNA vectors of the present invention. Examples of these host cells include cells which are capable of regeneration to a plant listed herein.

[0106] Accordingly, this invention provides transgenic plants or seeds therefrom or progeny thereof or their seeds carrying one or two vectors encoding an AEP and a polypeptide precursor. The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, Weissbach and Weissbach, eds., Academic Press, Inc., San Diego, California, USA (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil.

[0107] The regeneration of plants containing the subject vector or vectors introduced by Agrobacterium tumefaciens from leaf explants can be achieved as described by Fraley et al. (1983) Proc. Natl. Acad. Sci. USA., 80:4803-4807. In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed. This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transformant shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil to allow the production of roots. These procedures will vary depending upon the particular plant species employed, as is apparent to one of ordinary skill in the art.

[0108] A population of progeny can be produced from the first and second transformants of a plant species by methods well known in the art including cross fertilization and asexual reproduction. Transgenic plants embodied in the present invention are useful for production of desired peptides. The cyclic peptide may be used to protect the plant from pathogen infection or infestation or be extracted for use ex planta.

[0109] The present invention further contemplates a business model for producing cyclic peptides. In this regard, enabled herein is a system for generating and extracting a cyclic peptide from a plant, the system comprising maintaining a genetically modified plant which comprises cells expressing a recombinant nucleic acid encoding an AEP with peptide cyclization activity wherein the system requires introducing a nucleic acid molecule which is capable of expressing a linear polypeptide precursor of the cyclic peptide, into cells of the plant, regenerating a plant and growing the plant or its progeny under conditions sufficient for cells of the plant to produce the cyclic peptide and then extracting the cyclic peptide.

[0110] The business model enables a plant or part of a plant to be harvested and the cyclic peptide extracted. The business model is a useful method for generating cyclic peptides in a system that can be scaled up for mass production. Plant material can be harvested and subject to processing to extract the cyclic peptides. For example, tobacco plantations can be readily adapted for the mass production of genetically modified plants which produce the cyclic peptides for extraction. This also applies to many other crop plants. [0111] The extracted cyclic peptides have any of a range of useful properties including antipathogen, therapeutic, pain relieving, uterotonic and/or pharmacological activity. An example of a therapeutic activity including the treatment of obesity, cancer, cardiovascular disease, infectious disease and immune disease, is neurotension antagonism. The peptide may also be formulated into an agronomically acceptable composition for topical application to plants or seeds.

[0112] Agronomically acceptable carriers are used to formulate the peptides herein disclosed for the practice of the instant method. Determination of dosages suitable for systemic and surface administration is enabled herein and is within the ordinary level of skill in the art. With proper choice of carrier and suitable manufacturing practice, the compositions such as those formulated as solutions, may be administered to plant surfaces including above-ground parts and/or roots, or as a coating applied to the surfaces of seeds.

[0113] Agronomically useful compositions suitable for use in the system disclosed herein include compositions wherein the active ingredient(s) are contained in an effective amount to achieve the intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.

[0114] In addition to the active ingredients, these compositions for use against plant pathogens may contain suitable agronomically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used in the field, in greenhouses or in the laboratory setting.

[0115] Anti-pathogen formulations include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the peptides may be prepared as appropriate oily suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Further components can include viscosifiers, gels, wetting agents, ultraviolet protectants, among others.

[0116] Preparations for surface application can be obtained by combining the active cyclic peptides with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain powders for direct application or for dissolution prior to spraying on the plants to be protected. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose or starch preparations, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0117] Further enabled herein is a genetically modified plant or its genetically modified progeny or seed of the plant or its progeny or seed of the progeny having cells which comprise a recombinant nucleic acid encoding an AEP with peptide cyclization activity and encoding a linear polypeptide precursor of a cyclic peptide wherein the cells produce the cyclic peptide.

[0118] The AEP includes protein having at least 80% similarity to any one or more of SEQ ID NO:2, 4, 6 and/or 8, such as 80% similarity to SEQ ID NOs:2, 4 and/or 8 or 80% similarity to SEQ ID NO:6. As above, reference to "at least 80%" includes 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 100%.

[0119] Genetically modified plant contemplated herein include tobacco, N. benthamiana, canola, potato, bush bean, corn, soybean, wheat, alfalfa, barley, castor bean, clover, cotton, flax, oat, oilseed rape, rice, rye, ryegrass, safflower, sorghum, sugarbeet, sunflower, tomato, lettuce, celery, broccoli, cauliflower, cucurbits, chickpea, sugarcane, banana, onions and an ornamental flowering plant. In an embodiment, the plant is selected from the group consisting of N. benthamiana, tobacco, canola, potato and bush bean. Arabidopsis is also a useful test system and is contemplated for use herein.

[0120] The genetically modified plants may also comprise a multi-gene expression vector encoding the AEP and polypeptide precursor.

[0121] Taught herein is a genetically modified plant or its genetically modified progeny or seed of the plant or its progeny or seeds of the progeny having cells which comprise a recombinant nucleic acid encoding an AEP with peptide cyclization activity and encoding a linear polypeptide precursor of a cyclic peptide wherein the cells produce the cyclic peptide; wherein the recombinant nucleic acid encoding each of the AEP and the polypepeptide precursor is expressed in a single nucleic acid construct; and wherein the single construct is a multi-gene expression vehicle (MGEV) consisting of a polynucleotide comprising two or more segments, each segment encoding a functional protein, each segment being joined to the next in a linear sequence by a linker segment encoding a linker peptide, the segments all being in the same reading frame operably linked to a single promoter wherein at least one segment encodes the AEP and at least one other encodes the polypeptide precursor wherein the AEP comprises an amino acid sequence having at least 80% similarity to any one or more of SEQ ID NOs:2, 4, 6 and/or 8, or at least 80% similarity to any one or more of SEQ ID NOs:2, 4 and/or 8 or at least 80% similarity to any one or more of SEQ ID NO:6.

[0122] Taught herein is a genetically modified plant or its genetically modified progeny or seed of the plant or its progeny or seeds of the progeny having cells which comprise a recombinant nucleic acid encoding an AEP with peptide cyclization activity. The recombinant nucleic acid encoding the AEP maybe present or introduced into a plant cell by any type of vector including but not limited to via a MGEV. The MGEV contains two or more segments wherein at least one segment comprises the recombinant nucleic acid or the recombinant nucleic acid may be linked to another nucleic acid encoding a second AEP or another peptide or polypeptide via a linker nucleotide sequence within the MGEV. The MGEV may be in a viral vector, microbial vector (e.g. an Agrobacterium vector) or a plant vector (e.g. a hybrid of a viral and microbial vector).

[0123] The at least 80% similarity includes 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

EXAMPLES

[0124] Aspects disclosed herein are further described by the following non-limiting Examples.

Materials and Methods

Gene constructs

[0125] cDNAs encoding OaAEPl _b (SEQ ID NO: l) and Oakl (SEQ ID NO: 13) were cloned into the pAM9 vector to incorporate the 35S-cauliflower mosaic virus promoter and terminator sequences (Tabe et al. (1995) J Anim Sci 73:2752-2759) before transfer into the binary vector, pBIN19 (Bevan (1984) supra). The pBIN19 expression vectors were then transformed into Agrobacterium tumefaciens (strain LBA4404) by electroporation.

Agrobacterium-mediated transient gene expression in plants

[0126] Agrobacterium cells were spread as a bacterial lawn on agar plates containing yeast mannitol medium (Vincent (1985) A manual for the practical study of root-nodule bacteria IPB Handbook No. 15) supplemented with kanamycin (50 ug/mL) and streptomycin (100 ug/mL). The cells were grown in the dark at 28°C for three days. The lawn of bacteria was harvested and resuspended to an OD ₆ oo of 1.0 in infiltration buffer (10 mM MgCl ₂ and 10 uM acetosyringone). The resuspended bacteria were incubated at room temperature for 2-4 h. Nicotiana benthamiana plants were grown from seed in either a glasshouse or growth cabinet at 25 °C for approximately six weeks. Bush bean cotyledons (Phaseolus vulgaris cv. Royal Burgundy) were grown for 10 days and lettuce leaves (Lactuca sativa cv. Green Cos) for approximately 4 weeks in a growth cabinet at 25 °C. Leaves were infiltrated with the resuspended Agrobacterium by gently pressing a 1 mL syringe (without a needle) against the underside of the leaf. The infiltrated area of the leaf was outlined with a permanent marker. Plants were grown for a further four days before the infiltrated areas were excised.

[0127] Infiltrated leaf segments were weighed, placed in microfuge tubes with a ball bearing and ground to a fine powder in liquid nitrogen using a mixer mill (30 s ^"1 , 2 x 15 s). Proteins were extracted with 50% (v/v) acetonitrile containing 0.1% (v/v) TFA using 1 uL per mg of tissue (wet weight) together with a 5mg of insoluble PVPP. Samples were centrifuged and the supernatants were then analyzed by MALDI-TOF mass spectrometry.

MS to track cyclization of linear peptides

[0128] Leaf extracts were desalted and concentrated using C18 ziptips and then mixed 1: 1 with a saturated solution of -cyano-4-hydroxycinnamic acid before they were spotted onto a sample plate, and air dried. Mass analysis was performed in positive ion reflector mode on a Bruker ultraflex III MALDI TOF/TOF mass spectrometer (Bruker AXS GmbH, Karlsruhe, Germany). Two hundred spectra at each of 10 randomly selected positions were accumulated per spot between 1000 and 5000 Da using an MS positive ion reflectron mode acquisition method. Calibration was conducted using a mixture of peptide standards (Bruker Daltonics). Data were acquired and processed using Bruker flex-Analysis software.

[0129] For relative quantification of cyclic and acyclic peptides, the total integrated peak area corresponding to assigned peptides was taken as 100% of the expressed peptides. The percentage of cyclic and acyclic peptides within the sample could then be calculated.

OaAEPl-3 cloning

[0130] Full length AEP transcripts from the O. affinis transcriptome assembly were used to design a set of primers. A single degenerate forward primer (OaAEPdegen-F, 5`-ATG GTT CGA TAT CYC GCC GG-3` - SEQ ID NO:9) was manually designed to amplify all sequences due to the variability at a single nucleotide position within the 5` region of each full length transcript at the start codon. Three reverse primers, designed with the aid of Primer3, successfully amplified AEP sequences (OaAEPl-R, 5`- TCA TGA ACT AAA TCC TCC ATG GAA AGA GC -3` - SEQ ID NO: 10; OaAEP2-R, 5`- TTA TGC ACT GAA TCC TTT ATG GAG GG -3` - SEQ ID NO: 11; OaAEP3-R 5`- TTA TGC ACT GAA TCC TCC ATC G -3` - SEQ ID NO: 12).

[0131] To clone expressed OaAEPs, total RNA was extracted from O. affinis leaves and shoots using TRIzol (Life Technologies) and was reverse transcribed with Superscript III reverse transcriptase (Life Technologies) according to the manufacturer's instructions. Target sequences were amplified from the resulting cDNA using Phusion High Fidelity Polymerase (New England BioLabs) and the primers described above under the recommended reaction conditions. Gel extracted PCR products were dA-tailed by incubation with Invitrogen Taq Polymerase (Life Technologies) and 0.5 μL 10 mM dA in the supplied buffer. The processed products were cloned into pCR8-TOPO (Life Technologies) and transformed into E. coli. Purified DNA from clones that were PCR positive for an AEP insert were sent for Sanger sequencing at the Australian Genome Research Facility. Coding sequences have been deposited in Genbank (accession codes: OaAEPl (KR259377), OaAEP2 (KR259378), OaAEP3 (KR259379).

[0132] In parallel, genomic DNA was extracted from O. affinis leaf tissue using a DNeasy Plant Mini Kit according to the manufacturer's instructions. PCR amplification from this DNA used primers specifically targeting the OaAEPl nucleotide sequence. Gel extracted product was dA tailed as above, cloned into TOPO (Life Technologies) and transformed into E. coli. DNA from PCR-positive clones was sent for sequencing to the Australian Genome Research Facility. The AEP sequence identified using this method (OaAEPl _b ) was subsequently expressed as a recombinant protein.

EXAMPLE 1

Co-expression of Oakl and OaAEP1 _b in N. benthamiana

[0133] A 1: 1 mixture of resuspended Agrobacterium (OD ₆ oo of 1.0) carrying Oakl (SEQ ID NO: 13 which encodes kB l) and OaAEPl _b (SEQ ID NO: l) [Figures 3B and 3A respectively] was used to infiltrate N. benthamiana leaves as described in the Materials and Methods. Proteins were extracted and analyzed by MALDI-TOF mass spectrometry as described in the Materials and Methods. N. benthamiana leaves infiltrated with Oakl alone were included as a control.

Results

[0134] In the N. benthamiana control, where no OaAEPl _b was present, approximately 10% + 1% of the linear cyclic kB l precursor was converted into cyclic product. The remainder is present as linear kB l product (38% + 3%) or as various linear forms with one or more amino acids derived from the C-terminal propeptide still present. When Oakl was transfected together with OaAEPl _b there was 89% +1% conversion of the linear precursor to cyclic product. The results shown are an average of a minimum of three replicates + standard error.

EXAMPLE 2

Expression of linked Oakl and OaAEPl _b in N. benthamiana

[0135] The sequence of Oakl (minus the signal sequence) was linked to the full length sequence of OaAEPl _b using the cleavable six -residue linker, EEKKND (SEQ ID NO: 18) to generate the construct defined by SEQ ID NO:20 , from the multidomain Nicotiana alata proteinase inhibitor (Heath et al. (1995) supra) [Figure 4A]. Alternatively, the sequence of OaAEPl _b (minus the signal sequence) is linked to the full length sequence of Oakl (SEQ ID NO:22) [Figure 4B]. These constructs were made by splice overlap PCR (Horton et al. (1990) supra) and are used in transient expression assays as described in the Materials and Methods.

Results

[0136] The construct OaAEPl _b -EEKKND-Oakl (Figure 4A) when transfected into N. benthamiana resulted in 100% cyclic kalata B l product.

EXAMPLE 3

Expression of OaAEPl _f , linked to multiple peptide domains

[0137] Two (or more) kalata B l domains each flanked by the N-terminal repeat the C- terminal propeptide are linked to the full length sequence of OaAEPl _b with (Figure 5 A) or without (Figure 5B) the cleavable five-residue MGEV linker, EEKKN, from the multidomain Nicotiana alata proteinase inhibitor (Heath et al. (1995) supra). These constructs are made by splice overlap PCR (Horton et al. (1990) supra) and are used in transient expression assays as described in the Materials and Methods.

EXAMPLE 4

Effect on cyclization of amino acid substitutions at the enzyme recognition sites

[0138] Oakl variants were generated from the wild-type Oakl construct using the Phusion site-directed mutagenesis kit (Finnzymes). Amino acid substitutions within the N-terminal Ρ -Ρ4" site and the C-terminal P4 -P4' site of the kalata B l precursor (Figure 1) were made to test the sequence requirements for efficient processing and cyclization of the peptide substrate by OaAEPl . All constructs were subcloned into the pAM9 vector and then cloned into the binary vector, pBIN19. The pBIN19 expression vectors were transformed into Agrobacterium and used in transient expression assays as described in the Materials and Methods.

Results

[0139] The effect on the % cyclized kalata B 1 product of various amino acid substitutions or deletions in the precursor are shown in Table 2. Only the sequence of the first (GLP) and last (TRN) amino acids of kalata B 1 (the central amino acids of the sequence being represented by " ") and the CTPP (GLPSLAA) are shown. The results shown are an average of a minimum of three replicates + standard error. Table 2

EXAMPLE 5

OaAEPl _f , and Oakl double stack in N. benthamiana

[0140] The Oakl plant transcription unit was amplified with 3' and 5' Xbal restriction sites by PCR. The PCR product was digested with Xbal and then ligated into the OaAEPl _b pBIN19 expression vector that had been digested with Xbal and treated with Antarctic phosphatase (NEB). The inserted Oakl plant transcription unit was checked by restriction digest to ensure that it was oriented in the same direction as the OaAEPl _b gene. The construct (Figure 8A) was then transformed into Agrobacterium and used in transient expression assays as described in the Materials and Methods.

Results

[0141] Transient expression of Oakl-OaAEPl _b double stack in N. benthamiana resulted in 88% + 1% cyclic product.

EXAMPLE 6

Co-expression of Oakl and other AEPs in N. benthamiana

[0142] DNA sequences encoding OaAEPl _b , OaAEP2 (SEQ ID NO:5) O,aA EP3 (SEQ ID NO:7), CtAEP1 (butelase 1, Nguyen et al. (2014) supra)(SEQ ID NO:23), CiAEP2 (SEQ ID NO:25) and CtAEP6 (previously known as CiAEP5) [SEQ ID NO:27] were cloned into pAM9, transferred into pBIN19 and transformed into Agrobacterium. Oakl was then co- expressed with each of these AEPs individually in N. benthamiana as described in the Materials and Methods. For co-expression with CiAEPl, CiAEP2 and CiAEP6 the Oakl gene was modified by replacing the CTPP (GLPSLAA) with HV (SEQ ID NO:30), the recognition sequence for CtAEP1 (Nguyen et al. (2014) supra) [Figure 3C]. Oakl was co- infiltrated with the AEP in each case except for CtAEPl // Oakl-HV where the symbol "//" indicates the elements were cloned into a double stack construct (Figure 8B) for transient transformation into N. benthamiana. "Oakl or Oakl-HV were expressed in the absence of an exogenous AEP as a control. [0142] Results are shown in Table 3.

Table 3 EXAMPLE 7

Cyclization of cyclotide domains within the Cter M precursor

[0144] The Cter M peptide precursor, either wild type (SEQ ID NO:55) or with a kB l cyclotide domain replacing the Cter M cyclotide domain (CterM-kB l) [SEQ ID NO:57] (Figure 11) was transiently expressed in N. benthamiana together with an AEP derived from C. ternatea (either CtAEPl (SEQ ID NO:23), CtAEP2 (SEQ ID NO:25) or CtAEP6 (SEQ ID NO:27). Additionally, Cter M-kB l-GLP (SEQ ID NO:59) together with OaAEPl _b (SEQ ID NO: l).were transiently expressed in N. benthamiana. N.benthamiana leaves infiltrated with either Cter M alone or Cter M-kB 1 alone respectively were included as controls.

[0145] A 1: 1 mixture of resuspended Agrobacterium (OD ₆ oo of 1.0) carrying one of Cter M, CterM-kB l or CterM-kB l-GLP and one of CtAEPl, CtAEP2, CtAEP6 or OaAEPl _b was used to infiltrate N. benthamiana leaves as described in the Materials and Methods. Proteins were extracted and analyzed by MALDI-TOF mass spectrometry as described in the Materials and Methods.

[0146] Results are shown in Table 4. Both the native Cter M and kB l cyclotide domains within the Cter M precursor were able to be cyclized by CtAEP 1 but not CtAEP2 or CtAEP6. Neither cyclic nor linear Cter M was detected in the Cter M alone control. When the recognition sequence GLP is added to the C-terminal end of kB l within the C-ter M precursor OaAEPl _b is able to efficiently cyclise kB l. Table 4

EXAMPLE 8

Precursor requirements for cyclic peptide production from the Oak 1 precursor

[0147] Various N-terminal truncations of Oakl were made and linked to OaAEPl _b at either the OaAEPlb C-terminal (Figure 10) or the OaAEPlb N-terminal (Figure 11) end via a MGEV linker in order to determine the minimum peptide precursor sequence requirement for cyclic kB 1 production.

[0148] These constructs were made by splice overlap PCR, (Horton et al. (1990) BioTechniques 8:528-535) and were used in transient expression assays in N. benthamiana as described in the Materials and Methods.

Results

[0149] When the AEP is positioned first in the construct (Figure 10) cyclic kB l production is abolished if the entire N-terminal region (ER signal peptide, N-terminal propeptide and N-terminal repeat) is deleted. At least the -NTR (11 residues DQVFLKQLQLK (SEQ ID NO:97)) is required for cyclic kBl production. This domain, as well as the NTPP, have previously been shown to be vacuolar targeting sequences (Conlan et al (2011) Amer. J. Botany 98(12 ):2018-2026). The -NTR may also be necessary for efficient cleavage that generates the proto-N-terminus of the cyclotide domain. Processing of the ER signal peptide in the plant cell can generate the proto-N-terminus of the cyclotide domain in a MGEV-linked construct when the AEP is second (Figure 11). The -NTR is not required. For the purposes of the present example, and without limiting the present invention to any one theory or mode of action, it is presumed that the vacuolar targeting signal from OaAEPl _b directs the linked precursor molecule to the vacuole. EXAMPLE 9

Precursor requirements for cyclic peptide production from the Cter M precursor

[0150] The Cter M precursor structure includes a cyclotide domain linked to an albumin 1 a-chain. Truncations of the albumin domain and spacer region were made (Figure 12) to determine the minimum requirement for cyclic peptide production. Processing of the ER signal peptide in the plant cell to generate the proto-N-terminus of the cyclotide domain is the strategy used by cyclotide precursors from Clitoria ternatea. These precursors, such as the Cter M precursor, lack the NTPP and NTR of O. affinis cyclotide precursors.

[0151] The cyclotide domain of the precursor was either the native Cter M (SEQ ID NO:56) or was substituted with the kB l cyclotide domain (SEQ ID NO:58).These constructs were made by splice overlap PCR, (Horton et al. (1990) supra), were co- infiltrated with CtAEPl in transient expression assays in N. benthamiana as described in the Materials and Methods.

Results

[0152] Cyclic kB l can be produced by replacing the Cter M cyclotide domain with kB l and co-expressing CtAEPl. This is further evidence that the NTPP and NTR are not necessary per se for cyclic kB l production and that the likely roles of at least the NTR (vacuolar targeting and providing residues for efficient cleavage to generate the proto-N- terminus) are replaceable by alternative elements and strategies. Truncated Cter M constructs that each lack at least the albumin- 1 a-chain did not produce cyclic Cter M, indicating that this domain contains a vacuolar targeting signal. EXAMPLE 10

CycHzation of linked peptide domains by OaAEPs

[0153] The ability of OaAEPs to cyclise linked peptide domains was tested. The enzymes used were OaAEPl _b (SEQ ID NO:2), OaAEP2 (SEQ ID NO:6) and OaAEP3 (SEQ ID NO:8) and the linked peptides were Oak2a (which encodes kB2 linked to kB3 (SEQ ID NO:51) [Figure 7A]) and Oak4 (which encodes three linked kB2 domains (SEQ ID NO:53) [Figure 7B]). These genes were individually cloned into pBIN19 expression vectors, transformed into Agrobacterium for use in transient expression assays.

[0154] A 1: 1 mixture of resuspended Agrobacterium (OD ₆ oo of 1.0) carrying one of Oak2a or Oak4 and one of OaAEPl _b , OaAEP2 or OaAEP3 was used to infiltrate N. benthamiana leaves as described in the Materials and Methods. Proteins were extracted and analyzed by MALDI-TOF mass spectrometry as described in the Materials and Methods. N. benthamiana leaves infiltrated with either Oak2a alone or Oak4 alone respectively were included as controls.

[0155] Results are shown in Table 5. OaAEPl _b and OaAEP3 are able to cleave and cyclise linked kalata precursor peptides from both the Oak2a and Oak 4 constructs. The production of cyclized kB2 and kB3 from the Oak2a linked construct occurred with equal efficiency.

Table 5

EXAMPLE 11

CycHzation by OaAEPl _b of a naturally cyclic trypsin inhibitor

[0156] The sequence of the sunflower trypsin inhibitor 1 (SFTI-1) [Mylne et al. (2011) supra] I or a grafted sunflower trypsin inhibitor (SFTI-FCQR) which has potential application as a prostate cancer inhibitor (Swedberg et al. (2009) Chem Biol 16: 633- 643)were used to replace kBl within the full length sequence of Oakl (Figure 9 and SEQ ID NOs:70 and 72). The resulting constructs were synthesized and codon usage was optimized for expression in Nicotiana. The constructs were then co-expressed with OaAEPl _b in N. benthamiana as described in the Materials and Methods. Results showed that cyclic SFTI-1 and cyclic grafted SFTI-FCQR products were detected

EXAMPLE 12

CycHzation by OaAEPlb of a bracelet-type cyclotide

[0157] The mature sequence of cycloviolacin 013 (Ireland et al. (2006) Biochem. J. 400: 1-12) is amplified from a full length cDNA and is used to replace kB l within the full length sequence of Oakl [SEQ ID NO:32] (Figure 9). This construct is made by splice overlap PCR (Horton et al. (1990) supra) and used in transient expression assays with OaAEPl _b as described in the Materials and Methods.

EXAMPLE 13

Cyclization by O AEPl _b of grafted kalata Bl peptides

[0158] Three residues from a bracelet cyclotide (KNK) were substituted into loop 5 of kB l, a Mobius cyclotide (SEQ ID NO:62); alternatively two charged residues (DK) wre substituted for residues that form part of a surface-exposed hydrophobic patch in loop 5 of kB l (SEQ ID NO: 64). These grafted kB l molecules were originally synthesized chemically to show that the cylotide framework could tolerate residue substitutions (Clark et al. (2006) Biochem. J. 394: 85-93). The sequence of an anti-multiple sclerosis peptide MOG3 (RSPFSRV) was grafted onto loop 5 of kB l (SEQ ID NO:66) [Wang et al. (2014) ACS Chem. Biol. 9: 156-163]. A kB l variant in loop 4 (T20K) [SEQ ID NO:68] is also a potential treatment for multiple sclerosis (Thell et al. (2016) Proc Natl Acad Sci USA 113: 3960-3965). These constructs were made by splice overlap PCR (Horton et al. (1990) supra) and used in transient expression assays with OaAEPl _b and/or OaAEP3 as described in the Materials and Methods.

[0159] The sequence of an anti-angiogenesis peptide (RRKRRR) is grafted onto loop 3 of kB l (SEQ ID NO:33) [Gunasekera et al. (2008) J. Med. Chem. 51: 7697-7704] (Figure 9). This construct is made by splice overlap PCR (Horton et al. (1990) supra) and used in transient expression assays with OaAEPl _b as described in the Materials and Methods.

Results

[0160] The results are shown in Table 6.

EXAMPLE 14

CycHzation by OaAEPl , of cyclic peptides with N or D at the proto-C -terminus

[0161] Asparaginyl endopeptidases preferentially cleave their substrates at a C-terminal asparagine although a C-terminal aspartate is also permissible. The effect of a C-terminal N or D residue was investigated by substituting an aspartic acid residue for the proto-C- terminus asparagine residue of Oakl-MOG3 (SEQ ID NO:66) to create Oakl-MOG3D (SEQ ID NO:94). Conversely the native C-terminal aspartate residue of Oakl-SFTI (SEQ ID NO:70), was mutated to an asparagine residue to create Oakl-SFTI-N (SEQ ID NO: 96). These constructs were made by splice overlap PCR (Horton et al. (1990) supra) and used in transient expression assays with and without co-expressing OaAEPl _b. Methods were as described in the Materials and Methods.

[0162] Results are shown in Figure 13. Cyclic MOG3 was not detected when Oakl- MOG3 alone was transiently expressed in N. benthamiana with linear MOG3 (li ⁿ _MOG3 ) ^an d linear MOG3 minus the N-terminal G residue (lin _MOG3 -G) the predominant products. In contrast, cyclic MOG3D was produced when the proto-C -terminal asparagine residue was changed to an aspartic acid residue (Oakl-MOG3D) suggesting that this residue is a better substrate for cyclization by endogenous AEPs; lin _MOG3D -G was not detected but there was a prominent lin _MOG3D +G peak. This species is MOG3 with the C-terminal G (derived from the C-terminal GLPSLAA (CTPP)) still attached. The difference in linear products between the two constructs suggests that the asparagine residue is preferred by endogenous hydrolyzing AEPs in N. benthamiana. Although precursors with either residue are presumably cleaved by endogenous AEPs resulting in both lin _MOG3 and lin _MOG3D , the prominent lin _MOG3 -G from Oakl-MOG3 suggests that the pool of lin _MOG3 was larger, allowing aminopeptidase cleavage of the N-terminal glycine residue. Conversely, the prominent lin _MOG3D +G peak suggests that the precursor was less likely to be hydrolyzed at the aspartate residue, allowing carboxypeptidases to cleave residues at the C-terminal. Co- expression of OaAEPl _b results in an increase in proportion of both cyclic MOG3 and

MOG3D although the proportion of cyclic product is higher in the latter. [0163] The sunflower trypsin inhibitor, SFTI, naturally has an aspartic acid residue at its proto-C-terminus. Co-expression of OaAEPl _b with SFTI in the Oakl precursor (Oakl-

SFTI) in N. benthamiana results in the production of cyclic SFTI. However no product (cyclic nor linear) is detected when an asparagine residue is substituted for the aspartic acid residue. It is presumed for the purposes of the present Example that the precursor is hydrolyzed by endogenous AEPs and subsequently digested by other proteases.

[0164] Therefore the production of cyclic peptides in N. benthamiana may be improved by having an aspartic acid residue at the proto-C-terminus in place of an asparagine residue.

EXAMPLE 15

Cyclization by OaAEPl _b of a non-cyclic peptide

[0165] The sequence of -conotoxin, Vcl.l (Clark et al. (2010) supra), with a six-residue linker (GGAAGN) is used to replace kB l within the full length sequence of Oakl (SEQ ID NO:34). This construct is made by multiple rounds of PCR using overlapping primers and used in transient expression assays with OaAEPl _b as described in the Materials and Methods. Alternatively, Vcl. l without a linker is used to replace kB l within the full length sequence of Oakl (SEQ ID NO:35) but retaining the N-terminal GLP and C-terminal TRN tripeptides from the ends of the kalata B l sequence (Figure 9).

EXAMPLE 16

Co-expression of OaAEPl _f , in bush bean and lettuce

[0166] Resuspended Agrobacterium (OD ₆ oo of 1.0) carrying a construct (constructs used are listed in Table 7) was used to infiltrate bush bean cotyledons (Phaseolus vulgaris cv. Royal Burgundy) and lettuce leaves (Lactuca sativa cv. Green Cos) as described in the Materials and Methods. Where two constructs were co -infiltrated (eg. OaAEPl _f + Oakl) a 1: 1 mixture of Agrobacterium was prepared. Proteins were extracted and analyzed by MALDI-TOF mass spectrometry as described in the Materials and Methods.

[0167] Results are shown in Table 7

Table 7

EXAMPLE 17

Stable transformation in Nicotiana

[0168] Stably transformed tobacco (Nicotiana tabacum) or Nicotiana benthamiana plants were produced by Agrobacterium-mediated transformation (Svab et al. (1975) In "Methods in Plant Molecular Biology -A Laboratory Manual", P. Maliga et al., eds. Cold Spring Harbor Press: 55-77.). Constructs used for transformation were Oakl, OaAEPl _b , the Oakl//OaAEPlb double stack and the empty vector control (N. tabacum) and OaAEPlb, OaAEP3 and the empty vector control (N. benthamiana). Proteins were extracted from leaves and analyzed by MALDI-TOF mass spectrometry as described in the Materials and Methods. The OaAEPl _b and the OaAEP3-expressing plants were used in transient expression assays using Agrobacterium transformed with Oakl or other sequences encoding peptides for cyclization.

[0169] Results Fourteen N. tabacum plants were produced from the stable transformation with Oakl // OaAEPl _b double stack. Leaves were analyzed by MALDI-TOF for kB l. Ten plants produced masses corresponding to predominantly cyclic kB l.

[0170] Twelve N. benthamiana plants were produced from the OaAEPl stable transformation and eight were tested for expression of OaAEPl _b and the ability to generate cyclic kB l by transient expression of the Oakl as described in the Materials and Methods. Six plants produced masses corresponding to predominantly cyclic kB l.

[0171] Fifteen plants were produced from the OaAEP3 stable transformation and eight plants produced masses corresponding to >79% cyclic kB l. EXAMPLE 18

Identification of cyclizing AEPs by substrate specificity

[0172] AEP activity has traditionally been tracked by monitoring cleavage of the fluorescent substrate Z-AAN-MCA (where Z is carboxybenzyl; MCA is 7-amido-4- methylcoumarin) [Saska et al. (2007) supra; Rotari et al. (2001) Biol. Chem. 382:953- 959]. Cleavage C-terminal to the Asn liberates the fluorophore which then fluoresces to report substrate cleavage. However, neither butelase-1 (Nguyen et al. (2014) supra) nor rOaAEPl _b (Figure 14) were active against this substrate. Furthermore, two AEP active site inhibitors had limited efficacy against rOAaEPl _b at high concentrations (Figure 15). They are Ac-YVAD-CHO, which is routinely used to identify AEP activity (Hatsugai et al. (2004) Science 305(5685): 855-858) and Ac-STRN-CHO, which represents the P1-P4 residues of the C-terminal kB l cleavage site. These traditional routes of identifying AEP activity will therefore likely be ineffective for identification of AEPs with cyclizing ability.

[0173] IQF peptides that incorporate the P1-P4 as well as the Ρ -Ρ4' residues are, however, effectively targeted by rOa AEPl . These peptides contain a fluorescence donor/quencher pair, with fluorescence observed upon the spatial separation of this pair following enzymatic cleavage. Activity against such IQF reporter peptides without corresponding activity against the generic substrate (Z-AAN-MCA) may allow rapid identification of members of the AEP family likely to have cyclizing ability. In the IQF peptide format, rOaAEPl _b displayed a requirement for a bulky hydrophobic residue at the P2' position that was not shared by a hydrolyzing AEP, human legumain (rhuLEG) [Figures 16A and 16B]. Such P2' specificity could also be used to predict cyclization ability and or to select AEPs with different sequence requirements in the substrate to be cyclized. [0174] Those skilled in the art will appreciate that aspects of aspects described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that these aspects include all such variations and modifications. These aspects also include all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

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