Methods for production of insecticidal neuropeptides

Abstract

The invention relates to recombinant expression constructs comprising a polynucleotide encoding at least one insecticidal neuropeptide, or a precursor peptide thereof, operably linked to a polynucleotide comprising a promoter suitable for expression in a microorganism. The invention also relates to insecticidal neuropeptide precursors, improved methods of their production and use in plant protection. Composition comprising concatemers of insecticidal neuropeptides or precursor peptides thereof are provided.

Description

[0001] METHODS FOR PRODUCTION OF INSECTICIDAL NEUROPEPTIDES BACKGROUND [1] Protection of agriculturally important crops from pathogenic insects (e.g. flies, caterpillars, moths, beetles, and the like) is crucial in improving crop yields. Insect infestations are a particular problem in many of the worlds primary agricultural territories such as South America and Asia, where such infestations can result in spoilage and contamination of food or feed products. Unfortunately, modern growing methods, harvesting and storage systems can promote insect infestations. Losses of crop yields worldwide due to insect infestations are estimated to be between 20-40%, costing the global economy around $220 billion per year. Solutions to improve protection of crops against undesirable insect pests are highly desirable. [2] Many insects contain hormonal or signalling peptide molecules which control their homeostasis and behaviour. In particular, many insects rely on so-called neuropeptides which control multiple physiological functions. Insect neuropeptides can be grouped into a number of distinct classes based on the receptors that they target and bind to, such as kinin, pyrokinin, CAPA, and AKH. [3] In recent years it has been suggested that such insect neuropeptides could be manipulated or modified to produce analogues of the neuropeptides which interfere with the intended binding of the neuropeptides with their cognate receptors. Thereby altering the signalling within the insect, and the insect behaviour to reduce the insect ability to infest a host plant. This can be achieved by reducing insect fecundity, reducing feeding or overall fitness, or by ultimately killing the insect. Such insecticidal analogues of insect neuropeptides have been described in at least: WO2020 / 115076, WO2021 / 245429, WO2023 / 099922 for example. [4] These insecticidal neuropeptides have been suggested as a promising alternative to broad spectrum chemical insecticides that are typically relied upon by farmers to control insect infestations in crops. They have the advantage that they are biological so do not cause contamination of the field, and they are targeted to a specific insect receptor and therefore to particular insect species whilst leaving other non-pest insect species, such as important pollinators, unaffected. Furthermore, such receptors are not found in animals, therefore the insecticidal neuropeptides have no toxicity to humans or animals. [5] Given the significant advantages associated with insecticidal neuropeptides compared with traditional chemical insecticides, it would be desirable to provide such peptides at a commercially viable level to farmers. However, currently it is difficult to produce the insecticidal neuropeptides to such a level. Production is limited to chemical peptide synthesis which is despite the significant progress in developing green and sustainable methodologies, is still considered slow and expensive, it produces low yields and toxic byproducts / waste due to the strong solvents and hazardous reagents involved in the synthesis process, making the products unsuitable for application to crops. [6] Recombinant processes of producing proteins and peptides from microbes are already used for many biologic therapeutics such as insulin, calcitonin, ecallantide, or teduglutide. Such recombinant processes are more environmentally friendly and have lower costs than chemical synthesis, they are also more effective for producing a biological product which does not contain toxic contaminants. Therefore, a bioproduction method for making insecticidal neuropeptides could provide a viable way to produce enough of these peptides for commercial use. However, there are challenges associated with recombinant techniques including the difficulty of producing peptides which contain non-natural modifications, the fact that short peptides can be degraded by microbial proteases, finding the correct host and vectors, and isolating the final peptides from the culture. These challenges have so far hindered the exploration of recombinant production of insecticidal peptides. [7] One or more aspects or embodiments of the present invention are intended to solve one or more of the above-mentioned problems in the art. STATEMENTS OF INVENTION [8] According to a first aspect of the present invention, there is provided a recombinant expression construct comprising a polynucleotide encoding at least one insecticidal neuropeptide, or a precursor peptide thereof, operably linked to a polynucleotide comprising a promoter suitable for expression in a microorganism selected from the group consisting of bacteria, yeasts, and fungi, which is heterologous to the polynucleotide encoding the at least one insecticidal neuropeptide or a precursor peptide thereof. [9] In one embodiment, said expression construct comprises: (i) a polynucleotide encoding two or more copies of a first insecticidal neuropeptide, or precursor peptide thereof; suitably between 2 to 10 copies of a first insecticidal neuropeptide, or precursor peptide thereof; or (ii) a polynucleotide encoding at least one copy of a first insecticidal neuropeptide, or a precursor peptide, and at least one copy of one or more additional insecticidal neuropeptides, or precursor peptides thereof, wherein the first insecticidal neuropeptide or a precursor peptide, is different from the or each additional insecticidal neuropeptide or precursor peptide thereof. Suitably between 1-10, suitably up to 10, additional insecticidal neuropeptides, or precursor peptides thereof. Suitably the polynucleotide encodes up to 10 insecticidal neuropeptides, or a precursor peptides thereof total. Suitably wherein each of the insecticidal neuropeptides, or a precursor peptides thereof may be the same or different.

[0010] In one embodiment, the polynucleotides encoding each insecticidal neuropeptide or precursor peptides thereof are separated by a polynucleotide sequence encoding a cleavage site, suitably a cleavage site for a protease, suitably wherein the cleavage site comprises the sequence: [Arg / Lys-Arg / Lys-(Glu-Ala)n] (SEQ ID NO: 58) wherein n is between 1 to 5, suitably wherein the cleavage site comprises a sequence selected from: KR, RR and KREAEA (SEQ ID NO: 59).

[0011] In one embodiment, the recombinant expression construct further comprises a polynucleotide encoding one or more of: (a) a targeting peptide, suitably selected from a vacuolar targeting peptide, an endoplasmic reticulum targeting peptide, and / or a plastid targeting peptide; (b) a secretion peptide; and / or (c) a polyadenylation or transcriptional termination signal; wherein the polynucleotides of (i), (ii) and / or (iii) are operably linked to the polynucleotide encoding the at least one insecticidal neuropeptide or a precursor peptide thereof.

[0012] In one embodiment, the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof, encodes a pre-pro-peptide. More specifically, the precursor peptide is a pre-pro-peptide. In one embodiment, the pre-pro-peptide is non-naturally occurring. Suitably the pre-pro-peptide comprises the sequence: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n wherein (a) S comprises at least one targeting peptide and / or a secretion peptide; (b) [SS] is an optional stuffer sequence; (c) [CC1] and [CC3] are optional consensus cleavage sites, [CC2] is present and is a consensus cleavage site; (d) NP is an insecticidal neuropeptide, a precursor, or a variant thereof; (e) AS is an optional C-terminal amidation signal; (f) n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and (g) i is a numerator indicating that the sequence of each NP unit and / or each: CC unit AS unit and / or SS unit in the construct is optionally different, optionally wherein the polynucleotide encoding the pre-pro-peptide further comprises a trailer sequence [Tr] at the 3' end thereof.

[0013] According to a second aspect of the present invention, there is provided a eukaryotic nuclear or plastid genome comprising a polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof, wherein the polynucleotide is heterologous to the nuclear or plastid genome and wherein the polynucleotide is operably linked to an endogenous promoter of the nuclear or plastid genome.

[0014] According to a third aspect of the present invention, there is provided cell comprising the recombinant expression construct of the first aspect or the genome of the second aspect, wherein the cell is optionally a bacterial, yeast, fungal (filamentous fungi), insect, or plant cell.

[0015] According to a fourth aspect of the present invention, there is provided a plant comprising a recombinant expression construct of the first aspect, or the genome of the second aspect, or the cell of the third aspect.

[0016] According to a fifth aspect of the present invention, there is provided a pre-pro-peptide comprising the sequence: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n wherein a. S comprises at least one targeting peptide and / or a secretion peptide; b. [SS] is an optional stuffer sequence; c. [CC1] and [CC3] are optional consensus cleavage sites, [CC2] is present and is a consensus cleavage site; d. NP is an insecticidal neuropeptide, a precursor, or a variant thereof; e. AS is an optional C-terminal amidation signal; f. n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and g. i is a numerator indicating that the sequence of each NP unit and / or each: CC unit AS unit and / or SS unit in the construct can be optionally different. In one embodiment, the pre-pro-peptide is non-naturally occurring. Suitably any of the features described in relation to the polynucleotide encoding the pre-pro-peptide apply equally to the pre-pro-peptide produced therefrom and described herein.

[0017] According to a sixth aspect of the present invention, there is provided a composition comprising one or more isolated insecticidal neuropeptides or chemically modified variants thereof, and at least 0.1% w / w of a precursor peptide thereof, and / or optionally a pre-pro-peptide thereof according to the fifth aspect.

[0018] According to a seventh aspect of the present invention, there is provided a process for producing insecticidal neuropeptide or a precursor thereof comprising the steps of: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect; (ii) culturing said host cell under suitable conditions for an insecticidal neuropeptide or precursor peptide, or pre-pro-peptide thereof to be produced by the host cell; (iii) optionally further processing any pre-pro-peptide to release an insecticidal neuropeptide or a precursor peptide thereof; and (iv) optionally further chemically and / or enzymatically modifying any precursor peptide to produce an insecticidal neuropeptide.

[0019] According to an eighth aspect of the present invention, there is provided a composition comprising an isolated insecticidal neuropeptide produced by the process of the seventh aspect, or chemically modified variants thereof, wherein the composition comprises at least 0.1% w / w of a precursor peptide thereof, and / or optionally a pre-pro-peptide thereof.

[0020] According to a ninth aspect of the present invention, there is provided a method for preventing or treating an insect infestation of a plant, the method comprising: contacting a plant, or part of said plant with an effective amount of the composition of the sixth aspect or the eighth aspect.

[0021] In one embodiment, the method for preventing or treating an insect infestation of a plant comprises administering to the plant or part of said plant with a composition of the sixth aspect or the eighth aspects, wherein the pre-pro-peptide comprises one or more consensus cleavage sites that are cleaved by the target insect following ingestion of the pre-pro-peptide to release one or more active insecticidal neuropeptides or precursors thereof.

[0022] Advantageously, the inventors have developed a recombinant process of producing insect neuropeptides from cultures of microbial host cells such as yeast in an efficient manner. The neuropeptides can be produced using an expression construct as described herein which is transcribed and expressed in the host cell, typically as a concatemer, to produce a pre-pro-peptide having multiple neuropeptide units (NPi) therein which may be the same or different neuropeptide sequences. The pre-pro-peptide design allows various desirable combinations of neuropeptides to be produced in a single culture. The pre-pro-peptide is a longer molecule so is not susceptible to general cellular cleavage mechanisms of the host, but which has built in specific peptidase cleavage sites in order to split the pre-pro-peptide into the desired precursor neuropeptides. In order to facilitate this cleavage, the pre-pro-peptide suitably comprises a targeting signal peptide which directs the molecule to the Golgi within the host cells for cleavage by said peptidases into the individual precursor neuropeptides and for terminal amidation. The resulting neuropeptides or precursor neuropeptides are produced in the host cells at high yields, and can then be isolated from the host cells, by lysis or secretion into the media. Once isolated, the neuropeptides may be insecticidally active as is, and may be used directly in agricultural compositions. Alternatively, they may still be precursors which may optionally be chemically or enzymatically modified such as by amidation, in order to produce active insecticidal neuropeptides for such agricultural uses. Alternatively, target insect species may themselves activate the precursor neuropeptides following ingestion.

[0023] In a further aspect there is provided a method of method for preventing or treating an insect infestation of a plant, the method comprising: contacting a plant, or part of said plant with an effective amount of a composition comprising an insecticidal neuropeptide precursor (e.g. a pre-pro-peptide) comprising one or more insecticidal neuropeptide flanked by cleavage sites, wherein the cleavage sites are suitable for cleavage following ingestion of the insecticidal neuropeptide precursor by the insect infesting the plant (the target insect). Suitably the cleavage sites are cleaved by enzymes present in the target insect gut. Alternatively, the cleavage sites are suitable for cleavage under chemical or physical conditions encountered in the target insect gut. In one embodiment, the cleavage sites comprise an intein which is activated under conditions encountered in the target insect gut. Suitably cleavage releases insecticidal neuropeptides which are active without further modification, or are active following modification that occurs within the target insect.

[0024] In some preferred embodiments, the insecticidal neuropeptide precursor (e.g., pre-pro- peptide) comprises a single insecticidal neuropeptide, and a signal peptide and / or a purification tag. In some preferred embodiments, the insecticidal neuropeptide precursor comprises at least one cleavage site adapted for chemical cleavage; thus the insecticidal neuropeptide precursor is suitably chemically cleaved at at least one cleavage site, suitably all cleavage sites, during production. In some preferred embodiments, the insecticidal neuropeptide precursor is adapted for chemical conversion of an amino acid (e.g. cysteine) provided at the C-terminus of the insecticidal neuropeptide to an amidated form; thus the insecticidal neuropeptide is chemically amidated at the C-terminus, suitably as part of a chemical cleavage process. Simultaneous cleavage and amidation of the C-terminus can be achieved, for example, when the cleavage site is or comprises a cysteine. In some particularly preferred embodiments, the insecticidal neuropeptide precursor is produced in Bacillus or Aspergillus.

[0025] Further features of the aspects of the invention will now be described under the following headed sections. Any feature may be combined with any aspect in any workable combination. DEFINITIONS

[0026] As used herein, the terms "polypeptide", "protein", “peptide”, and “amino acid sequence” are used interchangeably, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. As used herein, amino acid residues will be indicated either by their full name or according to the standard three- letter or one-letter amino acid code.

[0027] As used herein the term ‘variant’ means a peptide having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to a peptide described herein, and which comprises one or more variations to said peptide sequence, wherein the or each variation may be a modification to said peptide sequence such as an amino acid insertion, deletion, substitution, and the like.

[0028] Amino acid residues in polypeptides are in certain instance referred to herein by one letter amino acid codes as follows: G- Glycine (Gly); P- Proline (Pro); A- Alanine (Ala); V- Valine (Val); L- Leucine (Leu); I- Isoleucine (Ile); M- Methionine (Met); C- Cysteine (Cys); F- Phenylalanine (Phe); Y- Tyrosine (Tyr); W- Tryptophan (Trp); H- Histidine (His); K- Lysine (Lys); R- Arginine (Arg); Q- Glutamine (Gln); N- Asparagine (Asn); E- Glutamic Acid (Glu); D- Aspartic Acid (Asp); S- Serine (Ser); or T- Threonine (Thr).

[0029] As used herein, the terms "nucleic acid molecule", "polynucleotide", “polynucleic acid”, “nucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0030] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term"and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0031] As used herein, the terms “comprising”, "comprise / s", “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0032] Where a term is provided in the singular, other embodiments described by the plural of that term are also provided.

[0033] As used herein, a polynucleotide is said to be “endogenous” to a given cell when it is found in a naturally occurring form and genomic location in the cell.

[0034] The term “Bacillus” means a genus of Gram-positive, rod-shaped bacteria, a member of the phylum Bacillota. Bacillus species can be either obligate aerobes which are dependent on oxygen, or facultative anaerobes which can survive in the absence of oxygen.

[0035] The term “Bacillus subtilis” means a Gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. It is one of the preferred bacteria in secreted enzyme production and used on an industrial scale by biotechnology companies.

[0036] The term “filamentous fungi” means a type of fungi characterised by long, thread-like multicellular structures known as hyphae. These hyphae form a network called a mycelium, which functions in nutrient absorption.

[0037] The term “endoproteinase” is used herein to refer to a peptidase capable of cleaving a peptide bond between two internal amino acid residues in a peptide sequence. Endoproteinases can also be referred to as “endoproteases” or “endopeptidases.” The proteolytic activity of an endoproteinase, endoprotease, or endopeptidase is thus different from the proteolytic activity of an “exopeptidase” which cleaves peptide bonds of terminal amino acid residues in a peptide.

[0038] The term “heterologous”, as used herein in the context of a second polynucleotide that is operably linked to a first polynucleotide, refers to: (i) a second polynucleotide that is derived from a source distinct from the source of the first polynucleotide; (ii) a second polynucleotide derived the same source as the first polynucleotide, where the first, second, or both polynucleotide sequence(s) is / are modified from its / their original form; (iii) a second polynucleotide arranged in an order and / or orientation or in a genomic position or environment with respect to the first polynucleotide that is different than the order and / or orientation in or genomic position or environment of the first and second polynucleotides in a naturally occurring cell; or (iv) the second polynucleotide does not occur in a naturally occurring cell that contains the first polynucleotide. Heterologous polynucleotides include polynucleotides that promote transcription (e.g., promoters and enhancer elements), transcript abundance (e.g., introns, 5’UTR, and 3’UTR), translation, or a combination thereof as well as polynucleotides encoding insecticidal peptides or precursors thereof, or targeting / secretion peptides In certain embodiments, a nuclear or plastid genome can comprise the first polynucleotide, where the second polynucleotide is heterologous to the nuclear or plastid genome. A “heterologous” polynucleotide that promotes transcription, transcript abundance, translation, or a combination thereof as well as polynucleotides encoding insecticidal peptides or precursors thereof, or targeting / secretion peptides can be autologous to the cell but, however, arranged in an order and / or orientation or in a genomic position or environment that is different than the order and / or orientation in or genomic position or environment in a naturally occurring cell. A polynucleotide that promotes transcription, transcript abundance, translation, or a combination thereof as well as polynucleotides encoding insecticidal peptides or precursors thereof, or targeting / secretion peptides can be heterologous to another polynucleotide when the polynucleotides are not operably linked to one another in a naturally occurring cell. Heterologous peptides include peptides that are not found in a cell or organism as the cell or organism occurs in nature. As such, heterologous peptides include peptides that are localised in a subcellular location, extracellular location, or expressed in a tissue that is distinct from the subcellular location, extracellular location, or tissue where the peptide or protein is found in a cell or organism as it occurs in nature. Heterologous polynucleotides include polynucleotides that are not found in a cell or organism as the cell or organism occurs in nature.

[0039] The term “intein” means is a segment of a protein that is able to excise itself and join the remaining portions (the exteins) with a peptide bond during protein splicing. Inteins are for example described in Anraku, Y; Mizutani, R; Satow, Y (2005). "Protein splicing: its discovery and structural insight into novel chemical mechanisms". IUBMB Life. 57 (8): 563–74. doi:10.1080 / 15216540500215499. PMID 16118114.

[0040] The phrase “operably linked” as used herein refers to the joining of nucleic acid or amino acid sequences such that one sequence can provide a function to a linked sequence. In the context of a promoter, “operably linked” means that the promoter is connected to a sequence of interest such that the transcription of that sequence of interest is controlled and regulated by that promoter. When the sequence of interest encodes a protein that is to be expressed, “operably linked” means that the promoter is linked to the sequence in such a way that the resulting transcript will be efficiently translated. If the linkage of the promoter to the coding sequence is a transcriptional fusion that is to be expressed, the linkage is made so that the first translational initiation codon in the resulting transcript is the initiation codon of the coding sequence. Alternatively, if the linkage of the promoter to the coding sequence is a translational fusion and the encoded protein is to be expressed, the linkage is made so that the first translational initiation codon contained in the 5 ‘untranslated sequence associated with the promoter and the coding sequence is linked such that the resulting translation product is in frame with the translational open reading frame that encodes the protein. Nucleic acid sequences that can be operably linked include sequences that provide gene expression functions (e.g., gene expression elements such as promoters, 5’ untranslated regions, introns, protein coding regions, 3' untranslated regions, polyadenylation sites, and / or transcriptional terminators), sequences that provide DNA transfer and / or integration functions (e.g., T-DNA border sequences, site specific recombinase recognition sites, integrase recognition sites), sequences that provide for selective functions (e.g., antibiotic resistance markers, biosynthetic genes), sequences that provide scoreable marker functions (e.g., reporter genes), sequences that facilitate in vitro or in vivo manipulations of the sequences (e.g., polylinker sequences, site specific recombination sequences) and sequences that provide replication functions (e.g., bacterial origins of replication, autonomous replication sequences, centromeric sequences). In the context of an amino acid sequence encoding a targeting or secretion peptide, or other peptide, “operably linked” means that the peptide is connected to the polyprotein sequence(s) of interest such that it provides a function. Functions of a targeting peptide include localisation of a protein or peptide of interest (e.g., an insecticidal neuropeptide or precursor thereof, or pre-pro-peptide) to an extracellular space or subcellular compartment.

[0041] The phrases “percent identity” or “sequence identity” as used herein refer to the number of elements (i.e., amino acids or nucleotides) in a sequence that are identical within a defined length of two DNA, RNA or protein segments in an alignment resulting in the maximal number of identical elements, and is calculated by dividing the number of identical elements by the total number of elements in the defined length of the aligned segments and multiplying by 100.

[0042] The term "about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 10% or less, preferably + / -5% or less, more preferably + / - 1% or less, and still more preferably + / - 0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier 'about' refers is itself also specifically, and preferably, disclosed.

[0043] “insecticidal” or “insecticidal activity”, as used interchangeably herein, means to interfere with the harmful activity of an insect, including but not limited to killing the insect, inhibiting the growth or activity of the insect, inhibiting reproduction of the insect, altering the behaviour of the insect, or repelling the insect.

[0044] For the purposes of the invention, the phrase “transgenic” refers to an organism or progeny thereof wherein the organism’s or progeny organism’s DNA of the nuclear or organellar genome contains an inserted exogenous DNA molecule of 10 or more nucleotides in length.

[0045] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference herein, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein. DETAILED DESCRIPTION

[0046] Insecticidal neuropeptide or a precursor peptide thereof

[0047] The present invention relates to an expression construct comprising a polynucleotide encoding at least one insecticidal neuropeptide, for use in methods of bioproduction of said insecticidal neuropeptides.

[0048] The term ‘insecticidal neuropeptide’ as used herein means a peptide with insecticidal activity which binds to a receptor located in insect target tissues. The term neuropeptide is known in the art, and such neuropeptides are chemical messengers made up of small chains of amino acids (peptides) that are synthesized and released by neurons. Neuropeptides typically bind to G protein-coupled receptors (GPCRs) to modulate neural activity and other tissues like the gut, muscles, and heart. In their natural context, neuropeptides are synthesized from large precursor proteins which are cleaved and post-translationally processed then packaged. Accordingly, ‘insecticidal neuropeptide’ as used herein, in preferred aspects and embodiments, particularly relates to a small peptide (preferably 5 to 30 amino acid) – including its variants, amidated version, and other chemical modifications thereof - with insecticidal activity which binds to a receptor located in insect target tissues, which peptide in its natural form is (i) endogenous to an insect, (ii) synthesized and released by neurons or neurosecretory cells (typically from a larger precursor protein), and (iii) acts as a chemical messenger to regulate physiological processes and behaviours in insects and other invertebrates. In some aspects and embodiments, an ‘insecticidal neuropeptide’ in the context of the present invention can be referred to as an ‘insecticidal insect neuropeptide’ or an ‘insecticidal insect-derived neuropeptide’. In some cases, an ‘insecticidal neuropeptide’ is a neuropeptide that is endogenous to a target insect (or a related insect in which the neuropeptide is active), and which is insecticidal to that insect when applied exogenously in a suitable manner. An ‘insecticidal neuropeptide’ in the present invention is, in preferred aspects and embodiments, not, and is not derived from, a venom or toxin from a venomous organism. Such venoms or toxins include venoms or toxins from spiders, scorpions, centipedes, and other arthropods, as well as microbes or other organisms.

[0049] The term ‘precursor neuropeptide’ as used herein means a peptide capable of forming an insecticidal neuropeptide, but which only has insecticidal activity after further processing, typically by modification, optionally at the N or C terminus, such as e.g. C-terminal amidation, or formation of N-terminal pyro group, such as a pyroglutamate, for example. In some preferred embodiments, the precursor neuropeptide may be a pre-pro-peptide.

[0050] The term ‘Pre-pro-peptide’ is known in the art, and as used herein means a peptide capable of forming an insecticidal neuropeptide which must be further processed but also contains signalling sequences. A pre-pro-peptide is therefore considered to be a form of a precursor neuropeptide. A pre-protein has a targeting or secretion peptide, and a pro-protein is inactive until further processing occurs, typically wherein an inhibitory sequence is removed by proteases. A pre-pro-protein therefore comprises both a targeting / secretion peptide and requires further processing, typically cleavage by proteases. Optionally the term ‘pre-pro-peptide’ as used herein may in some embodiments refer to a concatemer of said insecticidal neuropeptides or precursor neuropeptides, otherwise referred to as ‘NP’ units, separated by cleavage sites as defined further in the formula below.

[0051] Optionally in some embodiments, a pre-pro-peptide may be cleaved to form one or more precursor neuropeptides, which may then be further processed, suitably by modification, to form one or more insecticidal neuropeptides. Alternatively, a pre-pro-peptide may be cleaved to directly form one or more insecticidal neuropeptides.

[0052] Suitably the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof, is selected from the group consisting of: i. a wild type gene encoding an insect pre-pro-peptide or a variant thereof; ii. a synthetic gene encoding an insect pre-pro-peptide or a variant thereof; and iii. a synthetic gene encoding tandem repeats of the pre-pro-peptide defined in (i) or (ii), optionally separated by cleavage sites with or without intermediate sequences, optionally as defined according to the fifth aspect of the invention.

[0053] Suitably the wild type gene may comprise a complete or partial open reading frame of a wild type gene encoding a insect pre-pro-peptide.

[0054] Suitably the variants of the wild type gene or the synthetic gene may be codon optimised.

[0055] Whilst the expression construct of the invention may comprise a polynucleotide encoding any insecticidal neuropeptide, or a precursor peptide thereof, in particular any such pre- pro peptide as listed in (i) - (iii) above, in preferred embodiments the expression construct of the invention comprises a polynucleotide which encodes a pre-pro-peptide of the invention, suitably as defined in the fifth aspect. In a more preferred embodiment, the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof is that listed in (iii) and encodes a pre-pro-peptide, as defined in the fifth aspect of the invention. Suitably the pre-pro-peptide comprises tandem repeats of polynucleotides encoding insecticidal neuropeptides, or precursor peptides thereof. Suitably therefore the polynucleotide encoding the pre-pro-peptide may be regarded as a concatemer.

[0056] In one embodiment, the polynucleotide encodes a non-natural pre-pro-peptide, suitably which is synthetic.

[0057] In one embodiment, there is provided a recombinant expression construct comprising a polynucleotide encoding a pre-pro-peptide according to the invention, suitably according to the fifth aspect, operably linked to a polynucleotide comprising a promoter which is heterologous to the polynucleotide encoding the pre-pro-peptide.

[0058] In one embodiment, there is provided a eukaryotic nuclear or plastid genome comprising a polynucleotide encoding a pre-pro-peptide according to the invention, suitably according to the fifth aspect, wherein the polynucleotide is heterologous to the nuclear or plastid genome and wherein the polynucleotide is operably linked to an endogenous promoter of the nuclear or plastid genome.

[0059] Suitably therefore, the polynucleotide encoding the at least one insecticidal neuropeptide, or a precursor peptide thereof encodes one or more ‘NP’ units as described in the pre-pro-peptide formula below, wherein each NP unit comprises an insecticidal neuropeptide, precursor, or a variant thereof, optionally separated by cleavage sites with or without intermediate sequences. Suitably any insecticidal neuropeptide, precursor, or a variant thereof may be comprised in an NP unit of the pre-pro-peptide of the invention, and suitably therefore the polynucleotide may encode any insecticidal neuropeptide, or precursor peptide thereof.

[0060] Suitably the polynucleotide encodes two or more copies of a first insecticidal neuropeptide, or precursor peptide, or NP unit. Suitably the polynucleotide may encode up to 10 copies, suitably up to 6 copies, of a first insecticidal neuropeptide, or a precursor peptide, or NP unit. Suitably wherein the copies have the same sequence. Suitably in such embodiments the polynucleotide may be a homo-concatemer.

[0061] Alternatively, the polynucleotide may encode at least one copy of a first insecticidal neuropeptide, or a precursor peptide thereof, or NP unit, and at least one copy of one or more additional insecticidal neuropeptides, or precursor peptides thereof, or NP units, wherein the first insecticidal neuropeptide, or a precursor peptide thereof, or NP unit, is different from the or each additional insecticidal neuropeptide, or precursor peptide thereof or NP unit. Suitably therefore the first and additional insecticidal neuropeptides, or precursor peptides thereof, or NP unit have different sequences. Suitably in such embodiments the polynucleotide may be a hetero- concatemer. Suitably the polynucleotide may comprise between 1-10, suitably up to 10, additional insecticidal neuropeptides, or precursor peptides thereof, or NP units. In a preferred embodiment, such hetero-concatemers are composed of at least two different insecticidal neuropeptides, or precursor peptides thereof, or NP units. Suitably each insecticidal neuropeptide, or precursor peptide thereof, or NP unit may target the same pest or may be suitable to target different pests, sutiably they may target different pests on the same crop.

[0062] Suitably the polynucleotide encodes between 2-10, suitably 2, 3, 4, 5, 6, 7, 8, 9, or 10, suitably between 2-6 insecticidal neuropeptides, or a precursor peptides, or NP units thereof total. Suitably wherein each of the insecticidal neuropeptides, or a precursor peptides, or NP units thereof may be the same or different.

[0063] Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the pre-pro-peptide NP units is selected from any class of insect neuropeptide, such as for example: a kinin, a pyrokinin, a CAPA, or an AKH peptide.

[0064] Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the pre-pro-peptide NP units (NPi), is a kinin, suitably comprising the following motif: (S / Y / N / H)-(S / A / P / V / T)-W-G / A (SEQ ID NO: 60) which may optionally be modified at any position. Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the pre-pro-peptide NP units, comprises a kinin sequence selected from: KQRFHSWG (SEQ ID NO: 61), NSVVLGKKQRFHAWG (SEQ ID NO: 62), FHAWG (SEQ ID NO: 63), FHSWG (SEQ ID NO: 64), NSVVLGKKQRFHSWG (SEQ ID NO: 65), DPKYKFSSWG (SEQ ID NO: 66), AKFSSWG (SEQ ID NO: 67), ARFSSWA (SEQ ID NO: 68), SKFNSWA (SEQ ID NO: 69), AKFSSWA (SEQ ID NO: 70), ARFNSWA (SEQ ID NO: 71), VPFNSWA (SEQ ID NO: 72), PIFSSWG (SEQ ID NO: 73), GPDFYAWG (SEQ ID NO: 74), SKFNAWA (SEQ ID NO: 75), PAFSSWG (SEQ ID NO: 76), RQKTVFSSWG (SEQ ID NO: 77), ASDKHGRPKQTFSSWG (SEQ ID NO: 78), PAFSAWG (SEQ ID NO: 79), VRFSPWG (SEQ ID NO: 80), NFSPWG (SEQ ID NO: 81), KVKFSAWG (SEQ ID NO: 82), SFSPWG (SEQ ID NO: 83), FSPWG (SEQ ID NO: 84), YFSPWG (SEQ ID NO: 85), KVKFSVWG (SEQ ID NO: 86), KVKFSTWG (SEQ ID NO: 87), FSAWG (SEQ ID NO: 88), FSTWG (SEQ ID NO: 89), DPAFNSWG (SEQ ID NO: 90), RAFTSTGSSRSPAFSSWG (SEQ ID NO: 91), PASFSSWG (SEQ ID NO: 92), DAGFSSWG (SEQ ID NO: 93), SSGFSSWG (SEQ ID NO: 94), DPVFKSWG (SEQ ID NO: 95), DAAFSSWG (SEQ ID NO: 96), GSGFSSWG (SEQ ID NO: 97), DDDSDVSESGSKFSSWG (SEQ ID NO: 98), SFSSWG (SEQ ID NO: 99), GFSSWG (SEQ ID NO: 100), LDRSEGRVSKALFSSWG (SEQ ID NO: 101), WGQSSVGAVFSSWG (SEQ ID NO: 102), GAEFYSWG (SEQ ID NO: 103), FSSWG (SEQ ID NO: 104), GPAFSSWG (SEQ ID NO: 105), AAAFNSWG (SEQ ID NO: 106), NAAFSSWG (SEQ ID NO: 107), VPAFNSWG (SEQ ID NO: 108), TNTAFSSWG (SEQ ID NO: 109), PRFYSWG (SEQ ID NO: 110), GADFYAWG (SEQ ID NO: 111), and NAAFNSWG (SEQ ID NO: 112), which may optionally be modified at any position.

[0065] Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the pre-pro-peptide NP units (NPi), is a CAPA peptide, suitably comprising the following motif: (A / F)-(G / T / K)-(P / Hyp / Oic)-R-(I / V / L) (SEQ ID NO: 113), or A-S-G-(L / β HL / β HA / β HF)- (V / β HL / β HV / β HA / β HF)-(A / β A)-F-P-R-V (SEQ ID NO: 114), or L-(V / Y)-(A / Aib)-F- (P / A)-R-V (SEQ ID NO: 323), W-F-G-P-R-L (SEQ ID NO: 115), or P-F-P-R-(I / L / V) (SEQ ID NO: 324) which may optionally be modified at any position, and wherein Hyp is Hydroxyproline, Oic is Octahydroindole-2-carboxylic acid, β HL is beta-homoleucine, β HA is beta-homoalanine, β HF is beta-homo-phenylalanine, β HV is beta-homovaline, β A is beta-alanine, Aib is 2- aminoisobutyric acid. Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the NP units, comprises a CAPA sequence selected from: LVAFPRV (SEQ ID NO: 116), LYAFARV (SEQ ID NO: 117), LYAFPRV (SEQ ID NO: 118), SDSKNTALWFGPRL (SEQ ID NO: 119), SDSKNTFLWFGPRL (SEQ ID NO: 120), SDSKNTAAWFGPRL (SEQ ID NO: 121), DAGLFPFPRV (SEQ ID NO: 122), NGASGNGGLWFGPRL (SEQ ID NO: 123), ESVAGLIPFPRV (SEQ ID NO: 124), EGLIPFPRI (SEQ ID NO: 125), DGVLSLYPFPRV (SEQ ID NO: 126), QLYAFPRV (SEQ ID NO: 127), GSSGLIPMGRV (SEQ ID NO: 128), GSSGLISMPRV (SEQ ID NO: 129), GSSGMIPFPRV (SEQ ID NO: 130), ESGGSGEANGMWFGPRL (SEQ ID NO: 131), GSSSGLISMPRV (SEQ ID NO: 132), Q / ELYAFPRV (SEQ ID NO: 133), TESPGMWFGPRL (SEQ ID NO: 134), SVPQGVPGAGGPADNNGLWFGPRL (SEQ ID NO: 135), EAGGPSGLFPFPRI (SEQ ID NO: 325), Q / EGLVPFPRL (SEQ ID NO: 136), and EGLFPFPRI (SEQ ID NO: 137), which may optionally be modified at any position. Suitably the CAPA peptide may be any type of CAPA peptide, for example the CAPA peptide may be a CAPA-1, CAPA-2 or CAPA-3 peptide.

[0066] Suitably wherein in the final insecticidal neuropeptide, N-terminal Q / E amino acids are converted to pyroglutamate ([pyr]). Suitably therefore CAPA insecticidal neuropeptides may comprise a sequence selected from: [pyr]LYAFPRV (SEQ ID NO: 138) or [pyr]GLVPFPRL (SEQ ID NO: 139), which may optionally be modified at any position.

[0067] Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the pre-pro-peptide NP units (NPi), is an AKH, suitably comprising the following motif: (L / I / V)- (T / N)-(F)-(S / T)-(P / R / S / T)-(D / S / N / T / G)-W-[(G / T)-(G / Q)] (SEQ ID NO: 140) or (L / I / W)- (T / N)-(F)-(S / T)-(P / R / S / T)-(D / S / N / T / G)-W-[(G / T)-(G / Q)] (SEQ ID NO: 141), which may optionally be modified at any position, and wherein square bracket residues are optional. Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the NP units, comprises an AKH sequence selected from: Q / ELTFSPDW (SEQ ID NO: 142), Q / ELTFTSSWGG (SEQ ID NO: 143), Q / EITFSRDWTG (SEQ ID NO: 144), Q / ELTFSTW (SEQ ID NO: 145), Q / ETFSRDWTG (SEQ ID NO: 146), Q / EVNFTPTWGQ (SEQ ID NO: 147), Q / ELNFSPGW (SEQ ID NO: 148), Q / ELTSPNW (SEQ ID NO: 149), Q / EGWSTLGTFS (SEQ ID NO: 150), Q / EVNFSPGWGT (SEQ ID NO: 151), Q / ELNFSPGWGP (SEQ ID NO: 152), Q / ELTFTSSWG (SEQ ID NO: 153), Q / ELTFSSGWGN (SEQ ID NO: 154), Q / ELTFSSGW (SEQ ID NO: 155), Q / EVNFSPNW (SEQ ID NO: 156), and QLTFSPDW (SEQ ID NO: 206) which may optionally be modified at any position.

[0068] Suitably wherein in the final insecticidal neuropeptide, N-terminal Q / E amino acids are converted to pyroglutamate ([pyr]). Suitably therefore AKH insecticidal neuropeptides may comprise a sequence selected from: [pyr]LTFSPDW (SEQ ID NO: 157), [pyr]LTFTSSWGG (SEQ ID NO: 158), [pyr]LTFTPNW (SEQ ID NO: 159), [pyr]ITFSRDWTG (SEQ ID NO: 160), [pyr]LTFSTW (SEQ ID NO: 161), [pyr]TFSRDWTG (SEQ ID NO: 162), [pyr]VNFTPTWGQ (SEQ ID NO: 163), [pyr]LNFSPGW (SEQ ID NO: 164), [pyr]LTSPNW (SEQ ID NO: 165), [pyr]GWSTLGTFS (SEQ ID NO: 166), [pyr]VNFSPGWGT (SEQ ID NO: 167), [pyr]LNFSPGWGP (SEQ ID NO: 168), [pyr]LTFTSSWG (SEQ ID NO: 169), [pyr]LTFSSGWGN (SEQ ID NO: 170), [pyr]LTFSSGW (SEQ ID NO: 171), and [pyr]VNFSPNW (SEQ ID NO: 172), which may optionally be modified at any position.

[0069] Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the pre-pro-peptide NP units (NPi), is a pyrokinin, suitably comprising the following motif: (F / P / Q / R / Y)-X-(P / T)-(K / R)-L (SEQ ID NO: 173), wherein X is any amino acid, which may optionally be modified at any position. Suitably the insecticidal neuropeptide or a precursor peptide thereof, or one or more of the NP units comprises a pyrokinin sequence selected from: GDTTQSSNGGMWFGPRL (SEQ ID NO: 174), SPPYSPPFSPRL (SEQ ID NO: 175), AIMARPQVPRL (SEQ ID NO: 176), EQNVQSNGEPAYRVRTPRL (SEQ ID NO: 177), LRQLQSNGEPAYRVRTPRL (SEQ ID NO: 178), LRKLESNGEPAYRNVRTTRL (SEQ ID NO: 179), QLVAFRRL (SEQ ID NO: 180), VIFTPKL (SEQ ID NO: 181), NADEDQQQSVDFTPRL (SEQ ID NO: 182), GGSMTFSPRL (SEQ ID NO: 183), GSESTDSTSMWFGPRL (SEQ ID NO: 184), QATQFTPRL (SEQ ID NO: 185), KVPWTPTPRL (SEQ ID NO: 186), NIQLNGYTPRL (SEQ ID NO: 187), STSWFAPRL (SEQ ID NO: 188), FGRL (SEQ ID NO: 189), QAINARPQVPRL (SEQ ID NO: 190), AINAPQVPRL (SEQ ID NO: 191), SVPFKPRL (SEQ ID NO: 192), TGPSASSGLWFGPRL (SEQ ID NO: 193), FYAPFSPRL (SEQ ID NO: 194), Q / ETSFIPRL (SEQ ID NO: 195), SPPFAPRL (SEQ ID NO: 196), DHIPQDIYSPRL (SEQ ID NO: 197), QSMWFGPRL (SEQ ID NO: 198), HLVSAQFAPRL (SEQ ID NO: 199), Q / EPEAFTPRL (SEQ ID NO: 200), SLAYDDKVFENVEFTPRL (SEQ ID NO: 201), TMNFSPRL (SEQ ID NO: 202), and Q / EAIMARPQVPRL (SEQ ID NO: 205) which may optionally be modified at any position.

[0070] Suitably wherein in the final insecticidal neuropeptide, N-terminal Q / E amino acids are converted to pyroglutamate ([pyr]). Suitably therefore pyrokinin insecticidal neuropeptides may comprise a sequence selected from: [pyr]TSFIPRL (SEQ ID NO: 203) or [pyr]PEAFTPR (SEQ ID NO: 204), which may optionally be modified at any position.

[0071] Suitably by ‘modified at any position’ it is meant that any one or more of the amino acids given in the peptide sequence may be replaced with a synthetic amino acid or an amino acid analogue, or may be chemically modified at a side group thereof. Suitable synthetic amino acids or analogues may be: beta-residues, amino-iso-butyric acid (Aib), amino-hexanoic acid (Ahx), hydroxyproline (Hyp), or the like. Suitable chemical modifications include for example amidation, acetylation, methylation, pegylation, palmitoylation etc.

[0072] In one embodiment, one or more of the pre-pro-peptide NP units comprised in the pre- pro-peptide may comprise a glycine or cysteine residue. Suitably at the C-terminus thereof. Suitably which may later be converted to a C-terminal amide. Suitably therefore any of the above sequences when present as a precursor or as an NP unit in the pre-pro-peptide many comprise an additional C or G at the C-terminus thereof.

[0073] In one embodiment, the precursor neuropeptide, or one or more of the pre-pro-peptide NP units (NPi) comprised in the pre-pro-peptide, is selected from: SPPYSPPFSPRL (SEQ ID NO: 175) Q / EAIMARPQVPRL (SEQ ID NO: 205) NADEDQQQSVDFTPRL (SEQ ID NO: 182) GGSMTFSPRL (SEQ ID NO: 183) KVKFSAWG (SEQ ID NO: 82) RQKTVFSSWG (SEQ ID NO: 77) PAFSSWG (SEQ ID NO: 76) Q / ELTFTSSWGG (SEQ ID NO: 143) QLTFSPDW (SEQ ID NO: 206) Q / ELTFSPDW (SEQ ID NO: 142) SVPFKPRL (SEQ ID NO: 192) LRQLQSNGEPAYRVRTPRL (SEQ ID NO: 178) GDTTQSSNGGMWFGPRL (SEQ ID NO: 174) Suitably, each of said sequences when combined with the amidation signal, if present, may comprise an additional G or C residue at the C-terminus thereof. Such sequences are also envisaged as precursor neuropeptides of the invention.

[0074] In one embodiment, the precursor neuropeptide, or one or more of the pre-pro-peptide NP units (NPi) comprised in the pre-pro-peptide, is selected from: GDTTQSSNGGMWFGPRL (SEQ ID NO: 174) QAIMARPQVPRL (SEQ ID NO: 207) RQKTVFSSWG (SEQ ID NO: 77) PAFSSWG (SEQ ID NO: 76)

[0075] In one embodiment the insecticidal neuropeptide, suitably which is active, is selected from: SPPYSPPFSPRL-[nh2] (SEQ ID NO: 208) [pyr]AIMARPQVPRL-[nh2] (SEQ ID NO: 209) NADEDQQQSVDFTPRL-[nh2] (SEQ ID NO: 210) GGSMTFSPRL-[nh2] (SEQ ID NO: 211) KVKFSAWG-[nh2] (SEQ ID NO: 212) RQKTVFSSWG-[nh2] (SEQ ID NO: 213) PAFSSWG-[nh2] (SEQ ID NO: 214) [pyr]-LTFTSSWGG-[nh2] (SEQ ID NO: 215) [palm]-QLTFSPDW-[nh2] (SEQ ID NO: 216) [pyr]LTFSPDW-[nh2] (SEQ ID NO: 217) SVPFKPRL-[nh2] (SEQ ID NO: 218) LRQLQSNGEPAYRVRTPRL-[nh2] (SEQ ID NO: 219) GDTTQSSNGGMWFGPRL-[nh2] (SEQ ID NO: 220) RQKTVFSSWGG-[nh2] (SEQ ID NO: 260) RQKTVFSSWGG-[OH] (SEQ ID NO: 261) [Pyr]-LTFSPDW-[OH] (SEQ ID NO:262) [Pyr]-LTFTSSWGG-[OH] (SEQ ID NO: 263) Wherein [pyr] indicates pyroglutamate and [palm] indicates palmitoyl, and wherein [nh2] indicates an amide group.

[0076] In one embodiment the insecticidal neuropeptide, suitably which is active, is selected from: GDTTQSSNGGMWFGPRL-[nh2] (SB-P-45) (SEQ ID NO: 220) [pyr]AIMARPQVPRL-[nh2] (SB-P-47) (SEQ ID NO: 209) RQKTVFSSWG-[nh2] (SB-P-65) (SEQ ID NO: 213) PAFSSWG-[nh2] (SB-P-66) (SEQ ID NO: 214) wherein [pyr] indicates pyroglutamate, and wherein [nh2] indicates an amide group.

[0077] [pre-pro-]-peptide

[0078] As mentioned above, the invention relates primarily to expression constructs and polynucleotides encoding a ‘pre-pro-peptide’ of an insecticidal neuropeptide, but also to the pre- pro-peptide itself. In one embodiment, the pre-pro-peptide is non-natural.

[0079] The invention provides a polynucleotide encoding a pre-pro-peptide, wherein the pre- pro-peptide comprises the sequence: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n wherein a. S comprises at least one targeting peptide and / or a secretion peptide; b. [SS] is an optional stuffer sequence; c. [CC1] and [CC3] are optional consensus cleavage sites, [CC2] is present and is a consensus cleavage site; d. NP is an insecticidal neuropeptide, a precursor, or a variant thereof; e. AS is an optional C-terminal amidation signal; f. n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and g. i is a numerator indicating that the sequence of each NP unit and / or each: CC unit, AS unit and / or SS unit in the construct can be optionally different, optionally wherein the polynucleotide encoding the pre-pro-peptide further comprises a trailer sequence [Tr] at the 3' end thereof.

[0080] The invention also provides a pre-pro-peptide comprising the sequence: S-([SSi]- [CC2i]-[NPi]-[ASi])n wherein h. S comprises at least one targeting peptide and / or a secretion peptide; i. [SS] is an optional stuffer sequence; j. [CC2] is a consensus cleavage site; k. NP is an insecticidal neuropeptide, a precursor, or a variant thereof; l. AS is a C-terminal amidation signal; m. n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and n. i is a numerator indicating that the sequence of each NP unit and / or each: CC unit AS unit and / or SS unit in the construct can be optionally different, optionally wherein the polynucleotide encoding the pre-pro-peptide further comprises a trailer sequence [Tr] at the 3' end thereof.

[0081] The invention also provides a pre-pro-peptide comprising the sequence: S-[CC1i]- ([NPi]-[CC2i]-[SSi]-[CC3i])n wherein: o. S comprises at least one targeting peptide and / or a secretion peptide; p. [SS] is an optional stuffer sequence; q. [CC2] is a consensus cleavage site, [CC1] and [CC3] are optional consensus cleavage sites; r. NP is an insecticidal neuropeptide, a precursor, or a variant thereof; s. n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and t. i is a numerator indicating that the sequence of each NP unit and / or each: CC unit AS unit and / or SS unit in the construct can be optionally different, optionally wherein the polynucleotide encoding the pre-pro-peptide further comprises a trailer sequence [Tr] at the 3' end thereof.

[0082] Elements of the above pre-pro-peptide will now be discussed in more detail.

[0083] Suitably the pre-pro-peptide has a total length of less than 200 amino acids, in some cases less than 150 amino acids, in some cases less than 100 amino acids.

[0084] It will be appreciated that the consensus cleavage sites comprised in the pre-pro-peptide can comprise any suitable cleavage site, for example a cleavage site that is recognised by an enzyme, a sequence that is adapted for chemical cleavage, or it may comprise an intein sequence that provides for cleavage.

[0085] Targeting Peptide / Secretion Peptide

[0086] Suitably the pre-pro-peptide may comprise a targeting peptide and / or a secretion peptide, suitably in some embodiments, the pre-pro-peptide may comprise both a targeting peptide and a secretion peptide.

[0087] Suitable targeting peptides may be selected from: a vacuolar targeting peptide, an endoplasmic reticulum targeting peptide, a nuclear localisation signal, a nuclear export signal, a mitochondrial targeting peptide, a peroxisomal targeting signal, and / or a plastid targeting peptide.

[0088] In preferred embodiments the pre-pro-peptide comprises an endoplasmic reticulum targeting peptide which is usually regarded as a secretion peptide. The secretory pathway typically starts by translocation of transmembrane polypeptides and polypeptides intended for secretion into the lumen of the endoplasmic reticulum (ER). Proteins destined for either extra- cellular secretion, the plasma membrane, the lumen or membrane of either the (ER), Golgi or endosomes possess an amino-terminal secretion peptide, also known as a signal peptide, signal sequence or leader peptide. Suitably therefore the pre-pro-peptide comprises a secretion peptide or a signal peptide, the terms may be used interchangeably herein. In one embodiment, the secretion peptide is a Golgi secretion peptide.

[0089] Suitably the secretion peptide comprises 5 to 50 amino acids, suitably 10 to 40 amino acids, suitably 13 to 36 amino acids in length.

[0090] Suitably the secretion peptide comprises mostly hydrophobic amino acids. Such peptides typically have a common structure: a short, positively charged amino-terminal region (n-region); a central hydrophobic region (h-region); and a more polar carboxy-terminal region (c-region) containing the site that is cleaved by a peptidase.

[0091] Suitable secretion peptides may be derived from yeast, suitably the secretion peptides of the pre-pro-peptide are selected from yeast secretion peptides. Suitable examples of such secretion / signal peptides are: S. cerevisiae phosphatase (PHO5, DK3614), S. cerevisiae sucrose invertase (SUC, WO84 / 01153), and yeast aspartic protease 3 (YAP3, EP792367B1), the signal peptide sequence of S. cerevisiae SUC2 (EP0438200), a P. pastoris acid phosphatase (PHO1) signal sequence (U.S. Pat. No. 5,268,273), a P. pastoris PIR1 secretion signal peptide (U.S. Pat. No. 7,741,075), the pre-pro signal of PpPir1p protein from Pichia pastoris (Khasa et al. 2011 (Yeast. 28(3):213-26)), a hydrophobin signal sequence (WO2011073367A1 and Kottmeier et al. 2011 (Applied Microbiology and Biotechnology. 91:1, 133-141), any of the signal peptides described in (De Schutter et al. Nature Biotechnology doi: 10.1038 / nbt.15442009), residues 1- 21 of SEQ ID 8 as listed in US2011 / 0021378A1, a signal peptide of the P. pastoris Epx1 protein (EP2258855A1), any signal peptide described in EP2707382B1. A particularly useful signal peptide and propeptide sequence for secretion of proteins in yeast is derived from the S. cerevisiae alpha- factor, and is described in U.S. Patent Nos. 4,546,082, 4,588,684, 4,870,008, and 5,602,034. The S. cerevisiae alpha- factor signal peptide and propeptide sequence consist of amino acids 1-83 of the primary, unprocessed translation product of the S. cerevisiae alpha mating factor gene (GenBank Accession Number: P01149). Further secretion peptides are described in EP2390333B1, for example.

[0092] Further suitable signal peptides and associated linkers are described in EP4273249A1, EP0832256A1, for example.

[0093] On the endoplasmic reticulum (ER) luminal side the secretion / signal peptide is cleaved off by a peptidase. After successful folding of the nascent polypeptide by ER resident chaperones and foldases, the protein is further directed to exit the ER. This process may be supported by the presence of an additional leader peptide. The protein is then transported to the Golgi network and finally to the plasma membrane for secretion out of the cell. The leader peptide is cleaved off the protein by Golgi-resident proteases. Suitably the secretion peptide may comprise or encompass a cleavage site, sutiably which may be a consensus cleavage site as defined elsewhere herein, or which may suitably which may be a leader peptide. Suitably therefore the pre-pro-peptide may further comprise an additional leader peptide.

[0094] Suitably the leader peptide is selected from a yeast leader peptide, which is suitable to drive secretory expression in yeast species. Suitable examples of such leader peptides are: the mating factor alfa leader peptide (MFα) from S. cerevisiae, optionally which may be truncated as described in EP324274B1, the Kluyveromyces alfa-factor leader sequence as described in EP301669B1, and pre-sequence or pro-sequence of Trichoderma reesei hydrophobin (WO2011073367A1 and Kottmeier et al. 2011 (Applied Microbiology and Biotechnology. 91:1, 133-141), a leader sequence of the P. pastoris Epx1 protein (EP2258855A1),

[0095] In one embodiment the secretion peptide is an α-factor secretion peptide, suitably MFalpha1s, suitably comprising or consisting of the sequence: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN GLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 221), suitably which may be encoded by the nucleic acid sequence according to SEQ ID NO: 2. In one embodiment the secretion peptide comprises a sequence according to SEQ ID NO: 29 of EP2707382B1: Met Lys Leu Ser Thr Asn Leu Ile Leu Ala Ile Ala Ala Ala Ser Ala Val Val Ser Ala (SEQ ID NO: 222) or according to SEQ ID NO: 2 of US11359223B2: Met Lys Leu Ser Thr Asn Leu Ile Leu Ala Ile Ala Ala Ala Ser Ala Val Val Ser Ala (SEQ ID NO: 222).

[0096] Stuffer sequence

[0097] Filler or stuffer (SS) sequences can be located in the pre-pro-peptide at any desired position such that they do not prevent a function or activity of the peptide. Suitably the role of the stuffer sequence is to enhance the efficiency of expression of the pre-pro-peptide within a host cell.

[0098] Suitably, a filler or stuffer sequence is located outside of a 5' and / or 3' UTR that flanks the respective 5' and / or 3' termini of a heterologous peptide sequence. Suitably, a filler or stuffer sequence is positioned within a 5' and / or 3' UTR that flanks the respective 5' and / or 3' termini of a heterologous peptide sequence. Suitably, a filler or stuffer sequence is positioned adjacent to 5' and / or 3' UTR that flanks the respective 5' and / or 3' termini of a heterologous peptide sequence. Suitably in this case the heterologous peptide sequence is that of the NP insecticidal neuropeptide or a precursor thereof. Suitably a stuffer sequence is present between each element of the pre- pro-peptide. Suitably a stuffer sequence is present between each NP unit present in the pre-pro- peptide, and suitably between the secretion peptide / targeting peptide and the rest of the pre-pro- peptide.

[0099] Suitably a stuffer sequence may also be present between cleavage sites in the pre-pro- peptide. Suitably between CC1 and CC2, and / or suitably between CC2 and CC3 when present.

[0100] Suitably the stuffer sequence comprises between 1 and 5 amino acids, suitably 2 or 3 amino acids. Suitably the stuffer sequence is a nonsense sequence, suitably which does not code for any peptide or protein. The stuffer sequence may be for example: AEQGD (SEQ ID NO: 223), RNLKW (SEQ ID NO: 224), QPFTH (SEQ ID NO: 225), GYKHI (SEQ ID NO: 226), CVWSF (SEQ ID NO: 227), DYHLN (SEQ ID NO: 228), TLVIA (SEQ ID NO: 229), RSGYM (SEQ ID NO: 230), NIVKD (SEQ ID NO: 231), FAWLT (SEQ ID NO: 232), IYQPM (SEQ ID NO: 233), GACFR (SEQ ID NO: 234), HESNV (SEQ ID NO: 235), VTLWR (SEQ ID NO: 236), QMYKD (SEQ ID NO: 237), NRGHV (SEQ ID NO: 238), KTWVF (SEQ ID NO: 239), LACPI (SEQ ID NO: 240), WDNQS (SEQ ID NO: 241), or SIKGT (SEQ ID NO: 242).

[0101] Suitably the stuffer sequence may comprise one or more restriction enzyme sites. Suitably this allows the introduction of further sequences into the polynucleotide encoding the pre-pro-peptide at any time. For example for the introduction of polynucleotides encoding further NPi units, suitably to extend the pre-pro-peptide into a longer concatemer of NPi peptide units. Examples of such stuffer sequences, adjoining a methionine cleavage site are provided in SEQ ID NOs: 294-296. Examples of such stuffer sequences, adjoining a Factor Xa protease cleavage site are provided in SEQ ID NO:291.

[0102] Cleavage site

[0103] The terms “consensus cleavage site” or “cleavage site” are used interchangeably herein to mean a site at which cleavage occurs to allow component parts of a peptide of the present invention (e.g. a pre-pro-peptide or pro-peptide) to be separated. For example, to allow one or more insecticidal neuropeptides to be separated from the pre-pro-peptide such that it is either in an active form or in a precursor form which can be modified to an active form, e.g. by further modification. Various methods of cleavage are described herein, and the “consensus cleavage site” or “cleavage site” is selected so as to be suitable for the respective cleavage method envisaged (e.g., enzymatic-, chemical- or intein-based cleavage, or a combination thereof).

[0104] Suitably the pre-pro-peptide comprises up to three consensus cleavage sites (CC). Suitably the pre-pro-peptide comprises at least one CC site. Suitably this is [CC2] as shown in the formulae above. Suitably the pre-pro-peptide may comprise up to three CC sites, [CC1], [CC2], and [CC3] as shown in the formula above. In some embodiments, all three CC sites may be present.Suitably at least one cleavage site is present between each NP unit present in the pre- pro-peptide, to allow each insecticidal neuropeptide or precursor thereof present in the pre-pro- peptide to be separated after expression in the host cell. Suitably the at least one cleavage site is present at the N-terminus of each NP unit. Suitably the at least one cleavage site is contiguous with the N-terminal end of each NP unit. Suitably said cleavage site is CC2. Suitably a cleavage site may further be present between the secretion peptide and the rest of the pre-pro-peptide. Suitably said cleavage site is CC1. Suitably a cleavage site may further be present between each stuffer sequence and the rest of the pre-pro-peptide. Suitably said cleavage site is CC2. Suitably at least one cleavage site is present at the C-terminus of each NP unit, suitably after the amidation signal if present. Suitably said cleavage site is CC2 or CC3.

[0105] In one embodiment, the pre-pro-peptide comprises the following structure: S-([SSi]- [CC2i]-[NPi]-[ASi])n. In one embodiment, the pre-pro-peptide comprises the following structure: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi])n. In one embodiment, the pre-pro-peptide comprises the following structure: S-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n. In one embodiment, the pre-pro-peptide comprises the following structure S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]- [CC3i])n in which CC1, CC2, and CC3 are present.

[0106] In one embodiment, the pre-pro-peptide comprises the following structure: S-[CC1i]- ([SSi]-[CC2i]-[NPi]-[CC3i])n, or S-([SSi]-[CC2i]-[NPi]-[CC3i])n.

[0107] In one embodiment, the pre-pro-peptide comprises the following structure: S-[CC1i]- ([NPi]-[CC2i]-[SSi]-[CC3i])n. In one embodiment, the pre-pro-peptide comprises the following structure: S-([NPi]-[CC2i]-[SSi]-[CC3i])n. In one embodiment, the pre-pro-peptide comprises the following structure: S-[CC1i]-([NPi]-[CC2i])n, or S-([NPi]-[CC2i])n.

[0108] Suitably, the or each cleavage site (CCi) may comprise the same or a different sequence. Suitably therefore the pre-pro-peptide may be cleaved by one, or more than one protease enzyme. In one embodiment, the or each cleavage site (CCi) in the pre-pro-peptide comprises the same sequence and is cleaved by the same protease. Suitably the or each cleavage site (CCi) in the pre- pro-peptide may be a chemical cleavage site or an enzymatic i.e. protease cleavage site.

[0109] Suitably the or each cleavage site (CCi) is a consensus sequence, suitably which is recognised for cleavage by a protease. Suitably the or each cleavage site (CCi) is a dibasic site, optionally comprising additional consensus sequences. Suitably the or each cleavage site (CCi) comprises an arginine rich sequence. Suitably the cleavage site comprises a lysine rich sequence. Suitably the cleavage site comprises arginine and / or lysine amino acids. Suitably the or each cleavage site comprises the consensus sequence: Arg / Lys-Arg / Lys. In the native S. cerevisiae alpha mating factor gene the cleavage site sequence corresponds to amino acid residues 84-89 and is represented by the sequence Lys84-Arg85-Glu86-Ala87-Glu88-Ala89 (SEQ ID NO: 243). The sequence Lys-Arg corresponds to a KEX2 protease recognition site. Suitably therefore the cleavage site may comprise Arg / Lys-Arg / Lys-(Glu-Ala)-(Glu-Ala) (SEQ ID NO: 244). In one embodiment the cleavage site comprises one of the following sequences: RR, RK, KR, KK, KREA (SEQ ID NO: 245), RKEA (SEQ ID NO: 246), KKEA (SEQ ID NO: 247), RREA (SEQ ID NO: 248), KREAEA (SEQ ID NO: 59), RKEAEA (SEQ ID NO: 249), RREAEA (SEQ ID NO: 250), or KKEAEA (SEQ ID NO: 251). In one embodiment the or each cleavage site comprises one of: KR, RR, or KREAEA (SEQ ID NO: 59). Meanwhile the Glu-Ala-Glu-Ala (SEQ ID NO: 252) sequence corresponds to a duplicated dipeptidyl aminopeptidase or STE13 recognition site. Suitably therefore, the or each cleavage site (CCi) may also or solely comprise Glu, Ala, Pro, and / or Asp amino acids. Suitably the or each cleavage site (CCi) may comprise one or more repeats of the following sequences: EA, EP, DA, and DP. Suitably the cleavage site may comprise between 1 to 4 repeats of said sequences, such as EPEPEPEP (SEQ ID NO: 253) for example. In one embodiment, the or each cleavage site comprises the sequence EAEA (SEQ ID NO: 254). Suitably the listed cleavage sequences may be combined, suitably therefore the cleavage site comprises a KEX2 recognition site and / or a STE13 recognition site. In one embodiment, the cleavage site comprises the sequence KR followed by one or more repeats of the following sequences: EA, EP, DA, and DP.

[0110] In one embodiment the cleavage site comprises the sequence KREAEA (SEQ ID NO: 59). Suitably in such embodiments the KR sequence is the cleavage site for a KEX2 protease, which suitably leaves each cleaved precursor peptide with a C terminal KR. Suitably therefore after KEX2 cleavage, the precursor peptides comprise a KR sequence at the C terminal end thereof. Suitably this may be removed by contacting the pre-pro-peptide with a KEX1 protease. Suitably which ‘chews’ the KR sequence away. Suitably the KEX2 and the KEX1 protease are used in combination. Suitably the EAEA sequence is the STE13 recognition site. Suitably therefore after KEX2 cleavage, the precursor peptides comprise an EAEA sequence at the N terminal end thereof. Suitably STE13 removes the EAEA sequence. Suitably which ‘chews’ the EAEA sequence away. Suitably the KEX2, KEX1 and STE13 proteases are used in combination. Suitably whenever the cleavage site is KREAEA (SEQ ID NO: 59).

[0111] Suitably cleavage at the cleavage sites and amidation of the insecticidal precursor peptides produced from the pre-pro-peptide are carried out together by enzymes produced by a host cell. Suitably by an endogenous protease. Suitably this may take place intracellularly within the host cell or extracellularly. For example this may take place within the Golgi of the host cell. Suitably each cleavage site is a dibasic cleavage site or a minimal consensus cleavage site appropriate to the host organism. Suitably the cleavage site is recognized by an endogenous protease enzyme native to the host cell. Suitably the cleavage site is recognized by a Golgi- resident protease, suitably native to the host cell. For example, in S. cerevisiae, the KEX2 protease resident in the Golgi will cleave at the consensus cleavage site Arg / Lys-Arg / Lys.

[0112] In alternative embodiments, the or each cleavage site (CCi) may be recognised by a non- endogenous peptidase to the host cell, suitably which is not expressed in the host cell. Suitably in such embodiments, the or each cleavage site (CCi) may be recognised by an exogenous peptidase. Suitably the exogenous peptidase may be an endo- or exopeptidase, suitably with specific amino acid recognition cleavage sites, selected from for example: Factor Xa (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269), Enterokinase (Asp-Asp-Asp-Asp-Lys) (SEQ ID NO:270), Thrombin (Leu-Val-Pro-Arg-Gly-Ser) (SEQ ID NO: 326), Human rhinovirus 3C Protease (Leu- Glu-Val-Leu-Phe-Gln-Gly-Pro) (SEQ ID NO:271), TEV (Tobacco Etch Virus Protease) (Glu- Asn-Leu-Tyr-Phe-Gln-Gly) (SEQ ID NO:272), TVMV (Tobacco Vein Mottling Virus Protease) (Glu-Thr-Val-Arg-Phe-Gln-Gly-Ser) (SEQ ID NO:273), Adenain (Gly-Gly), Staphylolysin (Gly- Gly), Pepsin (Cleaves at the C-Terminus of Phe, Leu, Tyr and Trp), Thermolysin (cleaves at the C-terminus of Ile , Leu, Val, Ala, Met and Phe), elastase (cleaves at C-Terminus of Ala, Val, Ser, Gly, Leu and Ile) and Asp-N (cleaves at the N-Terminal Side of Asp and Glu). Suitably therefore the or each cleavage site may comprise any of these listed cleavage site sequences.

[0113] In one embodiment, the or each cleavage site (CCi) may comprise a Factor Xa cleavage site, suitably having the sequence (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269). In one embodiment the Factor Xa cleavage side may be encoded by any sequences selected from SEQ ID No:287-290.

[0114] In another embodiment, a single Cysteine residue may act as a cleavage site. Suitably a chemical cleavage site. Suitably a single Cysteine may be first converted to a Dehydroalanine (DHA) residue through reaction with alkylating agent 2,5-dibromohexanediamide (DBHDA), then reaction with Tetrahydroxydiboron (B2(OH)4) and ascorbate. Suitably this is adapted from the methodology of Mollner et al. (2022) Reductive site-selective atypical C,Z-type / N2-C2 cleavage allows C-terminal protein amidation | Science Advances. In one embodiment, the or each cleavage site (CCi) may comprise a Cysteine cleavage site. Where a cleavage site comprises a single Cysteine residue, it is preferred that there are no cysteine residues at locations other than intended Cysteine cleavage sites. In particular, in such embodiments, it is preferable that the one or more insecticidal neuropeptides do not comprise any Cysteine residues such that the insecticidal neuropeptides themselves are not cleaved. Accordingly, in one embodiment, the cleavage site is a Cysteine residue and there are no Cysteine residues in the one or more insecticidal neuropeptides, and optionally there are no Cysteine residues in the pre-pro-peptide at any position which is not a cleavage site.

[0115] In yet another embodiment, a single Methionine residue may act as a cleavage site. Suitably a chemical cleavage site. Suitably a single Methionine may be cleaved by reaction with CNBr. In one embodiment, the or each cleavage site (CCi) may comprise a Methionine cleavage site. Where a cleavage site comprises a single Methionine residue, it is preferred that there are no Methionine residues at locations other than intended cleavage sites. In particular, in such embodiments, it is preferable that the one or more insecticidal neuropeptides do not comprise any Methionine residues such that the insecticidal neuropeptides are not cleaved. Accordingly, in one embodiment, the cleavage site is a Methionine residue and there are no Methionine residues in the one or more insecticidal neuropeptides, and optionally there are no Methionine residues in the pre-pro-peptide at any position which is not a cleavage site.

[0116] In an alternative embodiment still, the or each cleavage site (CCi) may be a self-cleaving peptide or an intein. Suitable inteins are described further hereinbelow. Suitable self-cleaving peptides may be selected from: P2A, E2A, F2A, and T2A for example.

[0117] In some embodiments, more than one cleavage site may be used in combination. Suitably to achieve different stages of cleavage of the pre-pro-peptide into the final insect neuropeptides or precursors thereof. Suitably to achieve a first cleavage and then a second cleavage. In some embodiments, the pre-pro-peptide may comprise [CC2]-[SS]-[CC3] between each NPi unit. Suitably in such embodiments, [CC2] and [CC3] are different cleavage sites. Suitably [CC2] and [CC3] may be selected from any cleavage site describe herein, however there are some preferred combinations such as:

[0118] Suitably [CC2] may be an intein and [CC3] may be a peptidase cleavage site, suitably an endogenous or exogenous peptidase cleavage site as listed hereinabove, suitably an exogenous peptidase cleavage site, in some cases a Factor Xa cleavage site. Optionally a stuffer sequence may be present between each [CC2] and [CC3] in this embodiment.

[0119] Suitably [CC2] may be a cysteine and [CC3] may be a peptidase cleavage site, suitably an endogenous or exogenous peptidase cleavage site as listed hereinabove, suitably an exogenous peptidase cleavage site, in some cases a Factor Xa cleavage site. Optionally a stuffer sequence may be present between each [CC2] and [CC3] in this embodiment. Suitably in such an embodiment, the NPi units do not contain any cysteine residues, i.e. the peptide sequences do not contain any cysteine residues.

[0120] Suitably [CC2] may be a cysteine and [CC3] may be a methionine. Optionally a stuffer sequence may be present between each [CC2] and [CC3] in this embodiment. Suitably in such an embodiment, the NPi units do not contain any cysteine residues or any methionine residues, i.e. the peptide sequences do not contain any cysteine nor any methionine residues.

[0121] Trailer sequence

[0122] Suitably a polynucleotide encoding a trailer (Tr) sequence may be added to the polynucleotide encoding the pre-pro-peptide after the final ‘CC’ cleavage consensus site, suitably at the 3' end of the polynucleotide encoding the pre-pro-peptide.

[0123] Suitably the trailer sequence is a non-translated segment at the 3′ end of mRNA following the signal that terminates translation. Suitably therefore the trailer sequence is comprised in the 3'UTR. Suitably the trailer sequence is present in a polynucleotide encoding the pre-pro-peptide, suitably in an mRNA transcript encoding the pre-pro-peptide, but is not present in the expressed pre-pro-peptide itself. Suitably the polynucleotide encoding a trailer sequence encodes one or more regulatory elements, optionally one or more post-transcriptional modification sites. Suitably the polynucleotide encoding a trailer sequence comprises a cleavage and polyadenylation specificity factor (CPSF) site, suitably which comprises the sequence TTATTT, transcribed to AAUAAA in mRNA. Suitably the CPSF binds to said site in the trailer sequence of mRNA and directs polyadenylation of mRNA transcripts. Suitably the trailer sequence may therefore comprise a polyA tail for example. Suitably which may comprise between 50 and 250 A nucleotides. Suitably the polynucleotide encoding the trailer sequence comprises a terminator, suitably a transcriptional terminator sequence. Suitably the transcription terminator may be a CYC transcriptional terminator having a sequence according to SEQ ID NO: 3.

[0124] In one embodiment, the polynucleotide encoding the trailer sequence comprises the sequence: CCGGCCGGCCATTTAAATACAGGCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTA TGTCACGCTTACATTCACGCCCTCCTCCCACATCCGCTCTAACCGAAAAGGAAGGAG TTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTTAATAGTTATGTTAGTATTA AGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCGTGTACGCATG TAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAAT TTGC (SEQ ID NO: 255)

[0125] Neuropeptide Units

[0126] Suitably the pre-pro-peptide comprises one or more insecticidal neuropeptides or precursors thereof, or variant thereof, designated as ‘NPi’ units. Suitably each insecticidal neuropeptide or precursor thereof, i.e. NP unit, is less than 10 amino acids in length, suitably between 5 and 10 amino acids in length.

[0127] Suitably the pre-pro-peptide comprises a plurality of insecticidal neuropeptides or precursors or variants thereof, suitably a plurality of ‘NP’ units. Suitably the number of NP units within the pre-pro-peptide is designated as ‘n’. Suitably n is between 1-10, suitably n is at least 2, suitably n is between 2 to 6, suitably ‘n’ may be 2, 3, 4, or 6. In one embodiment n is 3. In one embodiment, n is 6. Suitably therefore the pre-pro-peptide comprises 3 or 6 insecticidal neuropeptides or precursors or variants thereof.

[0128] Suitably when the pre-pro-peptide comprises more than one NP unit, they are arranged in series as a concatemer of insecticidal neuropeptides or precursors or variants thereof. Suitably therefore the pre-pro-peptide comprises a concatemer of insecticidal neuropeptides or precursors, or variants thereof. Suitably a stuffer sequence and a cleavage consensus sequence are located between each NP unit of the pre-pro-peptide.

[0129] Suitably each NP unit contained within the pre-pro-peptide may be the same or may be different. Suitably therefore the sequence of each NP unit is identical, or the sequence of each NP unit is different. In some embodiments, the pre-pro-peptide may comprise a plurality of the same NP unit operable to produce a plurality of the same insecticidal neuropeptide thereby increasing the production efficiency of the peptide. In other embodiments, the pre-pro-peptide may comprise a plurality of different NP units operable to produce a desired mixture of insecticidal neuropeptides. Suitably the NP units may be chosen to produce a tailored mixture of any of the insecticidal neuropeptides described hereinabove. Therefore, the pre-pro-peptide design allows for maximum flexibility in producing the insecticidal neuropeptides in a host cell. Suitably, NP units may be chosen which provide beneficial effects in combination, for example NP units may be chosen which have a synergistic effect in insecticidal activity. This has the advantage that the host cell will produce a ready-made combination product as such.

[0130] Suitably the pre-pro-peptide comprises a combination of at least two different types of insecticidal neuropeptides, precursors or variants thereof, suitably therefore at least two different types of NP unit.

[0131] Suitably therefore the pre-pro-peptide comprises at least one NP unit of a first insecticidal neuropeptide or a precursor peptide, or variant thereof, and at least one NP unit of one or more additional insecticidal neuropeptides or precursor peptides or variants thereof. Suitably wherein the first insecticidal neuropeptide or a precursor peptide, or a variant thereof is different from the or each additional insecticidal neuropeptide or precursor peptide or a variant thereof.

[0132] In one embodiment, the pre-pro-peptide comprises at least one insecticidal neuropeptide or precursor thereof, or variant thereof in an NP unit which is an AKH insecticidal neuropeptide or precursor thereof, and at least one insecticidal neuropeptide or precursor thereof or variant thereof in an NP unit which is a pyrokinin insecticidal neuropeptide or precursor thereof.

[0133] In one embodiment, the pre-pro-peptide comprises at least one NP unit which is an AKH insecticidal neuropeptide or precursor thereof, or variant thereof and at least one NP unit which is a CAPA insecticidal neuropeptide or precursor thereof or variant thereof.

[0134] In one embodiment, the pre-pro-peptide comprises at least one NP unit which is an kinin insecticidal neuropeptide or precursor thereof, or variant thereof and at least one NP unit which is a pyrokinin insecticidal neuropeptide or precursor thereof or variant thereof.

[0135] In one embodiment, the pre-pro-peptide comprises at least one NP unit which is an kinin insecticidal neuropeptide or precursor thereof, or variant thereof and at least one NP unit which is an AKH insecticidal neuropeptide or precursor thereof or variant thereof.

[0136] In one embodiment, the pre-pro-peptide comprises at least one NP unit which is an kinin insecticidal neuropeptide or precursor thereof, or variant thereof and at least one NP unit which is a CAPA insecticidal neuropeptide or precursor thereof or variant thereof.

[0137] In one embodiment, the pre-pro-peptide comprises at least one NP unit which is a pyrokinin insecticidal neuropeptide or precursor thereof or variant thereof, and at least one NP unit which is a CAPA insecticidal neuropeptide or precursor thereof or variant thereof.

[0138] In one preferred embodiment, the pre-pro-peptide comprises at least one NP unit which is an AKH or precursor thereof or variant thereof, and at least one NP unit which is a pyrokinin insecticidal neuropeptide or precursor thereof or variant thereof.

[0139] Affinity Tag

[0140] Optionally, the pre-pro-peptide may further comprise one or more affinity tags, designated [A]. Suitably located at the N or C terminus of the pre-pro-peptide. Suitably the affinity tag comprises between 5-10 amino acids. Suitably the affinity tag aids in isolation and purification of the downstream insecticidal neuropeptide or precursor peptide, once released from the pre-pro-peptide. Suitably the affinity tag aids in isolation and purification of the insecticidal neuropeptide or precursor peptide from the host cell culture. Suitably by affinity chromatography. Suitable affinity tags are described elsewhere herein.

[0141] In one embodiment, the pre-pro-peptide may comprise the sequence: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n-[A]; or the sequence: S-[CC1i]-([SSi]-[CC2i]-[A]-[NPi]-[ASi]-[CC3i])n

[0142] In one embodiment, the pre-pro-peptide may comprise the sequence: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[CC3i])n – [A]; or the sequence S-[CC1i]-([SSi]-[CC2i]-[A]-[NPi]-[CC3i])n

[0143] In one embodiment, the pre-pro-peptide may comprise the sequence: S-[CC1i]-([NPi]-[CC2i]-[SSi]-[CC3i])n-[A]; or the sequence S-[CC1i]-([A]-[NPi]-[CC2i]-[SSi]-[CC3i])n.

[0144] Suitable affinity tags are known in the art, and may be selected from: a His-tag, an Arg- tag, a FLAG-tag, a Strep-tag, Glutathione-S-transferase tag, Twin-Strep Tag, Calmodulin Binding Peptide, Chitin-binding Tag, Maltose-binding Tag, and a Cellulose-binding Tag.

[0145] Suitably a reporter protein may be present between the affinity tag the rest of the pre- pro-peptide, suitably located at the N or C terminus of the pre-pro-peptide. Suitably the reporter protein and the affinity tag may be a contiguous sequence i.e. a fusion protein.

[0146] Optionally, a further cleavage consensus site (CC) may be present between the affinity tag and the rest of the pre-pro-peptide. Suitably said cleavage site is as defined above for the other cleavages sites. Suitably said further cleavage consensus site (CC) may be a protease recognition site linker. Suitably to enable removal of the affinity tag once the insecticidal neuropeptide or precursor peptide is isolated. Suitably in such embodiments, the pre-pro- peptide may comprise the sequence: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n-[CC4i]-[A]; S-[CC1i]-([SSi]-[CC2i]-[NPi]-[CC3i])n-[CC4i]-[A]; or S-[CC1i]-([NPi]-[CC2i]-[SSi]-[CC3i])n-[CC4i]-[A]

[0147] Preferred pre-pro-peptides

[0148] In some embodiments, the pre-pro-peptide comprises the formula: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n or S-[CC1i]-([NPi]-[CC2i]-[SSi]-[CC3i])n or S-[CC1i]-([NPi]-[CC2i])n

[0149] In some embodiments, the pre-pro-peptide comprises the formula: S-([SSi]-[CC2i]-[NPi]-[ASi])n, or S-([CC2i]-[NPi]-[ASi])n or S-([NPi]-[CC2i]-[SSi]-[CC3i])n or S-([NPi]-[CC2i])n

[0150] In some embodiments, the pre-pro-peptide comprises the formula: S-([KREAEA]-[NPi-Gly])n (SEQ ID NO: 256); or S-([KREAEA]-[NPi-Cys])n (SEQ ID NO: 257); or S-([KR]-[NPi-Gly])n; or S-([KR]-[NPi-Cys])n

[0151] In some embodiments, the pre-pro-peptide comprises the formula: [MFalpha1s secretion peptide]-([KREAEA]-[NPi-Gly])n (SEQ ID NO: 258); or [MFalpha1s secretion peptide]-([KREAEA]-[NPi-Cys])n (SEQ ID NO: 259); or [MFalpha1s secretion peptide]-([KR]-[NPi-Gly])n; or [MFalpha1s secretion peptide]-([KR]-[NPi-Cys])n

[0152] In some embodiments, the pre-pro-peptide comprises the formula: S-[CC1i]-([NPi]-[Cys])n S-[CC1i]-([NPi]-[Cys]-[SSi]-[Ile-(Glu or Asp)-Gly-Arg])n S-[CC1i]-([NPi]-[Cys]-[SSi]-[Met])n

[0153] In some embodiments, the pre-pro-peptide comprises the formula: S-([NPi]-[Cys])n S-([NPi]-[Cys]-[SSi]-[Ile-(Glu or Asp)-Gly-Arg])n S-([NPi]-[Cys]-[SSi]-[Met])n

[0154] In some embodiments, the pre-pro-peptide comprises the formula: [MFalpha1s secretion peptide]-[CC1i]-([NPi]-[Cys])n [MFalpha1s secretion peptide]-[CC1i]-([NPi]-[Cys]-[SSi]-[Ile-(Glu or Asp)-Gly-Arg])n [MFalpha1s secretion peptide]-[CC1i]-([NPi]-[Cys]-[SSi]-[Met])n

[0155] In some embodiments, of the above formulae, n is between 1-6, suitably 1, 3, or 6.

[0156] In some embodiments, each NP unit (NPi) in any of the above formulae may be selected from one of the following sequences: GDTTQSSNGGMWFGPRL (SEQ ID NO: 174) Q / EAIMARPQVPRL (SEQ ID NO: 205) RQKTVFSSWG (SEQ ID NO: 77) PAFSSWG (SEQ ID NO: 76)

[0157] In one embodiment of the above formulae, the pre-pro-peptide comprises one NP unit, suitably therefore n is 1 and the NP unit (NPi) consists of one of the following sequences: GDTTQSSNGGMWFGPRL (SEQ ID NO: 174), suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 6 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. QAIMARPQVPRL (SEQ ID NO: 207), suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 10 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. RQKTVFSSWG (SEQ ID NO: 77), suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 14, or 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. RQKTVFSSWG (SEQ ID NO: 77), suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 30 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. PAFSSWG (SEQ ID NO: 76), suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 40 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. PAFSSWG (SEQ ID NO: 76), suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 44 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0158] In one embodiment of the above formulae, the pre-pro-peptide comprises three NP units, suitably therefore n is 3 and each of the NP units (NPi) consists of the sequence RQKTVFSSWG (SEQ ID NO: 77). Suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. In one embodiment of the above formulae, the pre-pro-peptide comprises three NP units, suitably therefore n is 3 and each of the NP units (NPi) consists of the sequence RQKTVFSSWG (SEQ ID NO: 77). Suitably wherein the pre- pro-peptide comprises a sequence according to SEQ ID NO: 34, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0159] In one embodiment of the above formulae, the pre-pro-peptide comprises six NP units, suitably therefore n is 6 and each of the NP units (NPi) consists of the sequence RQKTVFSSWG (SEQ ID NO: 77). Suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 27, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. In one embodiment of the above formulae, the pre-pro-peptide comprises six NP units, suitably therefore n is 6 and each of the NP units (NPi) consists of the sequence RQKTVFSSWG (SEQ ID NO: 77). Suitably wherein the pre-pro- peptide comprises a sequence according to SEQ ID NO: 37, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0160] In one embodiment of the above formulae, the pre-pro-peptide comprises three NP units, suitably therefore n is 3, wherein the first NP unit (NPi) consists of the sequence RQKTVFSSWG (SEQ ID NO: 77), the second NP unit (NPi) consists of the sequence QAIMARPQVPRL (SEQ ID NO: 207), and the third NP unit (NPi) consists of the sequence GDTTQSSNGGMWFGPRL (SEQ ID NO: 174). Suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 18, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0161] In one embodiment of the above formulae, the pre-pro-peptide comprises three NP units, suitably therefore n is 3 and each of the NP units (NPi) consists of the sequence PAFSSWG (SEQ ID NO: 76). Suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 48, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0162] In one embodiment of the above formulae, the pre-pro-peptide comprises six NP units, suitably therefore n is 6 and each of the NP units (NPi) consists of the sequence PAFSSWG (SEQ ID NO: 76). Suitably wherein the pre-pro-peptide comprises a sequence according to SEQ ID NO: 51, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0163] In other embodiments of the invention in which the expression construct may comprise a complete or partial open reading frame of a wild type gene encoding an insect pre-pro-peptide or a codon optimized variant thereof, the expression construct may comprise a wild type gene encoding an insect pre-pro-peptide from P. pastoris, suitably which is codon optimised. In such an embodiment, the pre-pro-peptide comprises the sequence according to SEQ ID NO:54, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0164] In some embodiments, the polynucleotide encoding a pre-pro-peptide according to the invention may comprise or consist of a nucleic acid sequence selected from: SEQ ID NOs: 4, 8, 12, 16, 19, 22, 25, 28, 32, 35, 38, 42, 46, 49 and 52; or a nucleic acid sequence selected from: SEQ ID NOs: 5, 9, 13, 17, 20, 23, 26, 29, 33, 36, 39, 43, 47, 50, and 53, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or a nucleic acid sequence selected from: SEQ ID NOs: 285, 286, 292, 293, 297, 301-306, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0165] In some embodiments, the pre-pro-peptide according to the invention may comprise or consist of an amino acid sequence selected from: SEQ ID NOs: 6, 10, 14, 18, 21, 24, 27, 30, 34, 37, 40, 44, 48, 51 and 54, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0166] Expression Construct

[0167] The expression construct as disclosed herein is a nucleic acid, and may be DNA or RNA, in one embodiment the expression construct is double-stranded DNA.

[0168] Promoter

[0169] The present invention relates to an expression construct which comprises a polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof, preferably a pre-pro-peptide as defined herein, operably linked to a promoter, which is a heterologous promoter to the polynucleotide. Suitably the promoter is suitable for expression in a microorganism selected from the group consisting of bacteria, yeasts, and fungi.

[0170] Suitably by heterologous it is meant that the promoter is derived from a different genetic source than the polynucleotide. Suitable promoters for use in the expression construct may be derived from any organism as long as it is suitable for expression in the chosen host organism. Suitable promoters may be prokaryotic or eukaryotic promoters, preferably the promoter is a eukaryotic promoter. Suitable eukaryotic promoters may be derived from fungi, insects, plants, or mammals. Suitably, the promoter is selected based on their operability in a given host. In one embodiment, the promoter is a yeast promoter.

[0171] Suitable plant promoters may be selected from: Act1, Adh1, HSP18.2, ScBV, Ubi-1, RUBQ1 / RUBQ2, Gmubi, CaMV35S, nos, CmYLCV, KST1, Cula11 / Cula08, P OsCon1, TCTP, for example.

[0172] Suitable mammalian promoters may be selected from: CMV, EF1a, CAG, PGK, TRE, U6, UAS, for example.

[0173] Suitable bacterial promoters may be selected from: T7, Sp6, lac, araBad, trp, Ptac, for example.

[0174] Suitable yeast or fungal promoters may be selected from: AOX, GAP, ICL1, TEF1, ADH1, PGK1, DAS, FLD1, MOX, FMD, TPS, MET17, PYK, SNR52, TPI1, PHO5, G3P, FBA1, XPR2, RPS7, POT1, POX2, GAL1, GAL10, JUB1, SNR52, MET17, TDH3, TPI1, ENO1, PDC1, for example.

[0175] In one embodiment, the host cell is P. pastoris, and the promoter is selected from: AOX, GAP, ICL1, TEF1, ADH1, PGK1, DAS and FLD1.

[0176] Suitably the promoter may be constitutive or inducible, preferably the promoter is inducible such that expression of the insecticidal neuropeptide can be controlled by the presence of an inducer, suitably by contacting the host cell with an inducer. Examples of inducible promoters in yeast or fungi include the POX promotors which are inducible by the inducer oleic acid, the FMD promoter which is inducible with the inducer glucose, the MOX promoter which is inducible with the inducer methanol or glycerol, AOX promoters which are inducible with the inducer methanol.

[0177] In one embodiment, the AOX1 or AOX2 promoters of Pichia species can be used to express a heterologous protein such as an insecticidal neuropeptide or precursor thereof as described herein. Both the AOX1 and AOX2 promoters are particularly useful in Pichia species as both promoters provide for abundant expression of the linked heterologous gene when induced by addition of the inducer methanol to the growth medium. In one embodiment, the AOX1 promoter is used, suitably having a sequence according to SEQ ID NO: 1. In one embodiment, the Hansuela MOX, DHAS, or FMDH promoters can be used to express heterologous peptides such as an insecticidal neuropeptide or precursor thereof as described herein. The MOX, DHAS, or FMDH promoters are particularly useful in Hansuela species as these promoters provide for abundant expression of the linked heterologous gene when induced by addition of the inducer methanol or glycerol to the growth medium. In one embodiment, a Lactase promoter can be used to express heterologous genes such as an insecticidal neuropeptide or precursor thereof as described herein. Expression of heterologous genes that are operably linked to the Lactase promoter is achieved by growing a fungal species in the presence of the inducer galactose.

[0178] Suitably the promoter is located at the 5' end of the expression construct, suitably adjacent to the 5' end of the polynucleotide encoding the at least one insecticidal neuropeptide or a precursor thereof.

[0179] Targeting peptide / Secretion Peptide

[0180] Suitably the expression construct further comprises a polynucleotide encoding a targeting peptide and / or a secretion peptide. Suitably operably linked to the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably the polynucleotide encoding a targeting peptide and / or a secretion peptide is contiguous with the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably such that the at least one insecticidal neuropeptide or a precursor thereof is produced as a fusion protein with the targeting peptide and / or a secretion peptide. Suitably the polynucleotide encoding a targeting peptide and / or a secretion peptide is located at the 5' end of the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof, suitably between the promoter and the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof.

[0181] Suitable targeting peptides and / or a section peptides are described hereinabove.

[0182] 3'UTR

[0183] Suitably the expression construct further comprises a 3'UTR. Suitably the 3'UTR may be regarded as a trailer (Tr) sequence. Suitably the 3'UTR comprises a polyadenylation signal. Suitably the 3'UTR is operably linked to the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably the 3'UTR comprises a transcriptional termination signal. Suitably operably linked to the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof.

[0184] Suitably the 3'UTR is located at the 3' end of the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably the trailer sequence is as defined hereinabove.

[0185] Other transcription or translational control elements

[0186] Suitably the expression construct may comprise one or more further regulatory elements, suitably which are operable to control transcription or translation of the expression construct.

[0187] Suitable further regulatory elements may include: 5'UTR, introns, IRES, miRNA binding sites, activators, silencers, terminators, enhancers and the like. The expression construct may also comprise one or more restriction endonuclease sites suitable for inserting the desired polynucleotide for expression, for example: MlyI, FseI.

[0188] Optionally the expression construct comprises 5'UTR, which comprises an intron. An intron sequence increases the amount of the mature mRNA that accumulates in the cytosol of the host cell. Inclusion of a spliceable intron in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000- fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1 :1 183- 1200). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the construct.

[0189] In one embodiment, the expression construct comprises a terminator, suitably located at the 3’ end of the expression construct. Suitably the terminator is operably linked to the polynucleotide encoding the insecticidal neuropeptide or precursor thereof. Suitably the terminator is a transcriptional terminator, which may be CYC1 TT. Suitably as defined hereinabove.

[0190] Reporter Proteins

[0191] Optionally, the expression construct may further comprise one or more polynucleotides encoding a reporter protein. Suitably the polynucleotide encoding a reporter protein is operably linked to the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably the polynucleotide encoding a reporter protein is contiguous with the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably such that the at least one insecticidal neuropeptide or a precursor thereof is produced as a fusion protein with the a reporter protein. Suitably the polynucleotide encoding a reporter protein is located at the 5' end of the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof, suitably between the promoter and the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably the polynucleotide encoding a reporter protein is located 5’ of the first cleavage site [CC1i]. Suitably such that the reporter protein is expressed at the N terminus of the insecticidal neuropeptide or a precursor thereof. Alternatively the polynucleotide encoding a reporter protein is located at the 3' end of the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof, suitably between the final cleavage site of the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof and the stop codon(s).

[0192] Suitably the polynucleotide encoding a reporter protein further comprises polynucleotide encoding an affinity tag, suitably to enable isolation of the at least one insecticidal neuropeptide or a precursor thereof from cell culture. Suitably the polynucleotide encoding the affinity tag is located at the 5’ or at the 3’ end of the polynucleotide encoding a reporter protein, and is suitably contiguous with the polynucleotide encoding the reporter protein. Suitable affinity tags are discussed elsewhere herein. In one embodiment the affinity tag is a His-tag.

[0193] Suitably the reporter protein may be a fluorescent protein, a luminescent protein or other reporter system. Suitable fluorescent reporter proteins may be selected from any known in the art such as: GFP, eGFP, RFP, mCherry, YFP, eYFP, CFP, BFP, mOrange, tdTomato, mNeonGreen. Suitable luminescent reporter proteins may be selected from any known in the art, such as: luciferase, NanoLuc Luciferase, Gaussia Luciferase, Cypridina Luciferase, Click Beetle luciferase. Suitable other reporter systems that may be of use are Split GFP / Luciferase or Fluorescence Resonance Energy Transfer (FRET) Pairs. In one embodiment, the reporter protein is eYFP.

[0194] Amidation Enzyme

[0195] Optionally, the expression construct may further comprise one or more polynucleotides encoding an amidation enzyme. Suitably the polynucleotide encoding an amidation enzyme is operably linked to the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably the polynucleotide encoding an amidation enzyme is contiguous with the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably such that the at least one insecticidal neuropeptide or a precursor thereof is produced as a fusion protein with the amidation enzyme. Suitably the polynucleotide encoding an amidation enzyme is located at the 5' end of the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof, suitably between the promoter and the polynucleotide encoding at least one insecticidal neuropeptide or a precursor thereof. Suitably the polynucleotide encoding an amidation enzyme is located 5’ of the first cleavage site [CC1i]. Suitably such that the amidation enzyme is expressed at the N terminus of the insecticidal neuropeptide or a precursor thereof.

[0196] Suitably the amidation enzyme is a C-terminal [α-]amidation enzyme. Suitably the C- terminal α-amidation enzyme is a peptidylglycine-amidating monooxygenase (PAM) (in mammals), or may be the combination of a mono-oxygenase (PHM) and a lyase (PAL) (in insects). In one embodiment therefore, the amidation enzyme is a PAM, suitably derived from a mammalian species such as the rat.

[0197] Suitably said enzyme catalyses the conversion of a C-terminal glycine into a terminal amide group. Suitably the enzyme as part of the expression construct is co-expressed with the precursor peptides, and is able amidate each of the insecticidal neuropeptide precursors produced from the expression construct. Suitably this can occur within the host cell, and avoids the need for a separate amidation step after isolation of the precursor peptides.

[0198] Vector

[0199] The expression constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism in a form suitable for integration into the genomic DNA of the intended host cell, or in a form suitable for independent replication, maintenance and / or inheritance in the intended host cell. In one embodiment, the expression construct is suitable for integration into the genome of the host cell.

[0200] Suitably, the expression construct of the invention may be comprised upon a vector. In particular, the vector may be an expression vector, i.e., a vector that can provide for expression in vitro and / or in vivo, in a suitable host cell, host organism and / or expression system.

[0201] In a further aspect of the invention there is provided a vector comprising an expression construct of the first aspect.

[0202] Suitably the vector may be selected from: a plasmid, cosmid, YAC, phage, a viral vector or transposon. In one embodiment the vector is a plasmid.

[0203] Suitably the vector is a yeast vector. Suitable yeast vectors are selected from: yeast integrating plasmids, yeast replicating plasmids, yeast centromere plasmids, yeast episomal plasmids and yeast linear plasmids. Suitably the yeast vector may be a yeast shuttle vector.

[0204] In one embodiment the yeast vector is selected from: pYES2, pGADT7, pGBKT7, pRS316, pESC-URA, pC210, pYX212, pRS423, pDR195, pPICZ, pYEX-BX, pVT100U, pTEF1, pESC-LEU, pRS425, pRS426, pCM189, pUG6, pGREG505, pPIC3.5K, pFL61, and pYNR001, for example. In one embodiment the vector is a Pichia vector selected from: pPIC3.5k, pPIC9, pPIC9K, pPICZA, pPICZB, pPICZC, pPICZalpha,A, pPICZalphaB, pPICZalphaC, and pAO815, for example. These vectors provide a range of options for yeast expression systems, accommodating various experimental needs such as inducible promoters, selection markers, cloning convenience, and host specificity.

[0205] Regardless of the specific regulatory sequences employed, the expression construct is cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989). Once the isolated expression construct encoding the protein has been cloned into a vector, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation, depending upon the vector / host cell system as described below.

[0206] It is known that upon stable or transient integration of nucleic acids into host cells, only a minority of the cells take up the foreign DNA and, if desired, integrates it into the genome, depending on the vector used and the transfection technique used. Suitably, to identify and select these integrants, a gene coding for a selectable marker may also be introduced into the host cell together with the expression construct of the invention.

[0207] Suitably, a nucleic acid molecule encoding a selectable marker can be introduced into a host cell on the same vector that comprises the expression construct of the invention or used in the methods of the invention, or in a separate vector. Cells which have been stably transfected with the introduced nucleic acid can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).

[0208] Suitably therefore the vector may further comprise a "Selectable marker", "selectable marker gene" or “reporter gene”. Suitably which includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and / or selection of cells that are transfected or transformed with an expression construct of the invention. These marker genes enable the identification of a successful transfer of the nucleic acid molecules via a series of different principles. Suitable markers may be selected from: markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait, or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptll that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin, or genes conferring resistance to, for example, bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin), to herbicides (for example bar which provides resistance to Basta®; aroA or gox providing resistance against glyphosate, or the genes conferring resistance to, for example, imidazolinone, phosphinothricin or sulfonylurea), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source or xylose isomerase for the utilisation of xylose, or antinutritive markers such as the resistance to 2-deoxyglucose). Expression of visual marker genes results in the formation of colour (for example b-glucuronidase, GUS or b- galactosidase with its coloured substrates, for example X-Gal), luminescence (such as the luciferin / luceferase system) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). This list represents only a small number of possible markers. The skilled person will be familiar with such markers. Different markers are preferred, depending on the host organism and the selection method. In one embodiment, the selectable marker gene is AmpR conferring resistance to ampicillin.

[0209] Suitably in yeast host cells, a marker that introduces a metabolic trait is commonly used, suitably an auxotrophic marker. Suitable auxotrophic markers may be selected from: URA3, LYS2, LEU2, TRP1, MET15, ura4+, leu1+, ade6+, for example. Further commonly used auxotrophic markers are found in Brachmann et al. (1998) "Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications." Yeast 14:115-132. In one embodiment, the auxotrophic marker is the ADE2 gene conferring ability to catalyse purine biosynthesis in yeasts.

[0210] Genome

[0211] The invention further provides for a eukaryotic nuclear or plastid genome comprising a polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof, wherein the polynucleotide is heterologous to the nuclear or plastid genome and wherein the polynucleotide is operably linked to an endogenous promoter of the nuclear or plastid genome.

[0212] Suitably the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof is as defined herein above, and may comprise any of the further features of the expression construct identified hereinabove. In one embodiment, the eukaryotic nuclear or plastid genome comprises a polynucleotide encoding a pre-pro-peptide according to the invention, wherein the polynucleotide is heterologous to the nuclear or plastid genome and wherein the polynucleotide is operably linked to an endogenous promoter of the nuclear or plastid genome.

[0213] Suitably therefore the eukaryotic nuclear or plastid genome is a transgenic genome. Suitably the genome may be present extracellularly, or intracellularly, suitably the genome may be ex vivo, in vitro, or in vivo.

[0214] Suitably the eukaryotic genome may be the genome of any eukaryotic organism, suitably selected from: plants, animals, fungi, and insects. In one embodiment the eukaryotic genome is a fungal genome, suitably a yeast genome.

[0215] Suitably the genome is derived from a yeast selected from the following genera: Candida, Kluveromyces, Hansuela, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia. Suitable fungal cells include: Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma. Suitably the genome is derived from a yeast selected from the following species: Saccharomyces cerevisiae, Saccharomyces pombe and Pichia pistoris. In one embodiment, the genome is derived from Pichia pistoris (otherwise known as Komagataella phaffii).

[0216] Suitably the plastid genome may be the genome of any plastid, from any plant or algae, suitably known as a ‘plastome’, and which may suitably be selected from: a proplastid, chloroplast, etioplast, leucoplast, amyloplast, elaioplast, proteinoplast, chromoplast and gerontoplast, for example. In one embodiment, the plastid genome is a chloroplast genome.

[0217] Suitably the promoter is endogenous to the genome i.e. the promoter is from the same organism as the genome and is native to the genome. Suitable promoters are described hereinabove.

[0218] Suitably, in some embodiments, the genome is a yeast genome, therefore the promoter is a yeast promoter. Suitable yeast promoters or fungal promoters may be selected from: AOX, GAP, ICL1, TEF1, ADH1, PGK1, DAS, FLD1, MOX, FMD, TPS, MET17, PYK, SNR52, TPI1, PHO5, G3P, FBA1, XPR2, RPS7, POT1, POX2, GAL1, GAL10, JUB1, SNR52, MET17, TDH3, TPI1, ENO1, PDC1, for example.

[0219] In one embodiment, the genome is from P.pastoris, and the promoter is selected from: AOX, GAP, ICL1, TEF1, ADH1, PGK1, DAS and FLD1. In one embodiment, the genome is from P. pastoris, and the promoter is AOX1. Suitably as defined elsewhere herein.

[0220] Suitably, the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof is integrated into the genome to create a sequence within that genome that encodes the at least one insecticidal neuropeptide or a precursor peptide thereof, suitably stably integrated. Suitably, integration can occur at random sites in the genome or at specific sites in the genome.

[0221] Suitably the heterologous polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof can be inserted into an endogenous genomic region. Suitably wherein said endogenous genomic region comprises an endogenous promoter, suitably wherein the endogenous promoter is capable of driving expression of the inserted polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof. Suitably wherein the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof is inserted downstream of the endogenous promoter, suitably 3' of the endogenous promoter.

[0222] Suitably the genome may be modified to comprise the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof by transformation. Suitably by stable transformation. Suitable transformation techniques are known in the art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0223] Suitably a plastid genome may be transformed by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).

[0224] Suitably the genome may be modified to comprise the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof by homologous recombination between the polynucleotide and the genome. Integration can occur at random sites in the yeast host cell genome or at specific sites in the yeast host cell genome. Integration at specific sites in the yeast host cell genome is typically accomplished by homologous recombination between sequences contained in the expression vector and sequences in the yeast host cell genome. Homologous recombination is typically accomplished by linearizing the expression vector within the homologous sequence (for example, within the A0X1 promoter sequence of a Pichia expression vector when integrating the expression vector into the endogenous A0X1 gene in the Pichia host cell). Suitable method for integration by homologous recombination are described in EP1499739B1.

[0225] Suitably the genome may be modified to comprise the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof by site-specific integration. Integration at specific sites may be accomplished using gene editing technology. Suitable means of gene editing are known the art for example: using Zn-finger nucleases, meganucleases, TALENs, or CRISPR-Cas systems. Examples of methods for inserting heterologous DNA at specific sites in a genome with site-specific nucleases such as meganucleases or zinc-finger nucleases are at least disclosed in Voytas, 2013. Examples of methods for inserting heterologous DNA into a genome with clustered regularly interspaced short palindromic repeats (CRISPR)- associated (Cas)-guide RNA technology and a Cas endonuclease are at least disclosed by Svitashev et al., 2015; Murovec et al., 2017; Kumar and Jain, 2015; and in US Patent Appl. Pub. No. 20150082478. Examples of additional methods for editing genomes through use of Cpfl or Csml nucleases are disclosed in US Patent Application Publication 20180148735, for example.

[0226] Host cell

[0227] Host organisms or host cells that express or are capable of expressing one or more amino acid sequences are disclosed herein. Suitable examples of host organisms or host cells for expression of the insecticidal neuropeptides of the invention will be clear to the skilled person.

[0228] Suitably the host cell comprises the recombinant expression construct of the first aspect of the invention, the vector above, or the genome of the second aspect of the invention.

[0229] Suitably the host cell may be a bacterial, yeast, fungal (filamentous fungi), insect, animal, or plant cell, whether located in vitro or in vivo. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a plant cell. In one embodiment, the host cell is a yeast cell or a fungal cell.

[0230] Suitable host plant cells include those from any of the plant genera and species listed below in the plant section. In some embodiments therefore, the plants which the present invention is intended to protect from insect infestations are themselves the host organisms comprising host cells capable of expressing the insecticidal neuropeptides described herein. Suitably a plant or part thereof as described herein may comprise a host cell according to the invention, and express the insecticidal neuropeptides described herein.

[0231] Suitable host yeast cells include those from the following genera: Candida, Kluveromyces, Hansuela, Pichia, Komagataella, Saccharomyces, Schizosaccharomyces, and Yarrowia. Suitable fungal cells include: Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma. In one embodiment the host cell is a Pichia / Komagataella cell.

[0232] Suitable host cells include those from the following species: Saccharomyces cerevisiae, Saccharomyces pombe, Hanseula polymorpha, Yarrowa lipolytica, and Pichia / Komagataella pastoris.

[0233] Suitably the host cell may be a strain of Saccharomyces cerevisiae selected from: BY4716 W303, S288c, A634A, CEN.PK, BJ5464, ∑1278b, SK1, BY4741, BY4742, and BY474, for example.

[0234] Suitably the host cell may be a strain of Yarrowa lipolytica selected from: W29, E150, E129, YB423, and CX161-1B, for example.

[0235] Suitably the host cell may be a strain of Pichia / Komagataella pastoris selected from: Y-11430, X-33, NRRL-Y 11430, SMD1163, SMD1165, SMD1168, GS115, KM71, and SMD1168, for example. In one embodiment, the host cell is a Pichia pastoris cell.

[0236] Additional suitable host cells can be bacteria, preferably selected from the genera Bacillus, Geobacillus or Parageobacillus. In one preferred embodiment the host cells is from the genera Bacillus. Preferred Bacillus-like hosts are selected from the group of: Bacillus subtilis, Bacillus thuringiensis, Bacillus licheniformis, Bacillus coagulans, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus brevis, Bacillus pumilus, Bacillus polymyxa, Bacillus / Geobacillus stearothermophilus, Geobacillus thermoleovorans, Geobacillus kaustophilus, Parageobacillus thermantarcticus, Parageobacillus thermoglucosidasius, Parageobacillus toebii and Parageobacillus galactosidasius. In one embodiment, the host cell is a Bacillus subtilis. Suitably therefore in a further aspect of the invention there is provided a Bacillus subtilis strain for use in the expression of an insect neuropeptide or precursor thereof of the invention, suitably an insect neuropeptide pre-pro-peptide as described herein, or for use in a process of the invention.

[0237] Suitably any Bacillus subtilis strain may be used. Suitably the stain may be a modified strain, suitably comprising one or more genetic modifications compared to a wild type strain. In one embodiment the Bacillus subtilis strain has been modified to reduce expression of, silence, or delete at least one gene encoding an endogenous endoprotease.

[0238] Methods for production in Bacillus licheniformis strains are for example described in Novozymes A / S WO 01 / 90393, Novozymes A / S WO 02 / 00907, EP2213745A1, WO2003093453A1.

[0239] In a preferred embodiment the host cell is a fungi, suitably a filamentous fungi. Preferred filamentous fungi hosts are selected from the following: Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnapor the, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Schizophyllum, Talaroinyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma and / or Alternaria kikuchiana, Alternaria mali, Alternaria solani, Ashbya gossypii, otryti s cinerea, Cercospora beticola, Cercospora fuligena, Cercospora kaki, Cladosporum carpophilum, Cochliobolus heterostrophus, Colletotrichum fragariae, Colletotrichum heterostrophus, Colletotrichum lagenarium, Corynespora melonis, Diaporthe citri, Diplocar pon rosae, Elsinoe fawcetti, Erisyphe graminis, Leveillula taurica, Fusarium culmorum Fusarium nivale, Fusarium graminearum, Gloedes pomigena, Gloesporium kaki, Glomerella cingulata, Gymnosporangium yamadae, Leptothyrium pomi, Magnaporthe grise, Mycospha erella pomi, Mycosphaerella nawae, Neurospora crassa, Peronospora destructor, Peronospora spinaciae, Phaeoisariopsis vitis, Phyllactinia kakicola, Physalospora canker, Phytophthora citrophithora, Phytophthora investans, Phytophthora porri, Plasmopora viti cola, Podosphaera leucotricha, Podosphaera tridactyla, Pomophis sp., Pseudocercorsporella herpotrichoides, Pseudoperonospora cubensis, Puccinia allii, Puccinia recondita, Puccinia horiana, Pyricularia oryzae, Uncinula necator, Sclerotinia cinerea, Sclerot inia mali, Sclerotinia sclerotiorum, Septoria tritici, Sphaerotheca fuliginea, Sphaerotheca humuli, Sphaerotheca pannosa, Spaceloma ampelina, Stagnospora nodorum, Typhula ishikariensis, Typhula incarnata, Ustilago maydis, Venturia inaequalis, Venturia nashicola. Weitere bevorzugte Pilzstämme sind Aspergillus, Trichoderma, Neurospora, Fusarium, Beauveria, Pyrenophora teres, Saccharomyces (for example Saccharomyces cerevisiae), Pichia (like Pichia pastoris, Pichia methanolica), Magnaporthe, Pyrialeria or further fungi as disclosed in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995). In a preferred embodiment the fungi is a Magnaporthe species, wherein Magnaporthe species include Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe poae and Magnaporthe grisea, preferably Magnaporthe grisea.

[0240] Methods for production in filamentous fungi are for example described in WO2004050695A1 and EP1674577A1.

[0241] Expression can also be conducted in insect cells as described for example in EP1866427A4.

[0242] Suitably the host cell is transformed with the expression construct of the first aspect of the invention, the vector above, or with a polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof. Suitable methods of transformation are known in the art, and are relevant to particular host types. Transformation or transfection of nucleic acids into host cells may be accomplished by a variety of means known to the person skilled in the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0243] Suitable transformation methods for plants or plant cells include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium / polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363- 373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio / Technol 3, 1099-1 102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein TM et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491 -506, 1993), Hiei et al. (Plant J 6 (2): 271 -282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al (1984) Nucl. Acids Res. 12-8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0244] In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:1 -9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289] Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551 -558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316: 1 194-1 199], while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non- transgenic seeds by growing under the above- described selective conditions. In addition, the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229). The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

[0245] Generally, after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.

[0246] Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and / or genomic organisation. Alternatively, or additionally, expression levels of the newly introduced DNA may be monitored using Northern and / or Western analysis, both techniques being well known to persons having ordinary skill in the art.

[0247] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

[0248] Suitable transformation methods for yeast cells include transfection into yeast spheroplasts or electroporation. A general description of expression vectors and transformation systems for Saccharomyces is found in Kingsman et al (1985) Biotechnol Genet Eng Rev. 3:377- 416. Expression vectors and transformation systems useful for yeasts other than Saccharomyces are described in Reiser et al (1990) Adv Biochem Eng Biotechnol.;43:75-102. In one embodiment, the host cell is a yeast cell, preferably a Pichia cell, which is transformed with a vector, expression construct, or a polynucleotide of the invention by electroporation. One suitable technique for Pichia transformation is described in Wu & Letchworth, Biotechniques vol. 36, no. 1 Drug Discovery and Genomic Technologies.

[0249] Suitably therefore the host cell may be regarded as a transgenic cell. Suitably the host cell may be located within an organism, which suitably may be regarded as a transgenic organism comprising said host cell. Such organisms may be considered to be a further aspect of the invention. Suitably for example a transgenic yeast, or a transgenic plant. Suitably comprising the expression construct, genome or vector thereof, of the invention. Suitably capable of expressing said expression construct, genome or vector thereof, of the invention. Suitably to produce an insecticidal neuropeptide, or a precursor peptide thereof.

[0250] Compositions

[0251] The present invention further encompasses a composition comprising one or more isolated insecticidal neuropeptides or chemically modified variants thereof, and at least 0.1% w / w of a precursor peptide thereof, and / or optionally a pre-pro-peptide thereof according to the fifth aspect of the invention.

[0252] Suitably the composition may comprise an insecticidal neuropeptide produced by the process of the seventh aspect of the invention, or chemically modified variants thereof, wherein the composition comprises at least 0.1% w / w of a precursor peptide thereof, and / or optionally a pre-pro-peptide thereof.

[0253] Suitably the or each isolated insecticidal neuropeptide in the composition is an active insecticidal neuropeptide. In some embodiments, therefore, the isolated insecticidal neuropeptide may be amidated. Suitably at the C-terminus thereof. Suitably by ‘active’ it is meant that the neuropeptide displays insecticidal activity against at least one species of insect, which activity is defined hereinabove. Suitably the compositions of the invention may be regarded as insecticidal compositions, biocidal compositions or biostatic compositions. Optionally the or each isolated insecticidal neuropeptide in the composition may be chemically modified by any suitable chemical group at any position of the peptide. Suitably the insecticidal neuropeptide may comprise a C terminal or N terminal chemical modification. For example, the insecticidal neuropeptide may comprise an N-terminal lactamide group. Suitably in some cases in addition to a C-terminal amidation.

[0254] In a preferred embodiment the insecticidal neuropeptides are active without a C-terminal amidation. This is – surprisingly – the case for example for insecticidal neuropeptides of the AKH class. Such as SB-P-190 [Pyr]-LTFSPDW-[OH] (SEQ ID NO:262), SB-P-233 [Pyr]- LTFTSSWGG-[OH] (SEQ ID NO: 263).

[0255] Suitably the composition may comprise a combination of different isolated insecticidal neuropeptides, suitably the composition may comprise two or more different isolated insecticidal neuropeptides. Suitably the composition may comprise two, three, four, five or more different isolated insecticidal neuropeptides. Optionally the isolated insecticidal neuropeptides may be derived from the same pre-pro-peptide. Optionally the combination of different isolated insecticidal neuropeptides may be derived from the same pre-pro-peptide. Suitably wherein they are present as a concatemer.

[0256] Suitable combinations of insecticidal neuropeptides which may be expressed from a pre- pro-peptide are described above. Suitably therefore the composition may comprise any of the same combinations of insecticidal neuropeptides as defined in relation to the pre-pro-peptide. Suitably the composition may comprise any combination of insecticidal neuropeptides. Suitably the composition may comprise 2, 3, 4, 5, or more different insecticidal neuropeptides, suitably up to 10 different insecticidal neuropeptides. Suitably the composition may comprise a synergistic combination of insecticidal neuropeptides. In one embodiment, the composition comprises at least one pyrokinin insecticidal neuropeptide and at least one AKH insecticidal neuropeptide. In one embodiment, the composition comprises at least one CAPA insecticidal neuropeptide and at least one kinin insecticidal neuropeptide.

[0257] Suitably the insecticidal neuropeptides may be present in the composition in any proportion. For example the composition may comprise a first insecticidal neuropeptide and a second insecticidal neuropeptide. Suitably the composition may comprise a similar amount of a first insecticidal neuropeptide and a second insecticidal neuropeptide, suitably therefore an amount of a first insecticidal neuropeptide relative to an amount of a second insecticidal neuropeptide of about 1:1. Suitably the composition may comprise a greater amount of a first insecticidal neuropeptide compared to a second insecticidal neuropeptide or vice versa. Suitably the amount of a first insecticidal neuropeptide relative to an amount of a second insecticidal neuropeptide may be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:3, 2:5, 2:7, 2:9, 3:4, 3:5, 3:7, 3:8, 3:10, 4:5, 4:7, 4:9, 4:10; 5:6, 5:7, 5:8, 5:9, 6:7, 6:10, 7:8, 7:9, 7:10, 8:9, 9:10, or vice versa, for example.

[0258] Suitably the composition comprises an effective amount of the or each isolated insecticidal neuropeptide. Suitably the effective amount is a concentration of about 0.1, 0.5, 1.0, or 5 pg / ml to about 1, 5, 20, 50, or 100 mg / ml. Suitably the effective amount is a concentration of about 0.1 pg / ml to about 100 mg / ml. Suitably the effective amount is a concentration of about 0.5 pg / ml to about 50 mg / ml. Suitably the effective amount is a concentration of about 1 pg / ml to about 20 mg / ml. Suitably the effective amount is a concentration of about 5 pg / ml to about 5mg / ml.

[0259] Suitably the composition comprises between 0.1% and 1% of a precursor peptide thereof and / or optionally a pre-pro-peptide thereof, suitably between 0.1-0.5%, suitably between 0.1- 0.4%, suitably between 0.1-0.3%, suitably between 0.1-0.3% suitably between 0.1-0.2%, suitably around 0.1% of a precursor peptide and / or optionally a pre-pro-peptide thereof. Suitable precursors of active insecticidal neuropeptides, and pre-pro-peptides are defined hereinabove.

[0260] Optionally, the composition may further comprise one or more agrochemicals. Suitably one or more agrochemicals known in the art. “Agrochemical”, as used herein, means suitable for use in the agrochemical industry (including agriculture, horticulture, floriculture and home and garden uses, but also products intended for non-crop related uses such as public health / pest control operator uses to control undesirable insects and rodents, household uses, such as household fungicides and insecticides and agents, for protecting plants or parts of plants, crops, bulbs, tubers, fruits (e.g. from harmful organisms, diseases or pests); for controlling, promoting or increasing, the growth of plants; and / or for promoting the yield of plants, crops or the parts of plants that are harvested (e.g. its fruits, flowers, seeds etc.). Examples of such substances will be clear to the skilled person and may for example include compounds that are active as insecticides (e.g. contact insecticides or systemic insecticides, including insecticides for household use), herbicides (e.g. contact herbicides or systemic herbicides, including herbicides for household use), fungicides (e.g. contact fungicides or systemic fungicides, including fungicides for household use), nematicides (e.g. contact nematicides or systemic nematicides, including nematicides for household use) and other pesticides or biocides (for example agents for killing insects or snails); as well as fertilizers; growth regulators such as plant hormones; micro- nutrients, safeners, pheromones; repellents; insect baits; and / or active principles that are used to modulate (i.e. increase, decrease, inhibit, enhance and / or trigger) gene expression (and / or other biological or biochemical processes) in or by the targeted plant (e.g. the plant to be protected or the plant to be controlled), such as nucleic acids (e.g., single stranded or double stranded RNA, as for example used in the context of RNAi technology) and other factors, proteins, chemicals, etc. known per se for this purpose, etc. Examples of such agrochemicals will be clear to the skilled person; and for example include, without limitation: glyphosate, paraquat, metolachlor, acetochlor, mesotrione, 2,4-D,atrazine, glufosinate, sulfosate, fenoxaprop, pendimethalin, picloram, trifluralin, bromoxynil, clodinafop, fluroxypyr, nicosulfuron, bensulfuron, imazetapyr, dicamba, imidacloprid, thiamethoxam, fipronil, chlorpyrifos, deltamethrin, lambda-cyhalotrin, endosulfan, methamidophos, carbofuran, clothianidin, cypermethrin, abamectin, diflufenican, spinosad, indoxacarb, bifenthrin, tefluthrin, azoxystrobin, thiamethoxam, tebuconazole, mancozeb, cyazofamid, fluazinam, pyraclostrobin, epoxiconazole, chlorothalonil, copper fungicides, trifloxystrobin, prothioconazole, difenoconazole, carbendazim, propiconazole, thiophanate, sulphur, boscalid and other known agrochemicals or any suitable combination(s) thereof. Conventional insecticides which may be present in the composition are described in Worthington and Walker (1983) The Pesticide Manual, Seventh Edition, British Crop Protection Council. US Patent No. 5,421,839, further contains a comprehensive summary of the many active agents with which substances such as the present insecticidal neuropeptides can be formulated.

[0261] Suitably the composition described herein may be regarded as an agrochemical or agricultural composition. An “agrochemical or agricultural composition” as used herein means a composition for agrochemical use, as further defined, comprising at least one active substance, suitably wherein said active substance is an insecticidal neuropeptide. “Agrochemical use”, as used herein, not only includes the use of agrochemicals as defined above (for example, the insecticidal neuropeptides of the invention, or pesticides, growth regulators, nutrients / fertilizers, repellants, defoliants etc.) that are suitable and / or intended for use in field grown crops (e.g., agriculture), but also includes the use of agrochemicals as defined above (for example, the insecticidal neuropeptides of the invention, or pesticides, growth regulators, nutrients / fertilizers, repellents, defoliants etc.) that are meant for use in greenhouse grown crops (e.g. horticulture / floriculture) or hydroponic culture systems and even the use of agrochemicals as defined above, such as the insecticidal neuropeptides of the invention, that are suitable and / or intended for non-crop uses such as uses in private gardens, household uses (for example, herbicides or insecticides for household use), or uses by pest control operators (for example, weed control etc.).

[0262] Suitably the composition may further comprise one or more agriculturally, pharmaceutically, or veterinary-practicably acceptable additives, carriers, diluents, or excipients.

[0263] As a non-limiting example, the additives in the compositions disclosed herein may include but are not limited to diluents, solvents, preservatives, adjuvants, surfactants, wetting agents, spreading agents, oils, stickers, thickeners, penetrants, buffering agents, acidifiers, anti- settling agents, anti-freeze agents, photo-protectors, defoaming agents, biocides, siccatives, UV stabilisers, colorants, pigments, secondary thickeners, adhesives, and / or drift control agents. In one embodiment, the composition may comprise a protease inhibitor.

[0264] A “carrier”, as used herein, means any solid, semi-solid or liquid carrier in or on(to) which an active substance can be suitably incorporated, included, immobilized, adsorbed, absorbed, bound, encapsulated, embedded, attached, or comprised. Non-limiting examples of such carriers include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, a gel, weak ionic resin particles, liposomes, cochleate delivery vehicles, small granules, granulates, nano-tubes, bucky-balls, water droplets that are part of an water-in-oil emulsion, oil droplets that are part of an oil-in-water emulsion, organic materials such as cork, wood or other plant-derived materials (e.g. in the form of seed shells, wood chips, pulp, spheres, beads, sheets or any other suitable form), paper or cardboard, inorganic materials such as talc, clay, microcrystalline cellulose, silica, alumina, silicates and zeolites, or even microbial cells (such as yeast cells) or suitable fractions or fragments thereof.

[0265] A “diluent” used herein means any liquid or solid material used to reduce the concentration of the active ingredient. Typically a diluent is a solvent which may be a Liquid e.g. Oil, water, or alternatively the diluent may be a solid e.g. talc, soapstone, attapulgite.

[0266] An “excipient” used herein means any inert material provided in the composition to optimize delivery or uptake, optimise biological activity, stabilise the active ingredient, minimise user exposure, and simplify use of the composition, etc.

[0267] Formulation aids, such as carriers, inert materials, surfactants, solvents, and other additives are also well known in the art, and are described, for example, in Watkins, Handbook of Insecticide Dust Diluents and Carriers, Second Edition, Darland Books, Caldwell, N.J., and Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich.

[0268] Suitably the pH of the composition is between about 3.0 to about 9.0. Suitably the pH of the composition may be maintained by one or more buffers within the composition. Such compositions can be buffered using, for example, phosphate buffers or a Tris-EDTA buffer between about 1 mM and 1 M, about 10 mM to about 100 mM, or about 15 mM to about 50 mM. In the case of low buffer concentrations, a salt can be added to increase the ionic strength.

[0269] Suitably the composition may comprise a sodium salt at a concentration of at least l00 mM. Suitably the composition may comprise a sodium salt at a concentration between 100 mM and 500 mM, suitably between 200 mM and 400 mM, suitably around 300 mM.

[0270] Suitably the composition may comprise a calcium salt at a concentration of at least 2 mM. Suitably the composition may comprise a calcium salt at a concentration of between 2 mM and 10 mM, suitably between 4 mM and 8 mM, suitably around 5-6 mM.

[0271] Process of Production

[0272] The present invention further provides a method of producing an insecticidal neuropeptide or precursor thereof, suitably from a host cell. Suitably the method begins by providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect.

[0273] Upstream Bioprocess: Culturing and Fermentation

[0274] Suitably the second step in the method of production is culturing the host cell under suitable conditions for an insecticidal neuropeptide or precursor peptide or pre-pro-peptide thereof to be produced by the host cell. Suitably wherein the host cell is a host cell according to the third aspect of the invention. Suitably this step comprises culturing the host cell in a culture medium.

[0275] In a further aspect of the invention there is provided a culture vessel, such as a bioreactor, or medium comprising a host cell of the third aspect of the invention. Suitably the culture vessel or medium comprises a plurality of host cells according to the invention.

[0276] Suitable conditions for culturing the host cell will depend on the chosen host cell. Suitable culturing techniques and conditions are known in the art for a variety of different host cells. In some embodiments of the invention, the host cell is a yeast cell or fungal cell. Suitable media for culturing yeast or fungi may be selected from: YPD media, either broth or agar, CSM media, YNB media, BMGY media or BMMY media, for example.

[0277] One example of suitable culture media may be BMGY media and one example of suitable expression media may be BMMY media. Both can be prepared as follows: (1) 1 L BMGY or BMMY media may be made by first dissolving 10 g yeast extract, 20 g peptone and per 800 mL deionized (DI) water and autoclaving at 121 °C. One hundred mL of each of the following filter-sterilized components may then be added to create 1 L of BMGY or BMMY: 1 M potassium phosphate buffer (pH 6.0) prepared by dissolving 2.405 g K2HPO4(Fisher Chemical) and 11.73 g KH2PO4 (Sigma-Aldrich) in 100 mL DI water, and sterilizing through 0.2 µm membrane filtration; and 10X yeast nitrogen base (YNB) with ammonium sulfate, prepared by dissolving 13.4 g of the YNB powder (Invitrogen) in 100 mL DI water and sterilizing through 0.2 µm membrane filtration. Lastly, 100 mL of sterile glycerol (Fisher Bioreagents) is added to complete the media as BMGY media or 100 mL of 50% Methanol solution (Thermo Scientific Chemicals).

[0278] Suitable culturing for yeast cells at scale will require the preparation of seed cultures as inoculum. One example of suitable inoculum preparation begins with the provision of a colony host cell of the third aspect, suitably which is then used to inoculate a culture vessel, suitably containing a suitable culture media. Suitably the colony host cell is inoculated into a culture media at a concentration of about 1-10% of the target volume of the culture media. Suitably the colony host cells are then cultured at around 25-30 °C, suitably at 30 °C, at agitation equating 250-300 RPM, for 2-3 days until a suitable inoculum is formed. Suitably the inoculum has a measurable OD600 value, suitably between 2-20.

[0279] Suitably the inoculum is then inoculated into the culture media located in culture vessel such as a sterilized bioreactor. Suitably the culture vessel may be of any scale, up to 5000 L for example. Suitably the inoculum is inoculated into the culture media at a concentration of about 1-10% (v / v), suitably about 5% (v / v).

[0280] Suitable culture conditions for such cells may be as follows: suitably the yeast is cultured at around 25-30 °C, suitably around 30 °C for a growth phase and 24 °C for an induction phase. As such, the culture vessel therefore comprises a suitable heat exchange and / or temperature control system. Suitable culture vessel temperature control systems examples include a jacket system, external thermal block or cooling finger / coil.

[0281] Suitably the culture media is maintained at a pH of between about 5.0-7.0, suitably at around pH 6.0, suitably which may be controlled by the addition of about 30% (v / v) sterile ammonium hydroxide solution. As such, the culture vessel requires a pH probe and automated pH sensing and control system.

[0282] Suitable aerobic fermentation maintains the dissolved oxygen level at ≥20% in the culture media, suitably with supplied compressed air and oxygen, suitably the dissolved oxygen concentration is maintained during the growth phase at around 20-30%. Suitably the culture vessel therefore comprises a dissolved oxygen sensor and a suitable gas flow control system capable of maintaining the suitable dissolved oxygen concentrations in the culture media.

[0283] Suitably, the culture media is maintained with continuous mixing or agitation, for example at between 200-1500 RPM using an impeller system, for adequate culture mixing and to maintain the target dissolved oxygen concentrations. Suitably therefore the culture vessel also comprises an agitation system. While this could be managed manually, in this embodiment suitable programmable control systems may be integrated with the culture vessel to operate automatic control systems and maintain culture conditions suitable for the host cells.

[0284] Suitable culturing strategies consisting of one or more phases can be performed and adapted for different: host cells, suitable yields and / or expression levels of insecticidal neuropeptide or precursor peptide, or pre-pro-peptide or overall culture productivities. Suitably the culture may be operated as a batch, fed-batch, semi-continuous, continuous or a perfusion process. Suitably in some cases the culture media itself may be replaced or diluted. Suitably therefore media can be periodically or intermittently fed to or withheld from the culture vessel.

[0285] In one embodiment, the culture is a fed-batch, suitably multiphasic fed-batch. In one embodiment, the process comprises a batch phase and a fed-batch phase.

[0286] In one embodiment, the process of the invention may comprise providing a suitably inoculated culture media in a suitable culture vessel set to the suitable culture conditions. Suitably the host yeast cells are grown on suitable carbon source containing culture media, in one embodiment this is BMGY media comprising glycerol as a primary carbon source. Suitably the host cells are cultured as a batch culture until the glycerol is fully consumed, suitably indicated by a sharp increase in DO reading. In this embodiment, the batch growth phase of the process completes, and can then proceed to a fed-batch growth phase.

[0287] During the fed-batch growth phase, a suitable culture medium is periodically or intermittently fed to the culture vessel. Suitably the feeding culture medium may comprise a carbon source for the growth of yeast, such as glycerol, for example 50% (v / v) glycerol solution. Suitably this may be added automatically into the culture vessel, periodically or intermittently, when required to maintain a suitable parameter measurement and thus the culture vessel requires programmable calibrated peristaltic pumps to feed the media. In this embodiment, a suitable rate of feeding of between 5-15 mL / hr / L of culture media per initial fermentation media volume is chosen and the feeding culture media is periodically or intermittently added to the culture vessel.

[0288] Suitable heterologous host cell may express a polynucleotide according to the invention to produce insecticidal neuropeptides, precursor peptides, or pre-pro-peptides via regulated or constitutive gene expression systems. In suitable heterologous host cell systems where constitutive gene expression in the host cell is used to produce neuropeptides, precursor peptides, or pre-pro-peptides, feeding culture medium could continue at suitable rates and growth conditions, suitably for 40–70 hours of growth before ending the fermentation process. In suitable heterologous host cell systems where regulated gene expression in the host cell is used to produce an insecticidal neuropeptide, precursor peptide, or pre-pro-peptide, a suitable fed-batch growth phase is continued until a suitable high cell density wet cell weight between 50-300 g / L can be measured, suitably at around 150 to 220 g / L wet cell weight. In this embodiment, upon reaching suitable wet cell weight, the fed-batch growth phase of the method completes and can then proceed to a fed-batch induction phase.

[0289] At the initiation of a fed-batch induction phase, the feeding culture medium could be suitably switched with, or modified to become, a culture medium which may contain a suitable inducer molecule to induce expression and production from the host cell of an insecticidal neuropeptide or precursor peptide, or pre-pro-peptide. For example, in this embodiment, the expression system may comprise an AOX1 promoter to regulate gene expression, as discussed hereinabove, which is induced by a 100% (v / v) methanol solution fed to the culture vessel. Suitably, feeding culture media can be intermittently added or ‘pulsed’ to a target methanol concentration within the culture, for example between 0.5-2% (v / v), or another measured operational parameter. Suitable culture media can also be continuously fed at a fixed or variable rate or some combination thereof, for example at a continuous initial rate of 3.6 mL / hr / L of initial fermentation volume which increases stepwise to 10.5 mL / hr / L over the course of 6 hours. Suitable culture conditions for the host cell are managed to maintain a stable DO reading for the culture, suitably 20-30%. Such culture conditions are maintained through suitable combinations of rates of agitation, media feed, aeration, oxygen feeding and of culture vessel internal pressure, suitably managed manually or through automated control systems.

[0290] Suitably the insecticidal neuropeptide or precursor peptide, or pre-pro-peptide expressing host cell can then continue to be cultured in one or more suitable culture phases where the process is operated as a batch, fed-batch, semi-continuous, continuous or a perfusion process until such a time is reached whereby one or more of suitable cellular rates, phenotypes, productivities and / or product and / or biomass yields are achieved. The culturing process can then be suitably concluded. In one example of a fed-batch embodiment this is suitably between 24- 120 hours after the cells are induced.

[0291] Mid-Downstream Processing

[0292] Optionally, the method may comprise a step of further processing any pre-pro-peptide to release an insecticidal neuropeptide or a precursor peptide therefrom. Suitably this step is carried out after culturing, but before further modification of the peptides.

[0293] Suitably the further processing step comprises cleavage, suitably proteolytic cleavage or chemical cleavage of the pre-pro-peptide to release an insecticidal peptide or a precursor peptide thereof. Suitably cleavage takes place at each ‘CC’ cleavage consensus site present in the pre- pro-peptide.

[0294] Suitably the proteolytic cleavage is carried out by an enzyme. The cleavage could be conducted either in vivo in the host-strain by an endogenous (i.e., naturally occurring) or by an exogenous, for example a recombinantly expressed optionally heterologous, cleaving enzyme. Alternatively, the cleavage may be carried out ex vivo / in vitro outside the host strain by adding a cleaving enzyme to the secreted pre-pro-peptide. Optionally prior to addition of the cleaving enzyme, the secreted pre-pro-peptide is at least partially purified. Alternatively, the cleavage may be carried out chemically.

[0295] The person skilled in the art is aware of various cleaving enzymes including endoproteases and inteins.

[0296] Suitably the cleaving enzyme is a protease, preferably a dibasic-processing endoprotease, optionally which is endogenous to the host cell or recombinantly expressed within the host cell. Suitably therefore this step of the method comprises contacting the pre-pro-peptide with a protease enzyme, suitably contacting the pre-pro-peptide with a dibasic-processing endoprotease. Suitably under conditions to effect cleavage of the pre-pro-peptide, at CC sites.

[0297] The term “endoproteinase” is used herein to refer to a peptidase capable of cleaving a peptide bond between two internal amino acid residues in a peptide sequence. Endoproteinases can also be referred to as “endoproteases” or “endopeptidases.” The term “exoproteinase” is used herein to refer to a peptidase capable of cleaving a peptide bond between the penultimate or final amino acid residues in a peptide sequence. Exoproteinases can also be referred to as “exoproteases” or “exopeptidases.”

[0298] Suitably the protease is selected which is able to cleave at the consensus cleavage sites present in the pre-pro-peptide, suitably consensus sequences are provided elsewhere herein in relation to the pre-pro-peptide. Suitable dibasic-processing endoproteases may be selected from: Ste13, Kex2, XPR6, PC2, PC3 / PC1, PC4, furin / PACE, and PACE4. In one embodiment, the dibasic-processing endoprotease is Kex2.

[0299] In one embodiment the dibasic-processing endoprotease is endogenous to the host cell and is expressed in the Golgi. Suitably the pre-pro-peptide is targeted to the Golgi of the host cell by the targeting / secretion peptide therein, where it is then cleaved by resident endoproteases. In another preferred embodiment the concatemer pre-pro-peptide is not cleaved within the expressing host organism but after secretion and – optionally – after purification or isolation. In this context protein cleavage sites and cleavage enzyme / s are used which are not endogenously present in the expressing host organism, suitably said cleavage enzymes are exogenous to the host organism. Examples include but are not limited to: (a) Inteins. Any intein may be used, such as: P. abyssi DNA Pol II Intein WT (SEQ ID NO:264) as described in Mills, K. V., Manning, J. S., Garcia, A. M., & Wuerdeman, L. A. (2004). Protein splicing of a Pyrococcus abyssi intein with a C-terminal glutamine. Journal of Biological Chemistry, 279(20), 20685-20691. P. abyssi DNA Pol II Intein altered for amidation V1 (Q185A) (SEQ ID NO:265); P. abyssi DNA Pol II Intein altered for amidation V2 (Q185V) (SEQ ID NO:266) ; Mycobacterium xenopi GyrA intein (SEQ ID NO:267), as described in Southworth, M. W., Amaya, K., Evans, T. C., Xu, M. Q., & Perler, F. B. (1999); P. abyssi DNA Pol II Intein altered for amidation V3 (SEQ ID NO:298). Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. Biotechniques, 27(1), 110-120; and Mycobacterium xenopi GyrA intein truncated with linker (SEQ ID NO:268), as described in Albertsen, L., Shaw, A. C., Norrild, J. C., & Strømgaard, K. (2013). Recombinant production of peptide C-terminal α-amides using an engineered intein. Bioconjugate chemistry, 24(11), 1883- 1894. (b) Non-endogenous endo- and exopeptidases with specific amino acid recognition cleavage sites, for example: Factor Xa (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269), Enterokinase (Asp- Asp-Asp-Asp-Lys) (SEQ ID NO:270), Thrombin (Leu-Val-Pro-Arg-Gly-Ser) (SEQ ID NO: 326), Human rhinovirus 3C Protease (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) (SEQ ID NO:271), TEV (Tobacco Etch Virus Protease) (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) (SEQ ID NO:272), TVMV (Tobacco Vein Mottling Virus Protease) (Glu-Thr-Val-Arg-Phe-Gln-Gly-Ser) (SEQ ID NO:273), Adenain (Gly-Gly), Staphylolysin (Gly-Gly), Pepsin (Cleaves at the C-Terminus of Phe, Leu, Tyr and Trp), Thermolysin (cleaves at the C-terminus of Ile , Leu, Val, Ala, Met and Phe), elastase (cleaves at C-Terminus of Ala, Val, Ser, Gly, Leu and Ile) and Asp-N (cleaves at the N- Terminal Side of Asp and Glu).

[0300] In one embodiment, a modified intein-based protein splicing system is utilized to produce a neuropeptide with a C-terminal amide. The neuropeptide sequence is genetically encoded and fused to the N-terminus of the intein variant. Upon expression, an N-to-S acyl shift occurs at the N-terminal cysteine residue of the intein, transferring the neuropeptide to the cysteine R-group. The specific conditions for the N-acyl shift may vary depending on the intein variant used (e.g., temperature, pH, buffer composition, and co-factors known for facilitating intein splicing reactions). Following the acyl shift, the addition of a reducing agent and an ammonium source induces hydrolysis at the thioester bond, resulting in the release of the neuropeptide with a C-terminal amide modification. This approach provides a single-step process for peptide release and amidation.

[0301] In another alternate embodiment, cleavage of the pre-pro-peptide may be carried out chemically. Suitably using one or more chemicals which are capable of reacting with a cleavage site [CC] present in the pre-pro-peptide and cleaving at said cleavage site. Suitably the cleavage may be carried out ex vivo / in vitro outside the host strain by adding one or more cleaving chemicals to the secreted pre-pro-peptide. Suitably therefore this step of the method comprises contacting the pre-pro-peptide with one or more cleaving chemicals. Suitably under conditions to effect cleavage of the pre-pro-peptide, at CC sites.

[0302] Suitably the one or more chemicals are selected from those which are able to cleave at the consensus cleavage sites present in the pre-pro-peptide, suitably consensus sequences are provided elsewhere herein in relation to the pre-pro-peptide.

[0303] For example, one or more of the [CC] cleavage sites may comprise cysteine, suitably in which case the cleavage is carried out by one or more chemicals. Suitably the chemicals are alkylating agent 2,5 dibromohexanediamide (DBHDA), followed by B2(OH)4 and ascorbate, under suitable conditions. In some preferred embodiments, at cleavage sites that comprise a cysteine residue, the Cysteine residue is converted into a dehydroalanine (Dha) residue. This can be achieved, for example, using 2,5-dibromohexanediamide (DBHDA), but the skilled person can use any other suitable reagent. Subsequently, a boron-based chemical cleavage reaction (e.g., β-elimination or other boronate-mediated reactions) is used to cleave the peptide chain at the Dha residues. This is suitably performed using B2(OH)4, but the skilled person can use any other suitable reagent. This cleavage results in the release of individual peptides from the concatemer, where each upstream peptide acquires a C-terminal amide, while the downstream peptide is modified with a lactic acid / lactoyl moiety at its N-terminus. This single-step process achieves both peptide release and C-terminal peptide amidation. It will be apparent that in such methods cysteines should not be present in location where cleavage is undesirable (e.g. within an insecticidal neuropeptide).

[0304] In another example, one or more of the [CC] cleavage sites may comprise methionine, suitably in which case the cleavage is carried out by one or more chemicals. Suitably the chemical is cyanogen bromide (CNBr), under suitable conditions. It will be apparent that in such methods methionines should not be present in locations where cleavage is undesirable (e.g. within an insecticidal neuropeptide).Suitably the use of a cysteine as a [CC] site results in the release of each neuropeptide with a C-terminal amide modification. Similar to the use of an intein, this allows the direct single-step process for peptide release and amidation. Suitably this is also the case when more than one cleavage site is used in combination with a cysteine cleavage site, such as a cysteine and a protease site, a cysteine and a methionine site, for example.

[0305] Accordingly, in some preferred aspects of the invention, cleavage of the pre-pro- peptide or cleavage of a portion of the pre-pro-peptide directly results in modification of the one or more insecticidal peptides or precursors thereof. For example, cleavage can result in C- terminal amidation of the one or more insecticidal peptides or precursors thereof. Simultaneous cleavage and C-terminal amidation is an efficient approach for providing insecticidal peptides or precursors thereof which require C-terminal amidation for activity.

[0306] Modifying

[0307] Optionally, the method further comprises a step of chemically and / or enzymatically modifying any precursor peptide to produce an insecticidal neuropeptide. Suitably to produce an active insecticidal neuropeptide.

[0308] Suitably the further chemical and / or enzymatic modification may comprise N-terminal modification and / or C-terminal modification of the precursor peptide. Suitably the further chemical and / or enzymatic modification may comprise C-terminal amidation etc. Alternatively in some cases, the modification may comprise N-terminal deletion, suitably of one or several amino acids.

[0309] Optionally the further modification step may be carried out within the host cell, suitably by enzymes within the host cell which may be endogenous or heterologously expressed enzymes. Suitably therefore the method may comprise a step of culturing the host cell under suitable conditions for the precursor peptide to be enzymatically modified, optionally by enzymes within the host cell. Alternatively, the further modification step may be carried out outside the host cell, suitably by exogenous enzymes, which may optionally be added to the culture medium, or by chemical reagents which may be added to the culture medium.

[0310] In one embodiment, the further chemical and / or enzymatic modification step comprises amidating the precursor peptide to produce an insecticidal neuropeptide, suitably at the C- terminus. In some cases, the insecticidal neuropeptide is only active in the amidated form, for example the AKH family of insecticidal neuropeptides. Therefore, this step is required to convert the precursor peptide into the final insecticidal peptide. In other cases, the peptide will be active as it is produced, without requiring amidation, and therefore the step of modification is not required.

[0311] In some embodiments, the precursor peptide is chemically or photochemically amidated. In some embodiments, the precursor peptide is enzymatically amidated.

[0312] Suitably chemical amidation may be carried out by mixing Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU) and the peptide in a 1:1 ratio in organic solvent with 1.5 equivalents of diisopropylethylamine, and stirring for 3-5 minutes to allow activation of the carboxylic acid. Then, exposing to the peptide to ammonia, either by bubbling ammonia gas through the solution or by adding concentrated ammonia.

[0313] Suitably photochemical amidation may be carried out by any of the following means: employing Ru(bpy)3Cl2 as the photocatalyst under visible light, using 9-mesityl-10- methylacridinium tetrafluoroborate (Mes-Acr-MeBF4) under blue light, using a charge-transfer complex (CTC) under sunlight and then subsequent coupling with acids, using 4- dimethylaminopyridine-bromotrichloromethane (DMAP-BrCCl3) CTC under sunlight, a photocatalytic deoxygenative protocol employing amine-boranes and carboxylic acids, through the generation of oxyphosphonium ions by photoredox / cobaloxime catalysis, or by mixing the peptide with pyridine-CBr4 and exposing to light at a wavelength of 370 nm. In one embodiment, the photochemical process of amidation described in WO2023105074 (herein incorporated by reference) may be used. Suitably said process catalyses the conversion of a C-terminal cysteine into a terminal amide group. Suitably in such embodiments using the photochemical amidation described therein, each NP unit of the pre-pro-peptide comprises an additional cysteine, suitably at the C-terminus thereof. Suitably this is not the same as a Cysteine cleavage site described herein, which may from one or more of the [CC] sites in the pre-pro-peptide, and is present between NP units of the pre-pro-peptide. Suitably when a Cysteine cleavage site is present in the pre-pro-peptide, the peptide sequences thereof do not comprise any cysteine residues.

[0314] Suitably enzymatic amidation is carried out by a C-terminal [α-]amidation enzyme. Suitably the enzyme is endogenous to the host cell or recombinantly expressed within the host cell. Suitably the C-terminal α-amidating enzyme is a peptidylglycine-amidating monooxygenase (PAM) (in mammals), or may be the combination of a mono-oxygenase (PHM) and a lyase (PAL) (in insects). In one embodiment therefore, the enzymatic amidation is carried out by PHM and PAL enzymes Suitably said enzyme catalyses the conversion of a C-terminal glycine into a terminal amide group. Suitably in such embodiments using enzymatic amidation, each NP unit of the pre-pro-peptide comprises an additional glycine, suitably at the C-terminus thereof.

[0315] Suitably in such embodiments therefore, the pre-pro-peptide comprises an amidation signal [AS]. Suitably at the C-terminus of each NPi unit in the pre-pro-peptide. Suitably prior to any [CC] cleavage site. Suitably the amidation signal is a single cysteine or single glycine residue.

[0316] As discussed herein, in some preferred embodiments cleavage and modification can occur simultaneously, e.g. through various chemical- or intein-based approaches.

[0317] Isolating

[0318] Suitably the process further comprises a step of isolating the insecticidal neuropeptide or a precursor peptide thereof (suitably a pre-pro-peptide), from the culture medium, suitably from the culture medium of the completed fermentation of host cells. Suitably the isolating step may take place after the modification step, or may take place prior to the modification step. Suitably in some embodiments the isolating step may take place prior to the cleavage step. Suitably wherein the pre-pro-peptide is isolated. Suitably the isolating step may take place prior to any cleavage or modification step, e.g. where an insecticidal neuropeptide precursor, such as a pre-pro-peptide, is to be administered such the neuropeptide precursor is cleaved, and optionally further modified, following ingestion by the target insect.

[0319] Suitably isolating the insecticidal neuropeptide or a precursor peptide thereof may comprise separating the insecticidal neuropeptide or a precursor peptide thereof from the host cells. Suitably isolating the insecticidal neuropeptide or a precursor peptide thereof may comprise lysing the host cells to release the insecticidal neuropeptide or a precursor peptide thereof from the host cells into the culture media. Suitably the lysis step is carried out by high-pressure homogenizer, sonication, passing through mills, and the like. Suitably this is followed by clarification to remove cellular debris.

[0320] Suitably isolating the insecticidal neuropeptide or a precursor peptide thereof may comprise separating the insecticidal neuropeptide or a precursor peptide thereof from the culture media.

[0321] Suitably the isolation step is carried out by filtration, centrifugation, sedimentation, floatation of the culture media. Alternatively, if the insecticidal neuropeptide or a precursor peptide thereof is secreted from the host cells during culture, as in this embodiment, then the isolating step comprises separating the insecticidal neuropeptide or a precursor peptide thereof from the culture media and the host cells. In one embodiment, where the peptides are secreted from the host cells, centrifugation is used. Suitably this step may produce a supernatant comprising the desired peptides. In one embodiment the isolation step is carried out by ultrafiltration, suitably with a size exclusion column of either 10 kDa, 5 kDa or 3 kDa followed by a size exclusion column of less than or equal to 1kDa. Suitable filtration processes are described for example in EP1546187B1.

[0322] Suitably the method may also comprise a step of purifying the insecticidal neuropeptide or a precursor peptide thereof. Suitably from the obtained supernatant. Suitably to remove contaminants or impurities. Suitable means of purification are known in the art for example: ultrafiltration, chromatography, filtration, diafiltration precipitation and / or liquid / liquid extraction, and the like. In one embodiment purification is carried out by chromatography, which may be selected from one or more of (i) affinity, (ii) ion-exchange, (iii) hydrophobic interactions, (iv) size exclusion, and (v) mixed-mode chromatography. In some embodiments in which the purification step uses affinity purification, the insecticidal neuropeptide or a precursor peptide thereof further comprises an affinity tag. Suitably at the N or C-terminus thereof. Suitably the affinity tag is between 5-10 amino acids in length. Suitably it is cleavable. Suitably it is operable to be bound by a chromatography column carrying a suitable binding partner. Suitable tags may be selected from: a His-tag (for example encoded by a sequence according to SEQ ID NO: 276), an Arg-tag, a FLAG-tag, a Strep-tag, Glutathione-S-transferase tag, Twin-Strep Tag, Calmodulin Binding Peptide, Chitin-binding Tag (for example according to SEQ ID NO: 300),, Maltose- binding Tag, and a Cellulose-binding Tag. In one embodiment, the insecticidal neuropeptide or a precursor peptide thereof comprises a His-tag for IMAC affinity purification.

[0323] Suitably the purity of the isolated insecticidal neuropeptide thereof is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. Suitably, however, due to the biological production of the peptides, the isolated insecticidal neuropeptide comprises at least 0.1% w / w of a precursor peptide thereof, and / or optionally a pre-pro-peptide as discussed above. In some embodiments, compositions of the invention may comprise at least 0.1% w / w of precursor peptides and / or pre-pro-peptides. In some embodiments, compositions of the invention may comprise at least 0.1% w / w of precursor peptides.

[0324] Once isolated and optionally purified, the isolated insecticidal neuropeptide or a precursor peptide thereof may be further modified as described above.

[0325] Suitably the process produces one or more active isolated insecticidal neuropeptides, optionally which are not amidated. Suitably therefore the invention provides an isolated insecticidal neuropeptide produced by the process of the seventh aspect, and optionally a composition comprising said isolated insecticidal neuropeptide. Suitably wherein the isolated insecticidal neuropeptide is active. Suitably wherein the isolated insecticidal neuropeptide is amidated.

[0326] Suitably the process produces one or more insecticidal neuropeptides which are suitable for subsequent activation (e.g. by cleavage and / or modification) following ingestion by the target insect.

[0327] In one embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect; (ii) culturing said host cell under suitable conditions for an insecticidal neuropeptide to be produced by the host cell; (iii) optionally isolating the insecticidal neuropeptide from the culture; and (iv) optionally purifying the insecticidal neuropeptide.

[0328] In one embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect; (ii) culturing said host cell under suitable conditions for a precursor peptide to be produced by the host cell; (iiii) enzymatically modifying the precursor peptide to produce an insecticidal neuropeptide; (iv) optionally isolating the insecticidal neuropeptide from the culture; and (v) optionally purifying the insecticidal neuropeptide.

[0329] In one embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect; (ii) culturing said host cell under suitable conditions for a precursor peptide to be produced by the host cell; (iii) optionally isolating the precursor peptide from the culture; (iv) chemically modifying the precursor peptide to produce an insecticidal neuropeptide; and (v) optionally purifying the insecticidal neuropeptide.

[0330] In one embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be produced by the host cell; (iii) further processing the pre-pro-peptide to release an insecticidal neuropeptide; (iv) optionally isolating the insecticidal neuropeptide from the culture; and (v) optionally purifying the insecticidal neuropeptide.

[0331] In one embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be produced by the host cell; (iii) further processing the pre-pro-peptide to release a precursor neuropeptide; (iv) enzymatically modifying the precursor peptide to produce an insecticidal neuropeptide; (v) optionally isolating the insecticidal neuropeptide from the culture; and (vi) optionally purifying the insecticidal neuropeptide.

[0332] In one embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be produced by the host cell; (iii) further processing the pre-pro-peptide to release a precursor neuropeptide; (iv) optionally isolating the precursor peptide from the culture; (v) chemically modifying the precursor peptide to produce an insecticidal neuropeptide; and (vi) optionally purifying the insecticidal neuropeptide.

[0333] One embodiment of isolating, modifying and purifying insecticidal neuropeptides through affinity chromatography and excision with exogenous proteases

[0334] In some embodiments the pre-pro-peptide may be expressed with an affinity tag separated from the precursor peptide sequence by a protease recognition site linker. Suitably the affinity tag can be expressed to facilitate recovery and purification of the precursor peptides. Suitably the pre-pro-peptide may comprise a secretion signal, inducing movement of the pre-pro- peptide to the extracellular environment of the host cell. By having an affinity tag on the pre- pro-peptide sequence it can be pulled down or isolated from the cell culture by affinity chromatography, onto a suitable affinity resin (His / Nickel, protein A / IGG, chitin / chitin binding domain). The resin can then be treated with a protease of choice to liberate the precursor peptides from the bound pre-pro-peptide. Suitably said precursor peptides may then be subsequently recovered by size exclusion chromatography. Some suitable candidate proteases would be Kex2 (optionally in combination with Kex1 and / or Ste13), Factor Xa, Thrombin, HRV 3C, enterokinase, TEV, staphylolysin and adenain.

[0335] Proteases leave scar residues on the adjacent peptides in constructs expressing multiple tandem peptides in a single expression cassette. In some embodiments, novel proteases such as staphylolysin or adenain could be used as they leave an upstream ‘GG’ which is required in select insecticidal neuropeptides. Alternatively, scar residues left by an endoprotease could be removed through calibrated incubation with an exoprotease or amino dipeptidase such as Ste13. Suitably, after isolation, the precursor peptides could then be incubated with a PAM enzyme, as described above, resulting in C-terminal amidation prior to purification through size exclusion chromatography.

[0336] In one such an embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect, optionally wherein the expression construct comprises a polynucleotide encoding a secretion peptide and an affinity tag; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be secreted by the host cell; (iii) isolating the pre-pro-peptide from the host cell, optionally by affinity chromatography; (iv) contacting the pre-pro-peptide with a protease to release a precursor neuropeptide, optionally wherein the protease may be selected from: Kex2 (optionally in combination with Kex1 and / or Ste13), Factor Xa, Thrombin, HRV 3C, enterokinase, TEV, staphylolysin and adenain; (v) recovering the precursor peptide, optionally by size exclusion chromatography; (v) chemically modifying the precursor peptide, optionally wherein the modification is amidation of the precursor peptide, optionally by contacting with a PAM enzyme or by photoamidation, to produce an insecticidal neuropeptide; and (vi) optionally purifying the insecticidal neuropeptide.

[0337] One embodiment of isolating, modifying and purifying insecticidal neuropeptides using inteins

[0338] Inteins are peptide sequences naturally found within proteins which are functionally homologous to RNA introns but are self-splicing without reliance on cell machinery. They are located between N- and C- terminal protein exteins, which in the context herein comprises the rest of the pre-pro-peptide sequence on the N-terminal side of a given intein, and the rest of the pre-pro-peptide sequence on the C-terminal side of a given intein, respectively. Certain conditions cause the intein to excise itself from the protein, ligating the two exteins into a continuous sequence fused with a peptide bond. This is a four-step chemical reaction beginning with the transfer of the N-extein to the R group of the inteins N-terminal cysteine, this is known as the N-S acyl shift. This thioester bond undergoes transesterification with a nucleophilic residue at the N-terminus of the C-extein. Cyclisation of the asparagine at the C-terminus of the intein then results in intein cleavage (Prabhala et al, 2022). The self-excising property of inteins can be exploited to facilitate recombinant peptide purification and amidation. Suitable inteins are defined in SEQ ID NO: 264-268.

[0339] In some embodiments the pre-pro-peptide may be expressed with an affinity tag separated from the precursor peptide sequence by an intein, optionally by a modified intein. Intein cyclisation and cleavage is dependent on an N-terminal cysteine and a C-terminal asparagine. Mutating the C-terminal asparagine to an alanine / valine limits the excision / ligation of the two exteins (Albetsen et al, 2013, Aranko & Iwai, 2021), instead halting the reaction post N-S acyl shift. This confers a large advantage from the perspective of recombinant insecticidal neuropeptide production. In some embodiments the pre-pro-peptide may be expressed with an affinity tag separated from the precursor peptide sequence by a C-terminal valine modified intein. By having an affinity tag on the pre-pro-peptide sequence it can be pulled down or isolated by affinity chromatography, onto suitable affinity resin (His / Nickel, protein A / IGG, chitin / chitin binding domain). The resin can then be treated with a chemical to stimulate intein cleavage and suitably liberate the precursor peptides from the bound pre-pro-peptide. Suitably the precursor peptides may be eluted through the addition of thiols (DTT, β-mercaptoethanol) to stimulate intein cleavage. Suitably, eluting with a mixture of thiols and ammonium sulphate / ammonium carbonate further automatically amidates the precursor peptide upon N-terminal intein cleavage, reducing the number of steps in the process. Suitably such a process is shown herein in figures 30 and 31.

[0340] Inteins are diverse and can be induced to cleave under various conditions dependent on the evolutionary origin of the intein in question. Many inteins can be induced to initiate the N-S acyl shift through the addition of thiols (DTT, β-mercaptoethanol). Additionally, inteins identified from extremophilic organisms can be induced to initiate the N-S acyl shift through a shift in conditions including addition of 2-4 M NaCl (Aranko & Iwaï, 2021), a shift to elevated temperature (40-80 °C) (Mills et al, 2004), a shift to reduced temperature (4-18 °C) (Zeidler et al, 2004), or a change in pH. Mutant inteins displaying these characteristics are known, it is theoretically possible to modify them with a C-terminal alanine or valine to inhibit their ability to cyclise, excise and ligate the N- and C- terminal extein. Suitably therefore, the pre-pro-peptide may be isolated or pulled down by affinity chromatography, suitably an N-S acyl shift of the intein may be initiated by placing the loaded resin in conditions suited to the choice of intein, and finally the insecticidal neuropeptide may be eluted with the addition of ammonium sulphate / ammonium carbonate. This offers a method for purifying and amidating the insecticidal neuropeptide in one step. Suitably therefore no amidation signal is required in the pre-pro- peptide.

[0341] In one such an embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect, optionally wherein the expression construct comprises a polynucleotide encoding a secretion peptide and an affinity tag and wherein one or more of the cleavage sites are inteins; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be secreted by the host cell; (iii) isolating the pre-pro-peptide from the host cell, optionally by affinity chromatography; (iv) contacting the pre-pro-peptide with a chemical to stimulate intein cleavage and amidation of a precursor neuropeptide, optionally wherein the chemical is selected a mixture of thiols and ammonium sulphate / ammonium carbonate; (v) recovering the insecticidal neuropeptide, optionally by size exclusion chromatography.

[0342] In another such an embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect, optionally wherein the expression construct comprises a polynucleotide encoding a secretion peptide and an affinity tag and wherein one or more of the cleavage sites are inteins suitable for cleavage by a target insect following ingestion; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be secreted by the host cell; (iii) isolating the pre-pro-peptide from the host cell, optionally by affinity chromatography.

[0343] One embodiment of isolating, modifying and purifying insecticidal neuropeptides using Cysteines

[0344] In some embodiments the pre-pro-peptide may be expressed with an affinity tag separated from the precursor peptide sequence by a protease recognition site linker and a Cysteine cleavage site. Suitably the affinity tag can be expressed to facilitate recovery and purification of the precursor peptides. Suitably the pre-pro-peptide may comprise a secretion signal, inducing movement of the pre-pro-peptide to the extracellular environment of the host cell. By having an affinity tag on the pre-pro-peptide sequence it can be pulled down or isolated from the cell culture by affinity chromatography, onto a suitable affinity resin (His / Nickel, protein A / IGG, chitin / chitin binding domain). The resin can then be treated with one or more chemicals to liberate and elute the precursor peptides from the bound pre-pro- peptide.

[0345] In one embodiment, a C-terminal cysteine residue is incorporated into each peptide sequence within the concatemer (pro-peptide) sequence. This cysteine residue can be selectively converted to a dehydroalanine (DHA) residue through chemical modification using established methods (Chem. Sci., 2011, 2, 1666). Following this conversion, the concatemer structure remains intact, with DHA residues replacing the original cysteine residues. Subsequently, a boron-based chemical cleavage reaction (e.g., β-elimination or other boronate-mediated reactions) is used to cleave at the DHA residues. This cleavage results in the release of individual peptides from the concatemer, where each upstream peptide acquires a C-terminal amide, while the downstream peptide is modified with a lactic acid moiety at its N-terminus. This single-step process achieves both peptide release and C-terminal amidation. This offers a method for purifying and amidating the insecticidal neuropeptide in one step. Suitably therefore no amidation signal is required in the pre-pro-peptide.

[0346] Suitably in such an embodiment, the pre-pro-peptide is firstly treated with DBHDA to convert the Cysteine cleavage sites into DHA. Suitably the pre-pro-peptide is contacted with DTT (250 eq.) NaPi (100mM and 3M Gdn-Hcl of pH 8) RT for 30 minutes, then the pre-pro- peptide is reacted with DBHDA (80eq.), in DMF RT for 2 hours 37C for 2.5 hours and NaPi (100mM and 3M Gdn-HCl pH 8).

[0347] Secondly, the precursor peptides are then eluted by the addition of B2(OH)4 and ascorbate. Suitably the pre-pro-peptide is contacted with B2(OH)4200 eq. and ascorbate 50 eq. in PBS buffer up to 95% at 37C for 6 hours. Suitably, eluting with such a mixture further automatically amidates the precursor peptide upon cleavage, reducing the number of steps in the process. Suitably such a process is shown herein in figures 34 and 35.

[0348] In one such an embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect, optionally wherein the expression construct comprises a polynucleotide encoding a secretion peptide and an affinity tag and wherein one or more of the cleavage sites are Cysteines; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be secreted by the host cell; (iii) contacting the pre-pro-peptide with a chemical to convert Cysteine cleavage sites into DHA, optionally wherein the chemical is DBHDA; (iii) isolating the pre-pro-peptide from the host cell, optionally by affinity chromatography; (iv) contacting the pre-pro-peptide with a chemical to stimulate DHA cleavage and amidation of each precursor neuropeptide, optionally wherein the chemical is B2(OH)4and ascorbate; (v) recovering the insecticidal neuropeptide, optionally by size exclusion chromatography.

[0349] One embodiment of isolating, modifying and purifying insecticidal neuropeptides using Cysteines and Factor Xa

[0350] The cysteine / DHA system can be further optimised by incorporating a sequence with a specific endopeptidase or protease cleavage site. The inclusion of an endopeptidase / protease site such as a Factor Xa site enables precise cleavage, leaving a clean N-terminal residue at the beginning of the subsequent peptide sequence within the concatemer.

[0351] In some embodiments the pre-pro-peptide may be expressed with an affinity tag separated from the precursor peptide sequence by a protease recognition site linker and a Cysteine-Factor Xa cleavage site. Suitably the affinity tag can be expressed to facilitate recovery and purification of the precursor peptides. Suitably the pre-pro-peptide may comprise a secretion signal, inducing movement of the pre-pro-peptide to the extracellular environment of the host cell. By having an affinity tag on the pre-pro-peptide sequence it can be pulled down or isolated from the cell culture by affinity chromatography, onto a suitable affinity resin (His / Nickel, protein A / IGG, chitin / chitin binding domain). The resin can then be treated with Factor Xa peptidase to liberate and elute the precursor peptides from the bound pre-pro- peptide.

[0352] Suitably in such an embodiment, the pre-pro-peptide is firstly treated with a Factor Xa peptidase enzyme. Suitably to cleave at the Factor Xa cleavage sites (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269) in the pre-pro-peptide, thereby separating the pre-pro-peptide into precursor peptides. Suitably the pre-pro-peptide is contacted with Factor Xa at 200µg / ml RT for 2-24 hours. Suitably this step elutes the precursor peptides from the bound pre-pro-peptide.

[0353] Suitably then the precursor peptides are treated with DBHDA to convert the Cysteine cleavage sites into DHA. Suitably the precursor peptide is contacted with DTT (250 eq.) NaPi (100mM and 3M Gdn-Hcl of pH 8) RT for 30 minutes, then the precursor peptide is reacted with DBHDA (80eq.), in DMF RT for 2 hours 37C for 2.5 hours and NaPi (100mM and 3M Gdn-HCl pH 8).

[0354] Secondly, the precursor peptides are then treated with the addition of B2(OH)4and ascorbate. Suitably the precursor peptide is contacted with B2(OH)4200 eq. and ascorbate 50 eq. in PBS buffer up to 95% at 37C for 6 hours. Suitably, such a mixture further automatically amidates the precursor peptide upon cleavage, reducing the number of steps in the process. Suitably such a process is shown herein in figure 36. This offers a method for purifying and amidating the insecticidal neuropeptide in one step. Suitably therefore no amidation signal is required in the pre-pro-peptide.

[0355] In one such an embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect, optionally wherein the expression construct comprises a polynucleotide encoding a secretion peptide and an affinity tag and wherein one or more of the cleavage sites are Cysteine-Factor Xa cleavage sites; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be secreted by the host cell; (iii) isolating the pre-pro-peptide from the host cell, optionally by affinity chromatography; (iv) contacting the pre-pro-peptide with Factor Xa peptidase to cleave at the Factor Xa cleavage sites and elute each precursor peptide; (v) contacting each precursor peptide with a chemical to convert Cysteine cleavage sites into DHA, optionally wherein the chemical is DBHDA; (iv) contacting each precursor peptide with a chemical to stimulate DHA cleavage and amidation of each precursor neuropeptide, optionally wherein the chemical is B2(OH)4 and ascorbate; (v) recovering the insecticidal neuropeptide, optionally by size exclusion chromatography.

[0356] One embodiment of isolating, modifying and purifying insecticidal neuropeptides using Cysteines and Methionines

[0357] Alternatively, another effective strategy for the cysteine / DHA system involves incorporating a sequence with a methionine residue and performing chemical cleavage using cyanogen bromide (CNBr) . By designing a sequence containing a Cys-[SS]-Met wherein the SS sequence is an optional stuffer sequence therebetween, CNBr-mediated cleavage at methionine residues releases individual peptides from the concatemer. This process ensures that each peptide is C-terminally amidated and properly separated from adjacent sequences and leaves a clean N-terminal residue at the beginning of the subsequent peptide sequence within the concatemer.

[0358] In some embodiments the pre-pro-peptide may be expressed with an affinity tag separated from the precursor peptide sequence by a protease recognition site linker and a Cysteine-Methionine cleavage site. Suitably the affinity tag can be expressed to facilitate recovery and purification of the precursor peptides. Suitably the pre-pro-peptide may comprise a secretion signal, inducing movement of the pre-pro-peptide to the extracellular environment of the host cell. By having an affinity tag on the pre-pro-peptide sequence it can be pulled down or isolated from the cell culture by affinity chromatography, onto a suitable affinity resin (His / Nickel, protein A / IGG, chitin / chitin binding domain). The resin can then be treated with a chemical, CNBr, to liberate and elute the precursor peptides from the bound pre-pro-peptide.

[0359] Suitably in such an embodiment, the pre-pro-peptide is firstly treated with a chemical to cleave the Methionine cleavage sites in the pre-pro-peptide, thereby separating the pre-pro- peptide into precursor peptides. Suitably the pre-pro-peptide is contacted with CNBr in 0.5 M aqueous HCl. Suitably this step elutes the precursor peptides from the bound pre-pro-peptide.

[0360] Suitably then the precursor peptides are treated with DBHDA to convert the Cysteine cleavage sites into DHA. Suitably the precursor peptide is contacted with DTT (250 eq.) NaPi (100mM and 3M Gdn-Hcl of pH 8) RT for 30 minutes, then the precursor peptide is reacted with DBHDA (80eq.), in DMF RT for 2 hours 37C for 2.5 hours and NaPi (100mM and 3M Gdn-HCl pH 8).

[0361] Secondly, the precursor peptides are then treated with the addition of B2(OH)4and ascorbate. Suitably the precursor peptide is contacted with B2(OH)4200 eq. and ascorbate 50 eq. in PBS buffer up to 95% at 37C for 6 hours. Suitably, such a mixture further automatically amidates the precursor peptide upon cleavage, reducing the number of steps in the process. Suitably such a process is shown herein in figure 37. This offers a method for purifying and amidating the insecticidal neuropeptide in one step. Suitably therefore no amidation signal is required in the pre-pro-peptide.

[0362] In one such an embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect, optionally wherein the expression construct comprises a polynucleotide encoding a secretion peptide and an affinity tag and wherein one or more of the cleavage sites are Cysteine- Methionine cleavage sites; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be secreted by the host cell; (iii) isolating the pre-pro-peptide from the host cell, optionally by affinity chromatography; (iv) contacting the pre-pro-peptide with a chemical to cleave at the Methionine cleavage sites and elute each precursor peptide, optionally wherein the chemical is CNBr; (v) contacting each precursor peptide with a chemical to convert Cysteine cleavage sites into DHA, optionally wherein the chemical is DBHDA; (iv) contacting each precursor peptide with a chemical to stimulate DHA cleavage and amidation of each precursor neuropeptide, optionally wherein the chemical is B2(OH)4 and ascorbate; (v) recovering the insecticidal neuropeptide, optionally by size exclusion chromatography.

[0363] One embodiment of isolating and purifying insecticidal neuropeptides

[0364] Another approach for the present invention is the production and isolation of a neuropeptide precursor which is then administered such that it is subsequently activated in a target insect, e.g. as a result of the conditions encountered in the target insect gut.

[0365] This can be achieved using suitable cleavage sites that can be cleaved by enzymatic activity in the target insect, or by physical or chemical conditions in the target insect.

[0366] Suitably the neuropeptide precursors are described herein.

[0367] In one such embodiment, the process may comprise the following steps: (i) providing a host cell comprising a recombinant expression construct of the first aspect, the genome of the second aspect, or which is a cell of the third aspect, optionally wherein the expression construct comprises a polynucleotide encoding a secretion peptide and an affinity tag and wherein one or more of the cleavage sites are suitable for cleavage by a target insect following ingestion; (ii) culturing said host cell under suitable conditions for a pre-pro-peptide to be secreted by the host cell; (iii) isolating the pre-pro-peptide from the host cell, optionally by affinity chromatography. The isolated pre-pro-peptide can suitably be administered to a plant or part thereof. Upon ingestion by the target insect, the pre-pro-peptide is cleaved and becomes active. Suitably the one or more insecticidal neuropeptides released in such a method do not require further modification (e.g. amidation) to become active.

[0368] Methods of Preventing or Treating an Insect Infestation

[0369] The invention further provides a method for preventing or treating an insect infestation of a plant, an animal, or a building, the method comprising: contacting a plant, or part of said plant, an animal, or a part of said animal, or a building with an effective amount of the composition of the sixth or eighth aspects, or with an insecticidal neuropeptide produced by any of the methods described herein.

[0370] Suitably any such method steps below in the context of using the composition of the sixth or eighth aspects to prevent or treat insect infestations of a plant or part thereof, apply equally to methods of preventing or treating insect infections of animals or buildings, in particular household pests which are described hereinbelow.

[0371] Contacting

[0372] Suitably the method comprises as a first step, contacting the plant, or part of said plant with an effective amount of the composition. Suitably contacting may comprise any known technique of applying a composition to a plant or part thereof.

[0373] Suitably the composition can be contacted directly with the plant, e.g. with the surface of leaves, buds, roots, shoots, floral parts, seeds, etc., or contacted indirectly with the plant e.g. With the soil or the locus of growth of the plant.

[0374] For example, suitable techniques for applying the composition may include: watering, drenching, irrigating, vaporizing, dusting, spreading-on, spraying, atomizing, foaming, misting, fogging, culturing in hydroculture, culturing in hydroponics, coating, submerging, applying as a powder, and / or encrusting, optionally post-harvest.

[0375] The present invention also provides post-harvest treatment methods for preventing or reducing damage to a harvested plant or plant part caused by an insect, at least comprising the step of applying directly or indirectly to the harvested plant or to a harvested part of the plant, composition as disclosed herein, suitably with an effective amount of the composition. Suitably the harvested part is a fruit, flower, nut or vegetable, a fruit or vegetable with inedible peel. Suitably applying indirectly may comprise applying the compound or composition of the invention to the location in which the plant or a harvested part of the plant is stored, or in which it is intended to be stored. Suitably therefore there is provided a harvested plant or part thereof which has been contacted with the composition of the invention, and suitably therefore comprises a composition of the invention.

[0376] It is furthermore possible to apply the compositions by the ultra-low volume method, or to inject the composition itself into the soil, or directly into the plant.

[0377] In some embodiments, the composition may be used as a seed coating. Suitably, the composition is then applied to the plant seed prior to planting. Suitably therefore there is provided a seed comprising a coating wherein the coating comprises a composition of the invention.

[0378] In a further aspect of the invention there is provided a plant or plant part which is at least partially coated with a composition of the invention.

[0379] In other embodiments relating to household pests / insects, contacting may comprise applying a composition to a surface. Suitably to a surface with which the pest / insect will interact.

[0380] Effective amount

[0381] The terms "effective amount" and "effective dose", as used herein, mean the amount needed to achieve the desired result or results. Suitably the effective amount of the composition to be used in the method is the amount sufficient to have an insecticidal activity on a target insect. Suitably the effective amount of the composition to be used in the method is the amount sufficient to prevent or treat an insect infestation of a plant. This amount may vary with the insecticidal neuropeptide being used, the plant, the insect, and the conditions.

[0382] Suitably the effective amount is in the range of from about 0.1 pg / ml to about 100 mg / ml, or from about 5 pg / ml to about 5 mg / ml. Suitably an effective concentration of the insecticidal neuropeptide is generally between about 10−4M to 10−12M, more typically between about 10−5M to 10−10M, suitably in some cases about 10−6M.

[0383] Suitably prevention of an insect infestation may comprise inhibiting an insect infestation of a plant. Suitably treatment of an insect infestation may comprise reducing an insect infestation of a plant. Suitably, the method may be prophylactic. Thus, for example, the composition may be applied to the plant or plant part while the plant or part is free or substantially free of insects.

[0384] Suitably the compositions of the invention may inhibit insect infestation of a plant by up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Suitably the compositions of the invention may reduce insect infestation of a plant by up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Suitably compared to a control plant that has not received the composition of the invention.

[0385] Suitably treatment of an insect infestation may comprise reducing damage to the plant caused by the insect. Suitably treatment may encompass prevention. Suitably the composition of the invention may reduce damage to a plant caused by an insect infestation by up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Suitably in some cases the composition of the invention may completely reduce damage to a plant caused by an insect.

[0386] Suitable damage caused by insect infestations may encompass chewing of leaves or fruit, consumption of leaves or fruit, holes or notches in stems fruits or roots, bacterial or viral infection, leaf defoliation, skeletonizing, cutting of stems or roots. Suitably reduction in damage to a plant after contacting with a composition of the invention may be determined by visual inspection of the plant, imaging technologies. Suitably the compositions of the invention may prevent or treat an insect infestation by interfering with the harmful activity of an insect, including but not limited to: killing the insect inhibiting the growth or activity of the insect, inhibiting reproduction of the insect, altering the behaviour of the insect, or repelling the insect. Suitably the compositions of the invention may have a biostatic effect, biocidal effect, or an insecticidal effect. “Biostatic (effect)” or “biostatic use”, as used herein, includes any effect or use of a composition or combination as described herein (optionally comprised in a biostatic, or biocidal composition as defined herein) for controlling, modulating or interfering with the harmful activity of a pest, such as a plant pest or a plant pathogen. Suitably including but not limited to inhibiting the growth or activity of the insect, altering the behaviour of the insect, and repelling or attracting the insect in plants, plant parts or in other agro-related settings, such as for example for household uses or in soil. “Insecticidal activity” as used herein, means killing the insect or severely disabling the insect, which may be achieved in various different ways which are outlined below. Suitably the compositions of the invention may prevent or treat an insect infestation by increasing insect mortality. Suitably the composition of the invention may increase insect mortality up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Suitably in some cases the composition of the invention may completely kill an insect infestation. Suitably therefore, the invention may provide a method of increasing insect mortality comprising contacting a plant or part thereof with an effective amount of the composition of the invention.

[0387] Suitably the compositions of the invention may prevent or treat an insect infestation by decreasing insect feeding. Suitably the composition of the invention may decrease insect feeding by up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Suitably in some cases the composition of the invention may completely inhibit insect feeding. Suitably therefore, the invention may provide a method of reducing insect feeding comprising contacting a plant or part thereof with an effective amount of the composition of the invention.

[0388] Suitably the compositions of the invention may prevent or treat an insect infestation by decreasing insect fecundity or reproduction. Suitably the composition of the invention may decrease insect fecundity by up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Suitably in some cases the composition of the invention may completely inhibit insect reproduction. Suitably therefore, the invention may provide a method of reducing insect fecundity comprising contacting a plant or part thereof with an effective amount of the composition of the invention.

[0389] Plant

[0390] “Plant” as used herein, means an entire plant or a part thereof, including fresh fruit, vegetables and seeds. The plant or plant part may be a live plant or part thereof. Also, the term "plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores.

[0391] Suitably the plant or plant part may be any plant species or variety. Suitably the plant or plant part may be a monocot or a dicot. Suitably the plant or pant part may be an agronomically important species or variety. Suitably the plant or pant part may be a crop.

[0392] “Crop” as used herein means a plant species or variety that is grown to be harvested as food, livestock fodder, fuel raw material, or for any other economic purpose. As a non-limiting example, said crops can be maize, cereals, such as wheat, rye, barley and oats, sorghum, rice, sugar beet and fodder beet, fruit, such as pome fruit (e.g. apples and pears), citrus fruit (e.g. oranges, lemons, limes, grapefruit, or mandarins), stone fruit (e. g. peaches, nectarines or plums), nuts (e.g. almonds or walnuts), soft fruit (e.g. cherries, strawberries, blackberries or raspberries), the plantain family or grapevines, leguminous crops, such as beans, lentils, peas and soya, oil crops, such as sunflower, safflower, rapeseed, canola, castor or olives, cucurbits, such as cucumbers, melons or pumpkins, fibre plants, such as cotton, flax or hemp, fuel crops, such as sugarcane, miscanthus or switchgrass, vegetables, such as potatoes, tomatoes, peppers, lettuce, spinach, onions, carrots, egg-plants, asparagus or cabbage, ornamentals, such as flowers (e.g. petunias, pelargoniums, roses, tulips, lilies, or chrysanthemums), shrubs, broadleaved trees (e.g. poplars or willows) and evergreens (e.g. conifers), grasses, such as lawn, turf or forage grass or other useful plants, such as coffee, tea, tobacco, hops, pepper, rubber or latex plants.

[0393] In one embodiment, the plant or part thereof is selected from a plant which suffers from or attracts hemipteran insect infestations, for example: cereal crops such as wheat (Triticum spp.), oats (Avena spp), rye (Secale spp.), barley (Hordeum spp.), rice (Oryza spp.) and corn (Zea spp.); fruit and vegetable crops including apples (Malus spp); pears (Pyrus spp); strawberry (Fragaria spp.), blueberry (Vaccinum spp.), blackberry (Rubus spp.), raspberry (Rubus spp.), citrus (Citrus spp.), olive (Olea spp.), durian (Durio spp.), longan (Dimocarpus spp.), litchi (L. chinensis), persimmon (Diospyros spp.); beans and peas (including but not limited to Phaseolus, Vigna, Pisum, Lens, Glycine, Cicer, Cajanus, Arachis spp), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), lettuce (Lactuca spp.), brassicas (Brassica spp.) including oil seed rape, alliums (Allium spp.), tomato (Solanum spp.), pepper (Capsicum spp.), asparagus (A. officinalis), melon, squash, pumpkins (Cucumis spp.), and tubers (potato) (Solanum spp.), or a part thereof.

[0394] In one embodiment, the plant or part thereof is selected from a plant which suffers from or attracts aphid insect infestations, suitably M. persicae insect infestations, including Solanaceae, Cruciferae, and Leguminosae for example: cereal crops such as wheat (Triticum spp including winter wheat Triticum aestivum L); fruit and vegetable crops including peach (Prunus spp.), strawberry (Fragaria spp.), blueberry (Vaccinum spp.), blackberry (Rubus spp.), raspberry (Rubus spp.), brassicas (Brassica spp.) such as oil seed rape, lettuce (Lactuca spp.), tomato (Solanum spp.), pepper (Capsicum spp.), beans and peas (including but not limited to Vigna, Pisum spp), melon, squash, pumpkins (Cucumis spp.) , citrus (Citrus spp.), and tubers (potato) (Solanum spp.), or a part thereof. In one embodiment, the plant is a vegetable crop, suitably a brassica spp.

[0395] In one embodiment, the plant or part thereof is selected from a plant which suffers from or attracts dipteran insect infestations, for example: cereals (Triticum spp.); oats (Avena spp);, rye (Secale spp.); barley (Hordeum spp,) rice (Oryza spp.) and corn (Zea spp.); beans and peas (including but not limited to Phaseolus, Vigna, Pisum, Lens, Glycine, Cicer, Cajanus, Arachis spp); fruit crops including apples (Malus spp), pears (Pyrus spp), strawberry (Fragaria spp.), blueberry (Vaccinum spp.), blackberry (Rubus spp.), raspberry (Rubus spp.), cherry, plum, apricot, peach, nectarine (Prunus spp.), blackcurrant, redcurrant, whitecurrant, gooseberry (Ribes spp.), kiwi fruit (Actinidia spp), papaya (Carica spp.), avocado (Persea spp.), mango (Mangifera indica L), longan (Dimocarpus spp.), litchi (L. chinensis), grapes (Vitis spp.), fig (Ficus spp.), passionfruit (Passiflora spp.), Asian pears (Pyrus spp), citrus (Citrus spp.), and olive (Olea spp.); vegetable crops including alliums (Allium spp.), aubergine, tomato (Solanum spp.) and peppers (Capsicum spp.), lettuce (Lactuca spp.), brassicas (Brassica spp.) and courgette, melon, squash, pumpkins (Cucumis spp.); Apiaceae root crops including carrot (Daucus spp.), parsnip (Pastinaca spp.), or a part thereof.

[0396] In one embodiment, the plant or part thereof is selected from a plant which suffers from or attracts lepidoptera insect infestations, for example: cereal crops such as wheat (Triticum spp.), oats (Avena spp), rye (Secale spp.), barley (Hordeum spp.), rice (Oryza spp.) and corn (Zea spp.); fruit and vegetable crops including apples (Malus spp); pears (Pyrus spp); tree nuts (including for example almonds (P. amygdalus), pistachio (Pistacia vera), walnuts (Juglandaceae), hazlenuts (Corylus)); avocado, including Persea Americana (Lauraceaea), blueberry (Vaccinum spp.), citrus (Citrus spp.), olive (Olea spp.), durian (Durio spp.), longan (Dimocarpus spp.), litchi (L. chinensis), persimmon (Diospyros spp.); beans and peas (including but not limited to Phaseolus, Vigna, Pisum, Lens, Glycine, Cicer, Cajanus, Arachis spp), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.),lettuce (Lactuca spp.), brassicas (Brassica spp.) including oil seed rape, alliums (Allium spp.), tomato (Solanum spp.), pepper (Capsicum spp.), asparagus (A. officinalis), melon, squash, pumpkins (Cucumis spp.), and tubers (potato) (Solanum spp.), or a part thereof.

[0397] Insect

[0398] An "insect", as used here, is used in the broad popular sense and includes all species of the superphylum Panarthropoda (classification Systema Naturae, Brands, S.J. (comp.) 1989- 2005. Systema Naturae 2000. Amsterdam, The Netherlands, [http: / / sn2000.taxonomy.nl / ]), including the phyla Arthropoda, Tardigrada and Onychophora; in particular the phylum Arthropoda, and the class Insecta. Suitably the term insect includes all the different phases of the insect life cycle, such as, but not limited to eggs, larvae, nymphs, pupae and adults.

[0399] Suitably the insect may be any insect species. Suitably the insect may be any insect species which is capable of infesting a plant, suitably a plant-infesting insect. Suitably the insect species may also be any insect species which is capable of infesting an animal, or a building. In one embodiment, the insect is regarded as a pest or pathogen, suitably a plant pest or plant pathogen, or an animal pest or animal pathogen, or a household pest. The invention is not limited to crop pests such as insects, but also applies to household pests or insects, such as cockroaches, ants, etc., and disease vectors, such as malaria mosquitoes.

[0400] A “plant pest”, “plant pathogen” or “crop pest”, as used in the application interchangeably, refers to organisms that specifically cause damage to plants, plant parts or plant products, particularly plants, plant parts or plant products, used in agriculture. Note that the term “plant pest” or “crop pest” is used in the meaning that the pest targets and harms plants. Suitable plant pest examples include, but are not limited to, aphids, caterpillars, flies, wasps, and the like, nematodes (living freely in soil or particularly species that parasitize plant roots, such as root- knot nematode and cyst nematodes such as soybean cyst nematode and potato cyst nematode), mites (such as spider mites, thread-footed mites and gall mites) and gastropods (including slugs such as Deroceras spp., Milax spp., Tandonia sp., Umax spp., Arion spp. and Veronicella spp. and snails such as Helix spp., Cernuella spp., Theba spp., Cochlicella spp., Achatina spp., Succinea spp., Ovachlamys spp., Amphibulima spp., Zachrysia spp., Bradybaena spp., and Pomacea spp.).

[0401] In one embodiment, the insect may be selected from hemipteran, dipteran, blattodea, coleopteran, or lepidopteran insects.

[0402] Suitably the insect may be selected from the phylum Arthropoda, in particular from the class of the arachnids, for example Acarus spp., Aceria sheldoni, Acariformes spp., Aculops spp., Aculus spp., Amblyomma spp., Amphitetranychus viennensis, Argas spp., Boophilus spp., Brevipalpus spp., Bryobia praetiosa, Centruroides spp., Chorioptes spp., Dermanyssus gallinae, Dermatophagoides pteronyssius, Dermatophagoides farinae, Dermacentor spp., Eotetranychus spp., Epitrimerus pyri, Eutetranychus spp., Eriophyes spp., Halotydeus destructor, Hemitarsonemus spp., Hyalomma spp., Ixodes spp., Latrodectus spp., Loxosceles spp., Metatetranychus spp., Nuphersa spp., Oligonychus spp., Ornithodorus spp., Ornithonyssus spp., Panonychus spp., Parasitiformes spp.,Phyllocoptruta oleivora, Polyphagotarsonemus latus, Psoroptes spp., Rhipicephalus spp., Rhizoglyphus spp., Sarcoptes spp., Scorpio maurus, Stenotarsonemus spp., Tarsonemus spp., Tetranychus spp., Vaejovis spp., Vasates lycopersici. In one embodiment, the insect is a mite species such as an Acariformes spp. or Parasitiformes spp.

[0403] Still other examples are from the order of the Psocodea, suitably from the Anoplura (Phthiraptera), for example, Damalinia spp., Haematopinus spp., Linognathus spp., Pediculus spp., Ptirus pubis, Trichodectes spp. Still other examples are from the order of the Chilopoda, for example, Geophilus spp., Scutigera spp. Still other examples are from the order of the Collembola, for example, Onychiurus armatus. Still other examples are from the order of the Diplopoda, for example, Blaniulus guttulatus.

[0404] Suitably the insect may be selected from the order of the Diptera, for example, Aedes spp., Agromyza spp., Anastrepha spp., Anopheles spp., Asphondylia spp., Bactrocera spp., Bibio hortulanus, Calliphora erythrocephala, Ceratitis capitata, Chironomus spp., Chrysomyia spp., Chrysops spp., Cochliomyia spp., Contarinia spp., Cordylobia anthropophaga, Culex spp., Culicoides spp., Culiseta spp., Cuterebra spp., Dacus oleae, Dasyneura spp., Delia spp., Dermatobia hominis, Drosophila spp., Echinocnemus spp., Fannia spp., Gasterophilus spp., Glossina spp., Haematopota spp., Hydrellia spp., Hylemyia spp., Hyppobosca spp., Hypoderma spp., Liriomyza spp., Lucilia spp., Lutzomia spp., Mansonia spp., Musca spp., Nezara spp., Oestrus spp., Oscinella frit, Pegomyia spp., Phlebotomus spp., Phorbia spp., Phormia spp., Prodiplosis spp., Psila rosae, Rhagoletis spp., Sarcophaga spp., Simulium spp., Stomoxys spp., Tabanus spp., Tannia spp., Tetanops spp., Tipula spp. Still other examples are from the order of the Heteroptera, for example, Anasa tristis, Antestiopsis spp., Boisea spp., Blissus spp., Calocoris spp., Campylomma livida, Cavelerius spp., Cimex spp., Collaria spp., Creontiades dilutus, Dasynus piperis, Dichelops furcatus, Diconocoris hewetti, Dysdercus spp., Euschistus spp., Eurygaster spp., Heliopeltis spp., Horcias nobilellus, Leptocorisa spp., Leptoglossus phyllopus, Lygus spp., Macropes excavatus, Miridae, Monalonion atratum, Nezara spp., Oebalus spp., Pentomidae, Piesma quadrata, Piezodorus spp., Psallus spp., Pseudacysta persea, Rhodnius spp., Sahlbergella singularis, Scaptocoris castanea, Scotinophora spp., Stephanitis nashi, Tibraca spp., Triatoma spp. Still other examples are from the order of the Homoptera, for example, Acyrthosipon spp., Acrogonia spp., Aeneolamia spp., Agonoscena spp., Aleurodes spp., Aleurolobus barodensis, Aleurothrixus spp., Amrasca spp., Anuraphis cardui, Aonidiella spp., Aphanostigma pin, Aphis spp., Arboridia apicalis, Aspidiella spp., Aspidiotus spp., Atanus spp., Aulacorthum solani, Bemisia spp., Brachycaudus helichrysii, Brachycolus spp., Brevicoryne brassicae, Calligypona marginata, Carneocephala fulgida, Ceratovacuna lanigera, Cercopidae, Ceroplastes spp., Chaetosiphon fragaefolii, Chionaspis tegalensis, Chlorita onukii, Chromaphis juglandicola, Chrysomphalus ficus, Cicadulina mbila, Coccomytilus halli, Coccus spp., Cryptomyzus ribis, Dalbulus spp., Dialeurodes spp., Diaphorina spp., Diaspis spp., Drosicha spp., Dysaphis spp., Dysmicoccus spp., Empoasca spp., Eriosoma spp., Erythroneura spp., Euscelis bilobatus, Ferrisia spp., Geococcus coffeae, Hieroglyphus spp., Homalodisca coagulata, Hyalopterus arundinis, lcerya spp., Idiocerus spp., Idioscopus spp., Laodelphax striatellus, Lecanium spp., Lepidosaphes spp., Lipaphis erysimi, Macrosiphum spp., Mahanarva spp., Melanaphis sacchari, Metcalfiella spp., Metopolophium dirhodum, Monellia costalis, Monelliopsis pecanis, Myzus spp., Nasonovia ribisnigri, Nephotettix spp., Nilaparvata lugens, Oncometopia spp., Orthezia praelonga, Parabemisia myricae, Paratrioza spp., Parlatoria spp., Pemphigus spp., Peregrinus maidis, Phenacoccus spp., Phloeomyzus passerinii, Phorodon humuli, Phylloxera spp., Pinnaspis aspidistrae, Planococcus spp., Protopulvinaria pyriformis, Pseudaulacaspis pentagona, Pseudococcus spp., Psylla spp., Pteromalus spp., Pyrilla spp., Quadraspidiotus spp., Quesada gigas, Rastrococcus spp., Rhopalosiphum spp., Saissetia spp., Scaphoides titanus, Schizaphis graminum, Selenaspidus articulatus, Sogata spp., Sogatella furcifera, Sogatodes spp., Stictocephala festina, Tenalaphara malayensis, Tinocallis caryaefoliae, Tomaspis spp., Toxoptera spp., Trialeurodes spp., Trioza spp., Typhlocyba spp., Unaspis spp., Viteus vitifolii, Zygina spp.

[0405] Still other examples are from the order of the Hymenoptera, for example, Acromyrmex spp., Athalia spp., Atta spp., Diprion spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis, Solenopsis invicta, Tapinoma spp., Vespa spp. Still other examples are from the order of the Isopoda, for example, Armadillidium vulgare, Oniscus asellus, Porcellio scaber.

[0406] Suitably the insect may be selected from the order Blattodea, for example B. germanica, Blatta orientalis, Periplaneta America, Periplaneta spp., Supella longipalpa and Supella longipalpa; also those of the family termitidae.

[0407] Still other examples are from the order of the Isoptera, for example, Coptotermes spp., Cornitermes cumulans, Cryptotermes spp., Incisitermes spp., Microtermes obesi, Odontotermes spp., Reticulitermes spp.

[0408] Suitably the insect may be selected from the order of the Lepidoptera, for example, Acronicta major, Adoxophyes spp., Aedia leucomelas, Agrotis spp., Alabama spp., Amyelois transitella, Anarsia spp., Anticarsia spp., Argyroploce spp., Barathra brassicae, Borbo cinnara, Bucculatrix thurberiella, Bupalus piniarius, Busseola spp., Cacoecia spp., Caloptilia theivora, Capua reticulana, Carpocapsa pomonella, Carposina niponensis, Chematobia brumata, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocerus spp., Cnephasia spp., Conopomorpha spp., Conotrachelus spp., Copitarsia spp., Cydia spp., Dalaca noctuides, Diaphania spp., Diatraea saccharalis, Earias spp., Ecdytolopha aurantium, Elasmopalpus lignosellus, Eldana saccharina, Ephestia spp., Epinotia spp., Epiphyas postvittana, Etiella spp., Eulia spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Feltia spp., Galleria mellonella, Gracillaria spp., Grapholitha spp., Hedylepta spp., Helicoverpa spp., Heliothis spp., Hofmannophila pseudospretella, Homoeosoma spp., Homona spp., Hyponomeuta padella, Kakivoria flavofasciata, Laphygma spp., Laspeyresia molesta, Leucinodes orbonalis, Leucoptera spp., Lithocolletis spp., Lithophane antennata, Lobesia spp., Loxagrotis albicosta, Lymantria spp., Lyonetia spp., Malacosoma neustria, Maruca testulalis, Mamestra brassicae, Mods spp., Mythimna separata, Nymphula spp., Oiketicus spp., Oria spp., Orthaga spp., Ostrinia spp., Oulema oryzae, Panolis flammea, Parnara spp., Pectinophora spp., Perileucoptera spp., Phthorimaea spp., Phyllocnistis citrella, Phyllonorycter spp., Pieris spp., Platynota stultana, Plodia interpunctella, Plusia spp., Plutella xylostella, Prays spp., Prodenia spp., Protoparce spp., Pseudaletia spp., Pseudoplusia includens, Pyrausta nubilalis, Rachiplusia nu, Schoenobius spp., Scirpophaga spp., Scotia segetum, Sesamia spp., Sparganothis spp., Spodoptera frugiperda, Spodoptera spp., Stathmopoda spp., Stomopteryx subsecivella, Synanthedon spp., Tecia solanivora, Thermesia gemmatalis, Tinea pellionella, Tineola bisselliella, Tortrix spp., Trichophaga tapetzella, Trichoplusia spp., Tuta absoluta, Virachola spp.

[0409] Still other examples are from the order of the Orthoptera, for example, Acheta domesticus, Dichroplus spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Melanoplus spp., Pulex irritans, Schistocerca gregaria. Still other examples are from the order of the Siphonaptera, for example, Ceratophyllus spp., Ctenocephalides spp., Tunga penetrans, Xenopsylla cheopis. Still other examples are from the order of the Symphyla, for example, Scutigerella spp. Still other examples are from the order of the Thysanoptera, for example, Anaphothrips obscurus, Baliothrips biformis, Drepanothris reuteri, Enneothrips flavens, Frankliniella spp., Heliothrips spp., Hercinothrips femoralis, Rhipiphorothrips cruentatus, Scirtothrips spp., Taeniothrips cardamoni, Thrips spp. Still other examples are from the order of the Zygentoma (=Thysanura), for example, Lepisma saccharina, Thermobia domestica.

[0410] Suitably the insect may be selected from the order Hemiptera, for example: Aphidoidea (aphid superfamily), Aleyrodoidea (whiteflies), Coccoidea (scale insects), Phylloxeroidea (including Phylloxeridae or “phylloxerans”, and Adelgidae or woolly conifer aphids) or Psylloidea (jumping plant lice etc.). The aphids may be part of the family Aphididae, which contains sub-families Aiceoninae, Anoeciinae, Aphidinae, Baltichaitophorinae, Calaphidinae, Chaitophorinae, Drepanosiphinae, Eriosomatinae, Greenideinae, Hormaphidinae, Israelaphidinae, Lachninae, Lizeriinae, Macropodaphidinae, Mindarinae, Neophyllaphidinae, Phloeomyzinae, Phyllaphidinae, Pterastheniinae, Saltusaphidinae, Spicaphidinae, Taiwanaphidinae, Tamaliinae and Thelaxinae. The aphids may, for example, be of the genus Acyrthosiphon (e.g. Acyrthosiphon pisum), Aphis (e.g. Aphis gossypii, Aphis glycines), Diuraphis (e.g. Diuraphis noxia) Macrosiphum (e.g. Macrosiphum rosae, Macrosiphum euphorbiae), Myzus (e.g. Myzus persicae), Rhopalosiphum (e.g. Rhopalosiphum padi), or Sitobion (e.g. Sitobion avenae). The insects may be of the Aleyrodidae family, e.g. of the genus Bemisia (e.g. Bemisia tabaci) or Trialeurodes (e.g. Trialeurodes vaporariorum). The insects may be of the Psylloidea family, e.g. of the genus Pachypsylla (e.g. Pachypsylla venusta).As examples of hemipteran insects outside the sub-order Sternorryncha, the insects may be of the Cimicidae family, e.g. of the genus Cimex (bed bugs), e.g. Cimex lectularius. The insects may be of the Cicadellidae family, e.g. of the genus Cuerna (e.g. Cuerna arida), Graminella (e.g. Graminella nigrifrons) or Homalodisca (e.g. Homalodisca vitripennis). Also included in the Cicadellidae family are Amrasca biguttula. The insects may be part of the Delphacidae family, e.g. of the genus Nilaparvata (e.g. Nilaparvata lugens) or Sogatella (e.g. Sogatella furcifera). The insects may be of the Liviidae family, e.g. of the genus Diaphorina (e.g. Diaphorina citri). The insects may be part of the Miridae family, e.g. of the genus Pseudatomoscelis (e.g. Pseudatomoscelis seriatus), Lygus (e.g. Lygus hesperus) or Tupiocoris (e.g. Tupiocoris notatus). The insects may be of the Pentatomidae family, e.g. of the genus Acrosternum (e.g. Acrosternum hilare), Banasa (e.g. Banasa dimiata), Euschistus (e.g. Euschistus servus, Euschistus heroes), Halyomorpha(e.g. Halyomorpha halys), Murgantia (e.g. Murgantia histrionica), Nezara (e.g. Nezara viridula), Plautia (e.g. Plautia stali), or Podisus (e.g. Podisus maculiventris). The insects may be of the Pyrrhocoridae family, e.g. of the genus Pyrrhocoris (e.g. Pyrrhocoris apterus). The insects may be of the Reduviidae family, e.g. of the genus Rhodnius (e.g. Rhodnius prolixus), or Triatoma (e.g. Triatoma infestans). The insects may be of the Triozidae family, e.g. of the genus Acanthocasuarina (e.g. Acanthocasuarina muellerianae).

[0411] Suitably the insect may be selected from the order Coleoptera, for example: Carabidae spp, Cerambycidae spp, Coccinellidae spp., Curculionidae spp., Scarabaeidae spp., Tenebrionidae spp., Buprestidae spp., Chrysomelidae spp., Lampyridae spp., Bruchidae spp., Dermestidae spp., Elateridae spp., Scolytidae spp., Bostrichidae spp., Anobidae spp., Nitidulidae spp., Ptinidae spp., Silvanidae spp., Trogossitidae spp, and Laemophloeidae spp.

[0412] In one embodiment, the insect is selected from: Myzus persicae, Aphis fabae, Aphis gossypii, Acrythsiphon pisum, Amrasca biguttula, Rhopalosiphum padi, Nilaparvata lugens, Drosophila suzukii, Drosophila melanogaster, Blattella germanica, Plutella xylostella, Halyomorpha halys, Tribolium castaneum, and Spodoptera frugiperda. Household Pests

[0413] The insecticidal neuropeptides, or compositions thereof described herein may also have activity against domestic pests, such as cockroaches and termites (such as those of the family Termitidae). Of more than 3000 species, these may include the German cockroach (Blattella germanica), the Oriental cockroach (Blatta orientalis), the American cockroach (Periplaneta americana) and the brown banded cockroach (Supella longipalpa). Cockroaches are common domestic pests worldwide, and may carry various diseases. Control of these and other domestic pests (e.g. ants) is envisaged by treating surfaces where the insects run, but particularly with food baits containing the peptides and compositions thereof described herein, and with spray application of the peptides and compositions thereof described herein.

[0414] Therefore further aspects of the invention may include a bait comprising one or more insecticidal neuropeptides or compositions thereof as described herein, suitably which may be a bait for domestic pests. Further aspects of the invention may include a method of controlling domestic pests, reducing domestic pest populations, inhibiting domestic pest populations, increasing domestic pest mortality, the method comprising treating a surface (e.g. wood) which is contacted by the domestic pest with an insecticidal neuropeptide or composition thereof of the invention, or contacting the domestic pest with an insecticidal neuropeptide or composition thereof of the invention. Optionally treating or contacting may comprise a suitable means of application as described elsewhere herein, such as by spraying. Suitably therefore a sprayable formulation comprising an insecticidal neuropeptide or composition thereof of the invention is also envisaged. Suitable formulations are described elsewhere herein.

[0415] The invention will now be described with reference to the following figures and non- limiting examples. FIGURES Figure 1 shows a schematic representation of the single short peptide genetic construct SB-P-65 and how the final expression cassette of the peptide will appear after successful insertion into the pPinkα- HC plasmid. Figure 2 shows schematic representations of the single short peptide genetic constructs: SB-P-65 (M. persicae leucokinin), SB-P-65KR (SB-P-65 lacking amino acids ‘EAEA’ [SEQ ID NO: 254]), SB-P- 66 (M. persicae leucokinin), SB-P-47 (M. persicae pyrokinin) and SB-P-45 (M. persicae Capa peptide). Schematic features the: recognition sequence for restriction enzyme MlyI (R1 with vertical dashed line), ‘EAEA’ (SEQ ID NO: 254) amino acids to be removed by enzyme STE13 (grey rectangle), coding sequence for peptide, double stop codon for translational termination (hexagon) and recognition sequence for restriction enzyme FseI (R2 with vertical dashed line). Figure 3 shows schematic representations of pPinkα-HC-SB-P-65. A) Conceptual representation of pPinkα-HC-SB-P-65 around the insertion site featuring the: Mating Factor Alpha secretion signal sequence (MFα1s), KR and EAEA (SEQ ID NO: 254) amino acids comprising Kex2 recognition cut sites (KR↓ EAEA [SEQ ID NO: 59], marked with vertical dashed line), site / s hypothesised to be removed by enzyme STE13, coding sequence for SB-P-65, double stop codon for translational termination (Red hexagon), restored recognition sequence for restriction enzyme FseI (vertical dashed line) and transcriptional terminator (CycT). The diagram does not feature the AOX1 promoter, which is upstream from the MFα1s site. B) Conceptual representation of pPinkα-HC-SB-P-65KR. The difference between of pPinkα-HC-SB-P-65 and pPinkα-HC-SB-P-65KR is the absence of the Ste13 EAEA (SEQ ID NO: 254) recognition cut site. C) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-65 insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 4 shows schematic representations of pPinkα-HC-SB-P-45. A) Conceptual representation of pPinkα-HC-SB-P-45 around the insertion site featuring the: Mating Factor Alpha secretion signal sequence (MFα1s), KR and EAEA (SEQ ID NO: 254) amino acids comprising the Kex2 recognition cut site (KR↓ EAEA [SEQ ID NO: 59], marked with vertical dashed line), site hypothesised to be removed by enzyme STE13, coding sequence for SB-P-65, double stop codon for translational termination (hexagon), restored recognition sequence for restriction enzyme FseI (vertical dashed line) and transcriptional terminator (CycT). The diagram does not feature the AOX1 promoter, which is upstream from the MFα1s site. B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-45 insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 5 shows schematic representations of pPinkα-HC-SB-P-47. A) Conceptual representation of pPinkα-HC-SB-P-47 around the insertion site featuring the: Mating Factor Alpha secretion signal sequence (MFα1s), KR and EAEA (SEQ ID NO: 254) amino acids comprising the Kex2 recognition cut site (KR↓ EAEA [SEQ ID NO: 59], marked with vertical dashed line), site hypothesised to be removed by enzyme STE13, coding sequence for SB-P-47, double stop codon for translational termination (Red hexagon), restored recognition sequence for restriction enzyme FseI (vertical dashed line) and transcriptional terminator (CycT). The diagram does not feature the AOX1 promoter, which is upstream from the MFα1s site. B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-47 insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 6 shows schematic representations of pPinkα-HC-SB-P-66. A) Conceptual representation of pPinkα-HC-SB-P-66 around the insertion site featuring the: Mating Factor Alpha secretion signal sequence (MFα1s), KR and EAEA (SEQ ID NO: 254) amino acids comprising the Kex2 recognition cut site (KR↓ EAEA [SEQ ID NO: 59], marked with vertical dashed line), site hypothesised to be removed by enzyme STE13, coding sequence for SB-P-66, double stop codon for translational termination (hexagon), restored recognition sequence for restriction enzyme FseI (vertical dashed line) and transcriptional terminator (CycT). The diagram does not feature the AOX1 promoter, which is upstream from the MFα1s site. B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-66 insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 7 shows schematic representations of pPinkα-HC-SB-P-65x3. A) Conceptual representation of pPinkα-HC-SB-P-65x3 around the insertion site featuring the: Mating Factor Alpha secretion signal sequence (MFα1s), KR and EAEA (SEQ ID NO: 254) amino acids comprising Kex2 recognition cut sites (KR↓ EAEA [SEQ ID NO: 59], marked with vertical dashed line), site / s hypothesised to be removed by enzyme STE13 (grey rectangle), coding sequence for SB-P-65, double stop codon for translational termination (hexagon), restored recognition sequence for restriction enzyme FseI (vertical dashed line) and transcriptional terminator (CycT). The diagram does not feature the AOX1 promoter, which is upstream from the MFα1s site. B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-65x3 insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 8 shows a conceptual representation of pPinkα-HC-SB-P-65x6 featuring the: Mating FactorAlpha secretion signal sequence (MFα 1s), KR and EAEA (SEQ ID NO: 254) amino acidscomprising Kex2 recognition cut sites (KR↓ EAEA [SEQ ID NO: 59], marked with vertical dashed line), site hypothesised to be removed by enzyme STE13, coding sequence for SB-P-65 , double stop codon for translational termination (hexagon), restored recognition sequence for restriction enzyme FseI (vertical dashed line) and transcriptional terminator (CycT). The diagram does not feature the AOX1 promoter, which is upstream from the MFα1s site. Figure 9 shows schematic representations of pPinkα-HC-MultiMyzus. A) Conceptual representation of pPinkα-HC-MultiMyzus featuring the: Mating Factor Alpha secretion signal sequence (MFα1s), KR and EAEA (SEQ ID NO: 254) amino acids comprising Kex2 recognition cut sites (KR↓ EAEA [SEQ ID NO: 59], marked with vertical dashed line), site / s hypothesised to be removed by enzyme STE13, coding sequence for: SB-P-65, SB-P-47 (green rectangle), SB- P-65 and double stop codon for translational termination (hexagon), restored recognition sequence for restriction enzyme FseI (vertical dashed line) and transcriptional terminator (CycT). The diagram does not feature the AOX1 promoter, which is upstream from the MFα1s site. B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-MultiMyzus insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 10 shows a conceptual representation of pPinkα-HC-Prepropeptide featuring the: Mating Factor Alpha secretion signal sequence (MFα1s), Myzus persicae prepropeptide DNA sequence highlighting DNA sequences of peptides SB-P-65 and SB-P-66, native KR amino acids comprising Kex2 recognition cut sites (KR↓), double stop codon for translational termination (hexagon), a restored recognition sequence for restriction enzyme FseI (vertical dashed line) and transcriptional terminator (CycT). The diagram does not feature the AOX1 promoter, which is upstream from the MFα1s site. Figure 11 shows depictions of DNA sequence changes to SB-P-65 (A) and SB-P-66 (B) to yield peptides with C-terminal cysteines. Figure 12 shows (A) a schematic representation of pPinkα-HC-SB-P-65GC. (B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-65GC insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 13 shows (A) schematic representations of pPinkα-HC-SB-P-66GC. (B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-66GC insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 14 shows (A) schematic representations of pPinkα-HC-SB-P-66GCx3. (B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-66GCx3 insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 15 shows (A) schematic representations of pPinkα-HC-SB-P-66GCx6. (B) Image of SnapGene detailing the DNA sequence of the pPinkα-HC-SB-P-66GCx6 insertion site. Highlighted in blue is the expected product of a PCR using the 5’Alpha-FactorF and 3’Cyc1TermR primer pair. Figure 16 shows the construct design for expressing neuropeptides as C-terminal eYFP fusion proteins separated by Kex2 / Ste13 protease recognition sites. Fluorescent reporters are cloned between Mly1 and Stu1 and Kex2 / Ste13-peptide sequences are cloned between Stu1 and Fse1. Figure 17 shows the relative fluorescent values for culture supernatants harvests from induced growth of candidate producer strains. eYFP6 is a positive control and demonstrates fluorescence of ~45 AU, eYFP-ENTK-MF expressing strains show a range of fluorescence between 5-10 AU. eYFP-47C expressing strains show varied fluorescence with eYFP-47C 18 showing the highest level of protein secretion with a fluorescence measured at ~35 AU. Controls of BMMY media and H2O were used as negative controls. eYFP 6 spiked with MM and SB-P-44 peptides was used as a positive control. Figure 18 shows coomassie stained SDS gels show proteins separated from culture supernatants of strains expressing eYFP and eYFP-peptide fusion constructs. A clear eYFP band is observed at the expected size in culture supernatants of eYFP expressing strains. eYFP-66GC expressing strains show a doublet corresponding to cut and uncut fusion protein. The sizes of the band correspond to what we expect for cut eYFP at ~28500 Da and eYFP-66GC at ~29736 Da. Culture supernatants from strains expressing eYFP-44 show 2 bands corresponding to the expected sizes for cut and uncut fusion protein with a fusion protein band observed with ~34000 Da. Figure 19 shows the construct design for expressing multiple peptides in tandem with YFP, for native cleavage with Kex2 and Ste13. Figure 20 shows culture supernatant and cell lysates of cultures expressing eYFP and eYFP-peptide fusions. A single eYFP band is observed in extracts from cultures expressing an eYFP gene. A doublet is visible in cell cultures supernatants from strains expressing eYFP-47C, 1 band corresponds with cut eYFP and the second with intact eYFP-47C. Culture supernatants from cells expressing eYFP-MF show 4 bands corresponding to eYFP-peptide fusions which have been cleaved at each of the 3 available cut sites. Culture supernatants (S) are compared to cell pellet protein extracts (P), comparison of these samples confirms successful secretion of our target proteins. Figure 21 shows the construct design encoding for recombinant peptide synthesis with in vivo C- terminal amidation through the action of PAM enzymes. Kex2 / Ste13 cut sites between peptide encoding sequences result in peptide cleavage from the fusion protein. Figure 22 shows the activity of the rat PAM domains. The C-terminal peptidyglycine is α- hydroxylated by PHM, this reaction is dependent on ascorbate. Subsequently, the α-hydroxylated glycine has a glyoxylate cleaved from it, leaving the amidated peptide N-terminus. Figure from Prigge et al, 1999. Figure 23 shows SDS gel electrophoresis analysis of purified rat PAM and rat PAM-MM peptide fusions. Control lanes labelled RAT PAM and RAT PAM B shows SDS analysis of recombinant purified rat PAM enzyme. Comparison of rat PAM-MM fusions to the control lanes demonstrates that the PAM-peptide fusions are larger proteins suggesting the existence of fusion proteins carrying the neuropeptide sequences. Figure 24 shows LC-MS traces depicting parent SB-P-181 (▼), intermediate amidated SB-P-181 with a hydroxylated glycine (■) and the amidated product (*). Figure 25 shows the rate of SB-P-181 amidation measured through HPLC. After a 45-minute incubation at 37 °C the majority of SB-P-181 has been altered to have an alpha-hydroxylated C- terminal glycine. After 60 minutes the majority of this has been converted to C-terminally amidated SB-P-181 with full conversion occurring by 90 minutes. Figure 26 shows LC-MS spectra demonstrating the amidation of SB-P-181 in minimal media after a 2-hour incubation at 37 °C. The parent peptide is detected with a mass of 1254.6 Da. The intermediate containing the α-hydroxylated C-terminal glycine is detected with a mass of 1269.6 Da, as is the target amidated product with a mass of 1195.6 Da. Figure 27 shows a schematic demonstration of the proposed workflow for using altered and unaltered inteins to facilitate recovery and amidation of recombinant peptides from culture supernatant. Figure 28 shows the schematic details of the genetic design for the expression and secretion of two different recombinant neuropeptides with in vivo cleavage. SS represents a secretion signal to induce movement of the peptides to the extracellular environment. All proteolytic steps occur within the Pichia host, relying on Kex2, followed by Kex1 and Ste13 activity to allow for peptide separation within the secretory system of Pichia before secretion as separate peptides. Precursor peptides are generated originally with either a Glycine or Cysteine amidation signal at the C-terminus of the peptides. Glycine ended peptides can be amidated using a PAM enzyme while Cysteine ended peptides can be amidated using photochemical amidation techniques. Figure 29 shows the schematic details of the genetic construct design for the expression and secretion of two different recombinant neuropeptides which utilizes in vitro cleavage. SS represents a secretion signal to induce movement of the pre-pro-peptide concatemer to the extracellular environment. All proteolytic steps occur outside the Pichia host, relying on Kex2 supplied in vitro, followed by two other enzymes able to replicate Kex1 and Ste13 activity to allow for peptide separation outside of the cell. Precursor peptides are generated originally with either a Glycine or Cysteine amidation signal at the C-terminus of the peptides. Glycine ended peptides can be amidated using a PAM enzyme while Cysteine ended peptides can be amidated using photochemical amidation techniques. Figure 30 shows an expression and purification / amidation method for recovering recombinant neuropeptides. The integrated DNA construct encoding the pre-pro-peptide is expressed within the cell and the translated pre-pro-peptide is targeted for secretion. From the culture supernatant the pre- pro-peptide is isolated using affinity purification, an example is shown using chitin resin binding to a chitin binding domain (SEQ ID NO: 300). The target peptide is cleaved from the intein and amidated through the addition of a reducing agent and ammonium sulphate / carbonate. This liberates the amidated peptide while leaving the intein-CBD domain bound to the chitin resin. The amidated peptide can then be eluted from the resin and harvested directly. Suitable examples of such expression constructs are also shown in SEQ ID NOs: 301-305) Figure 31 shows an expression and purification / amidation method for recovering recombinant neuropeptides. The integrated DNA is expressed within the cell and the translated pre-pro-peptide is targeted for secretion. From the culture supernatant the pre-pro-peptide is isolated using affinity purification, an example is shown using chitin resin binding to a chitin binding domain (SEQ ID NO: 300). The target peptide is cleaved from the intein and amidated through the addition of a reducing agent and ammonium sulphate / carbonate. The amidated product is liberated from the eYFP fusion through treatment with factor Xa. The amidated peptide can then be purified through size exclusion chromatography. Figure 32 shows the generic design for the secreted production of concatemer pre-pro-peptides destined for intein mediated C-terminal amidation. The indicated monomer can be repeated indefinitely, allowing for numerous peptide ORFs to be expressed from a single gene. Upon expression the fusion protein is recovered on chitin resin through affinity capture to its chitin binding domain (CBD) (SEQ ID NO: 300). From here intein cleavage can be induced through the addition of a reducing agent and ammonium sulphate / carbonate to amidate the C-terminus of encoded peptides. A subsequent digestion with factor Xa liberates the peptides from upstream concatemer inteins. Suitable examples of such expression constructs are also shown in SEQ ID NO: 306) Figure 33 shows schematics detailing the generic construct design options for the expression of recombinant, C-terminally amidated peptides. The dotted line beneath the schematic represents the translated pre-pro-peptide and notes any amino acid residues of particular relevance to the design. ‘X’ represents any amino acid. SS represents a secretion signal to induce movement of the recombinant fusion protein to the extracellular environment. An 8xHis-tagged eYFP with amino acid linker sequence (SEQ ID NOs 274, 275 and 276) is placed at the N-terminus of each construct to facilitate identification of highly expressing strains. Each construct is completed with a duo of translational stop codons (SEQ ID NO:277) followed by a transcriptional terminator. A) Schematic of a DNA construct encoding for a pre-pro-peptide containing a single mature insect neuropeptide sequence delineated by a Cysteine residue that will be the eventual site of downstream peptide cleavage (see SEQ ID NOs 278-284). B) Schematic of a DNA construct encoding for a pre-pro-peptide containing tandem 1+…+n insect neuropeptide sequences delineated by a Cysteine residue that will be several sites of downstream peptide cleavage (see SEQ ID NOs: 285 and 286). C) Schematic of a DNA construct encoding for a pre-pro-peptide containing tandem 1+…+n insect neuropeptide sequences delineated by a stuffer sequence comprising a Cysteine residue and ending with an example of a Factor Xa Protease recognition sequence (IDGR), both of which will be sites of downstream peptide cleavage (see SEQ ID NOs:292 and 293). D) Schematic of a DNA construct encoding for a pre-pro-peptide containing tandem 1+…+n insect neuropeptide sequences delineated by a stuffer sequence comprising a Cysteine residue and ending with a Methionine residue, both of which will be sites of downstream peptide cleavage (see SEQ ID NO: 297). Figure 34 shows a flowchart schematic detailing the chemical peptide cleavage and purification / amidation method for recovering recombinant secreted neuropeptides from the Cysteine only singlet peptide DNA construct (Fig.33A). Notably, the peptide sequence cannot encode Cysteine resides. In a suitable approach, the translated and secreted pre-pro-peptide has a terminal Cysteine residue at the end of the target mature peptide amino acid sequence (see SEQ ID NOs: 278-284) . Adapting the methodology of Mollner et al. (2022) Reductive site-selective atypical C,Z-type / N2-C2 cleavage allows C-terminal protein amidation | Science Advances the Cysteine residues of the pre- pro-peptide in the culture supernatant are first converted to Dehydroalanine (DHA) residues through reaction with alkylating agent 2,5-dibromohexanediamide (DBHDA). In one potential approach, the DHA pre-pro-peptide is then bound to a Ni-NTA (nickel-nitrilotriacetic acid) resin via the 8x Histidine tag. Finally, the N-terminally active amidated insect neuropeptide is liberated from the resin bound pre-pro-peptide and eluted through a cleavage reaction with Tetrahydroxydiboron (B2(OH)4) and ascorbate. Eluted insect neuropeptides can then be further purified to a final product through a range of potential chromatographic techniques. Figure 35 shows a flowchart schematic detailing the chemical peptide cleavage and purification / amidation method for recovering recombinant secreted neuropeptides from the Cysteine only tandem peptide DNA construct (Fig. 33B). Notably, this DNA construct features two peptides per pre-pro-peptide, but the design could be expanded theoretically to number n peptides (see SEQ ID NOs: 285 and 286). Additionally, the peptide sequences cannot encode Cysteine resides. In a suitable approach, the translated and secreted pre-pro-peptide has terminal Cysteine residues at the end of the target mature peptide amino acid sequences. Adapting the methodology of Mollner et al. (2022) Reductive site-selective atypical C,Z-type / N2-C2 cleavage allows C-terminal protein amidation | Science Advances, the Cysteine residues of the pre-pro-peptide in the culture supernatant are first converted to Dehydroalanine (DHA) residues through reaction with alkylating agent 2,5- dibromohexanediamide (DBHDA). In one potential approach, the DHA pre-pro-peptide is then bound to a Ni-NTA (nickel-nitrilotriacetic acid) resin via the 8x Histidine tag. Finally, the N-terminally amidated active insect neuropeptides are liberated from the resin bound fusion protein and eluted through a cleavage reaction with Tetrahydroxydiboron (B2(OH)4) and ascorbate. Notably, the first peptide in the DNA sequence will be native sequence identical, whereas the 2nd-> nth peptides will have a lactic derivative group on the C-terminus of the N-terminally amidated peptide. Eluted insect neuropeptides can then be further purified to a final product through a range of potential chromatographic techniques. Figure 36 shows a flowchart schematic detailing the chemical peptide cleavage and purification / amidation method for recovering recombinant secreted neuropeptides from the Cysteine and Factor-Xa Protease tandem peptide DNA construct (Fig. 33C). Notably, this DNA construct features two peptides per pre-pro-peptide, but the design could be expanded theoretically to number n peptides. Additionally, the peptide sequence cannot encode Cysteine resides. In a suitable approach, the translated and secreted pre-pro-peptide has a stuffer sequence ‘X’ between peptide amino acid sequences featuring the same terminal Cysteine residues at the end of the target mature peptide as above, with an additional amino acid sequence which acts as recognition cleavage site sequence for a protease enzyme (See SEQ ID NOs: 292 and 293). In this embodiment, this is represented by a recognition sequence for the protease Factor Xa (Ile-Glu / Asp-Gly-Arg) (SEQ ID NO:269) see also SEQ ID NOs: 287-291. In one potential approach, the pre-pro-peptide is first bound to a Ni-NTA (nickel-nitrilotriacetic acid) resin via the 8x Histidine tag. The peptides with stuffer sequences are then liberated from the resin bound fusion protein through cleavage at the IDGR amino acid recognition sequence. Adapting the methodology of Mollner et al. (2022) Reductive site-selective atypical C,Z-type / N2-C2 cleavage allows C-terminal protein amidation | Science Advances, the Cysteine residues of the liberated precursor peptides are then converted to Dehydroalanine (DHA) residues through reaction with alkylating agent 2,5-dibromohexanediamide (DBHDA). Finally, the N-terminally amidated active insect neuropeptides are generated through a cleavage reaction with Tetrahydroxydiboron (B2(OH)4) and ascorbate. Eluted insect neuropeptides can then be further purified from the residual stuffer amino acid sequence to a final product through a range of potential chromatographic techniques. Figure 37 shows a flowchart schematic detailing the chemical peptide cleavage and purification / amidation method for recovering recombinant secreted neuropeptides from the Cysteine and Methionine tandem peptide DNA construct (Fig.33D). Notably, this DNA construct features two peptides per pre-pro-peptide, but the design could be expanded theoretically to number n peptides (see SEQ ID NO: 297). Additionally, the peptide sequence cannot encode Cysteine or Methionine resides. In a suitable approach, the translated and secreted pre-pro-peptide has a stuffer sequence ‘XX’ between peptide amino acid sequences featuring the same terminal Cysteine residues at the end of the target mature peptide as above, and a Methionine residue at the start of the next peptide to be cleaved by CNBr (See SEQ ID NOs: 294-296). In one potential approach, the pre-pro-peptide is first bound to a Ni-NTA (nickel-nitrilotriacetic acid) resin via the 8x Histidine tag. The precursor peptides with stuffer sequences are then liberated from the resin bound fusion protein through a cleavage reaction with CNBr. Adapting the methodology of Mollner et al. (2022) Reductive site-selective atypical C,Z- type / N2-C2 cleavage allows C-terminal protein amidation | Science Advances, the Cysteine residues of the liberated precursor peptides are then converted to Dehydroalanine (DHA) residues through reaction with alkylating agent 2,5-dibromohexanediamide (DBHDA). Finally, the N-terminally amidated active insect neuropeptides are generated through a cleavage reaction with Tetrahydroxydiboron (B2(OH)4) and ascorbate. Eluted insect neuropeptides can then be further purified from the residual stuffer amino acid sequence to a final product through a range of potential chromatographic techniques. Figure 38 shows samples eluted from chitin resin were ran alongside a synthetic SB-P-44 to verify that the eluted peptide was the same size as our expected product. Peptide was successfully eluted from CBD resin using a 50 mM DTT cleavage buffer and a 1M (NH4)2CO3, 50 mM DTT cleavage buffer designed to amidate the C-terminus of the cleaved peptide. A band identifying our product as the same size as the synthetic SB-P-44 is labelled with an arrow. Figure 39 - Schematic representation of peptide ‘SB-P-281’. The schematic details that SB-P-281 is a composite peptide comprised of SEQ ID NO: 143 (SB-P-86) connected via a non-native Cysteine residue to peptide SEQ ID NO: 207 (SB-P-47) with an additional non-native C-terminally amidated Cysteine amino acid (new SEQ ID NO: 318). Figure 40 - Image of Novex™ 10 to 20% Tricine Mini Protein gel with control sample results of the modified peptide cleavage process at the end of each of the three process stages (A-C in method). Lanes 2-4 represent the control sample of SB-P-281 in S2 supernatant which received all reagents apart from DBHDA. Lanes 5-7 represent the control sample of S2 supernatant with no SB-P-281. Lane 8 represents a blank well to delineate the samples from the standards. Lanes 9 and 10 represent the respective standards of SEQ ID NO: 143 (SB-P-86) and peptide SEQ ID NO: 207 (SB-P-47). Protein ladders are run in Lanes 1 and 11 with associate Kilodalton (kDa) sizes are printed alongside the image. Estimated peptide molecular weights (kDa): SB-P-281 (2.650), SB-P-86 (1.065) and SB- P-47 (1.361). Figure 41 - Image of Novex™ 10 to 20% Tricine Mini Protein gel with sample results of the modified peptide cleavage process at the end of each of the three process stages (A-C in method). Lanes 2-4 represent the sample of SB-P-281 reacted in S2 supernatant. Lanes 5-7 represent the sample of SB-P- 281 reacted in S2 Supernatant buffered with 100 mM NaPi (pH 8). Lanes 8-10 represent the sample of SB-P-281 reacted in 100 mM NaPi (pH 8) buffer. Lane 11 represents a blank well to delineate the samples from the standards. Lanes 12 and 13 represent the respective standards of SEQ ID NO: 143 (SB-P-86) and peptide SEQ ID NO: 207 (SB-P-47). Protein ladders are run in Lanes 1 and 14 with associate Kilodalton (kDa) sizes are printed alongside the image. Estimated peptide molecular weights (kDa): SB-P-281 (2.650), SB-P-86 (1.065) and SB-P-47 (1.361). Figure 42 - Representative mass spectra and ion mass-to-charge tables demonstrating the detection of SB-P-281 and Pyr-SB-P-281 after solubilisation and DTT treatment in the reaction buffers: S2 Supernatant (A), S2 Supernatant with 100 mM NaPi receiving all components (B), 100 mM NaPi (C) and S2 Supernatant with 100 mM NaPi receiving all components except DBHDA (D). Asterisks (*) identify detected mass-to-charge peaks concordant with the anticipated masses of the two tabulated variants of SB-P-281. Figure 43 - Representative mass spectra and ion mass-to-charge tables demonstrating the detection of SB-P-281,Pyr-SB-P-281 and Dha derivatives thereof after incubation with 50 mM DBHDA in the reaction buffers: S2 Supernatant (A), S2 Supernatant with 100 mM NaPi receiving all components (B), 100 mM NaPi (C) and S2 Supernatant with 100 mM NaPi receiving all components except DBHDA (D). No ions reflecting and variant of SB-P-281 were observed in the sample reacted with S2 Supernatant with 100 mM NaPi receiving all components (B), Asterisks (*) identify detected mass- to-charge peaks concordant with the anticipated masses of the six tabulated variants of SB-P-281. Figure 44 - Representative mass spectra and ion mass-to-charge tables demonstrating the detection of SB-P-86, Pyr-SB-P-86, Na+Pyr-SB-P-86 sodium adduct and Pyr-SB-P-281 after incubation with 100 mM B2(OH)4and 25 mM Sodium Ascorbate in the reaction buffers: S2 Supernatant (A), S2 Supernatant with 100 mM NaPi receiving all components (B), 100 mM NaPi (C) and S2 Supernatant with 100 mM NaPi receiving all components except DBHDA (D). Asterisks (*) identify detected mass-to-charge peaks concordant with the anticipated masses of the six tabulated variants of SB-P- 281. Figure 45 shows the different DNA designs for expression of recombinant peptide designed to be purified with a CBD affinity tag and amidated through intein cleavage in the presence of ammonium. Above is shown the construct design within hosts which are unable to secrete recombinant proteins. These fusion proteins must be encoded with an N-terminal methionine (M) as this is required to initiate protein translation. Below shows the fusion protein design when expression occurs in hosts capable of secreting recombinant protein through an N-terminal secretion signal. This is cleaved in secretory vesicles allowing the target peptide to have any sequence once recovered from culture supernatant. Figure 46 shows the induction of Met-51-intein-CBD in E. coli following addition of 4mM IPTG. Samples show that after 45 minutes protein production was strongly induced, with progressively higher levels of protein being measured over the course of the induction. Figure 47 - The gel shows the fractions collected during pulldown, cleavage and elution of recombinant M51 from E. coli. The first lane contains a chemically synthesized peptide 51 standard. In subsequent lanes we loaded the clarified lysate, flowthrough, wash, cleavage flush and elutions of our peptide. Figure 48 - Shown is an LC-MS trace of M51 elution 1. Peaks associated with different ions of M51 are indicated with an asterisk. The sample was loaded onto the mass analyser through a C18 column. Figure 49 - cAMP GPCR cell-based assay output – A, cAMP Standard curve using a 6-point 2x- dilution series starting at 50 nM. Each point represents n=2. B, Insect DH44 Receptor cell line tested against Forskolin using a 6-point 2x-dilution series starting at 100 µM in 384 well format. Each point represents n=2. C, Insect DH44 Receptor cell line tested against two positive control peptides and two P-44 containing samples using a 6-point 2x-dilution series starting at 5 µM in 384 well format. Each point represents n=1. Final DMSO concentration in assay is 1% (v / v) in Induction buffer (1x HBSS buffer pH 7.4, 500 µM IBMX). Figure 50 - Shown are the fractions of affinity capture of culture supernatant from Pichia pastoris expressing peptide 51-P. abyssi pol II intein-CBD domain after a 48-hour induction. A band of equivalent size to a synthetic 51 standard is visible in an elution following cleavage at 55°C. Figure 51 - Shown is a Coomassie stained gel containing fractions from a pulldown of peptide-intein fusion containing culture supernatant. Prior to intein cleavage the resin was split into 2 columns, one was cleaved at room temperature (RT) and the other at 55°C. A peptide band comigrates alongside the synthetic SB-P-51 standard only in the elution from the 55°C cleavage. Very little protein is visible in the lane run from the room temperature column, indicating the stability of the P. abyssi DNA pol II intein at this temperature. Figure 52 - HPLC chromatogram of an elution of SB-P-51. Our target peptide is the peak at 7.284 minutes. Comparison to a calibration curve identified a peptide concentration of 11.806g / L in the above sample. Figure 53 - Schematic details an insect neuropeptide pro-drug design unit concept which can liberate neuropeptides in situ in the insect gut where they can directly activate their target receptors. 'Promoiety' represents a variable carrier to promote the overall stability and / or solubility of the peptide pro-drug compound. 'Linker' represents a peptide and / or chemical sequence in the pro-drug which would cleave due to the pH of the target insect gut and / or protease enzymes of the insect gut to yield a liberated active peptide. All cleavage steps to liberate the peptides occur once ingested and within the insect host, utilizing the pH of the insect gut and / or native insect gut enzyme activity to allow for peptide separation from the prodrug within the gut of the insect host secretion where they can activate their target receptors. Figure 54 - The schematic details the genetic design to produce a ‘pro-drug’ style prepropeptide which liberates recombinant neuropeptides in situ in the insect gut. SS represents a secretion signal to induce movement of the peptides to the extracellular environment. Prepropeptide represents a variable chassis or filler of amino acids to promote the biostability, and ability to secrete the prepropeptide ‘pro-drug’. All proteolytic steps to liberate the peptides occur within the insect host, utilizing native Trypsin or Trypsin-like enzyme activity to allow for peptide separation from the prepropeptide prodrug within the gut of the insect host secretion where they can trigger activity. EXAMPLES 1. Neuropeptide DNA expression construct designs:

[0401] The overall design for the P. pastoris expression cassette to produce insecticidal peptides combines the P. pastoris PichiaPink™ Expression System (Invitrogen UK) plasmid pPinkα-HC with custom DNA genetic constructs. The pPinkα-HC plasmid contains an AOX1 promoter for tightly regulated methanol-induced expression of a gene of interest (GOI), an α-factor secretion signal for directing secreted expression of the recombinant protein, a Multiple Cloning Site (MCS) for cloning in of a GOI and a transcriptional terminator (CYC1 TT). It also contains a pUC origin for propagation and Ampicillin resistance gene for selection in E. coli.

[0402] These DNA genetic constructs contain P. pastoris codon optimised DNA sequences to encode insecticidal neuropeptides. These DNA constructs explore three main designs: Direct expression of single short peptides from single DNA copies, single insect peptides from tandem DNA copies, and expression of multiple different insect peptides from single DNA copies. 1.1 Peptide DNA construct design template

[0403] All insect neuropeptide DNA constructs have been designed to be cloned into the expression vector pPinkα-HC shuttle plasmid from the P. pastoris PichiaPink™ Expression System (Invitrogen UK).

[0404] The general designs for the individual for the insect neuropeptide genetic constructs to be cloned into the combine the: native structure of M. persicae leucokinin prepropeptide sequence, native yeast propeptide architecture seen the successful heterologous yeast peptide production by Vogt et al. (2022)1and the sequence analysis results of Bader, Krauke & Hube (2008)2which demonstrate that the presence of negatively charged amino acid residues after the ‘KR’ recognition site supports successful cleavage by P. pastoris, S. cerevisiae, C. albicans and C. glabrata Kex2 enzymes.

[0405] Practically, the genetic construct designs simplify a full prepropeptide down to the DNA coding sequences for the insect peptides, capped with a double translational stop codon (TAATAG) and preceded by DNA sequences encoding the amino acid sequence ‘KREAEA’ (SEQ ID NO: 59) (Fig. 1). This amino acid sequence introduces a ‘KR’ KEX2 protease cleavage site (either present in pPinkα-HC plasmid α-factor secretion signal or as separators for repeat units) and additional negatively charged ‘EAEA’ (SEQ ID NO: 254) amino acids hypothesised by Bader, Krauke & Hube (2008)2to aid in KEX2 cleavage. Importantly, Vogt et al. (2022)1suggest that the ‘EAEA’ (SEQ ID NO: 254) amino acids themselves should be removed by enzyme Ste13 as the peptide matures.

[0406] To enable cloning, the genetic construct is then flanked by an N-terminal MlyI restriction site and a C-terminal FseI restriction site.

[0407] Ultimately, the expression cassette fuses the peptide with an α-MF secretion signal pre- and pro-peptide under the regulatory control of a methanol inducible Alcohol oxidase I promoter (pAOX1) with a transcriptional terminator downstream of the FseI restriction site (CYC1tt). The successful insertion also places the peptide directly in frame with to the mating α-factor secretion signal (MFα1S) to ensure full processing and secretion of the peptide to the culture. 2. DNA Construct Design: Direct expression of single short peptide from single DNA copy

[0408] To demonstrate the feasibility of this proof of principle approach, neuropeptides SB-P-65, SB- P-45 and SB-P 47 were selected as a focus. These peptides each demonstrate experimentally validated binding affinity for their respective Kinin, Pyrokinin and Capa G-Protein Coupled Receptors (GPCRs) in agricultural pest Myzus persicae (the green peach aphid).

[0409] DNA sequences for each of neuropeptide family were ordered reflecting the design of Fig. 1 and codon-optimised for expression in yeast P. pastoris from the methanol-inducible expression cassette present on the pPinkα-HC plasmid.

[0410] For each peptide, the first strategy to be investigated is the direct expression of individual expression cassettes of the pPinkα-HC plasmid, simply containing a single cloned in copy of either the SB-P-65, SB-P-66, SB-P-45, or SB-P 47 peptide sequences (Fig. 2). 2.1 ‘SB-P-65’ and ‘SB-P-65KR’

[0411] The DNA construct ‘SB-P-65’ consists of a single DNA sequence for SB-P-65 in an expression cassette (Fig. 3A). The DNA construct ‘SB-P-65KR’ is the same as ‘SB-P-65’ but lacks the ‘EAEA’ (SEQ ID NO: 254) Ste13 recognition cut site (Fig. 4B). 2.2 ‘SB-P-45’

[0412] The DNA construct ‘SB-P-45’ consists of a single DNA sequence for SB-P-45 in an expression cassette (Fig. 4). 2.3 ‘SB-P-47’

[0413] The DNA construct ‘SB-P-47’ consists of a single DNA sequence for SB-P-47 in an expression cassette (Fig. 5). 2.4 ‘SB-P-66’

[0414] The DNA construct ‘SB-P-66’ consists of a single DNA sequence for SB-P-66 in an expression cassette (Fig. 6). 3. DNA Construct Design: Single insect peptides from tandem DNA copies 3.1‘SB-P-65x3’

[0415] The DNA construct of ‘SB-P-65x3’ consists of three repeats of the DNA sequence for SB-P- 65 with Kex2 and Ste13 recognition sequences (KR↓ EAEA) (SEQ ID NO: 59) between each DNA sequence unit (Fig. 7). 3.2 SB-P-65x6’

[0416] The DNA construct of ‘SB-P-65x6’ consists of six repeats of the DNA sequence for SB-P-65 with Kex2 and Ste13 recognition sequences (KR↓ EAEA) (SEQ ID NO: 59) between each DNA sequence unit (Fig. 8). 4. DNA Construct design: Expression of multiple different insect peptides from single DNA copies 4.1 ‘Multimyzus’

[0417] The DNA construct of ‘MultiMyzus’ consists of single units of the DNA sequences for SB-P- 65, SB-P-47 and SB-P-45 combined with Kex2 and Ste13 recognition sequences (KR↓ EAEA) (SEQ ID NO: 59) between each DNA sequence unit (Fig. 9). 4.2 ‘Natural Prepropeptide’

[0418] The DNA construct ‘Prepropeptide’ consists of a codon optimised complete naturally occurring prepropeptide gene from Myzus persicae utilising the native ‘KR’ peptide recognition sequence architecture. Within this prepropeptide are the DNA sequences for SB-P-65, SB-P-66 and other suspected, but unconfirmed, insect neuropeptides (Fig. 10). 5. Myzus persicae peptides SB-P-65 and SB-P-66 with -GC ends for ex-vivo photoamidation strategy.

[0419] Amidation of a neuropeptide’s C-terminus is predominantly required for peptide bioactivity. In higher eukaryotes, this C-terminal amidation is performed by a Peptidylglycine alpha-amidating monooxygenase (PAM). The PAM has two catalytic domains: oxygen-dependent and copper containing peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha- hydroxyglycine alpha-amidating lyase (PAL). Yeast however do not conventionally have a PAM or similar analogue. As a result, successful expression and secretion of the peptides from these genetic constructs in P. pastoris should result in a peptide with an extra and non-amidated terminal glycine.

[0420] Alongside the pursuit of a long-term all-in-one yeast strain with a form of PAM capable of producing and specifically amidating peptides, we are now also pursuing an amidated peptide production strategy involving a chemical photoamidation process.

[0421] As this process relies on a C-terminal cysteine instead of a glycine, the same gene constructs for key target peptides SB-P-65 and SB-P-66 were modified and reordered so that, when cloned into pPinkαHC using the same MlyI and FseI restriction sites, the terminal amino acid of both peptides is a cysteine instead of a glycine (Fig. 11). 6. DNA Construct Design: Direct expression of single -GC ended peptide from single DNA sequence constructs.

[0422] The single DNA constructs ‘SB-P-65GC’ and ‘SB-P-66GC’ consist respectively of single DNA sequences for SB-P-65GC and SB-P-66GC in the same pPinkα-HC expression cassette. 6.1 ‘SB-P-65GC’

[0423] The DNA construct ‘SB-P-65GC’ consists of a single DNA sequence for SB-P-65GC in the pPinkα-HC expression cassette (Fig. 12). 6.2 ‘SB-P-66GC’

[0424] The DNA construct ‘SB-P-66GC’ consists of a single DNA sequence for SB-P-66GC in the pPinkα-HC expression cassette (Fig. 13). 7. DNA Construct Design: Single -GC ended insect peptides from tandem DNA sequence constructs.

[0425] This same process was also applied to the tandem repeat versions of the DNA constructs and SB-P-65GCx3, SB-P-65GCx6, SB-P-66GCx3 and SB-P-66GCx6 were ordered as DNA fragments from ThermoFisher’s GeneArt. To avoid sequence repetition that would make these DNA sequences difficult to synthesize, DNA codon re-optimization was performed, and the original coding sequence of the singular DNA construct was varied in each case.

[0426] The tandem DNA constructs ‘SB-P-66GCx3’ and ‘SB-P-66GCx6’ consist respectively of triple and sextuple tandem repeats of the DNA sequence for SB-P-66GC in the pPinkα-HC expression cassette, with each additional repeat sequence separated by DNA sequences encoding a ‘KR’ Kex2 recognition cut site and ‘EAEA’ (SEQ ID NO: 254) Ste13 recognition cut site (Figs. 14 and 15). 7.2 ‘SB-P-66GCx3’

[0427] This construct is shown in Figure 14. 7.3 ‘SB-P-66GCx6’

[0428] This construct is shown in Figure 15. 8. Expression in Pichia pastoris yeast

[0429] To express the neuropeptide constructs in Pichia pastoris cells, the heterologous polynucleotide expression cassette encoding at least one insecticidal neuropeptide or precursor peptide needs to be transformed and inserted into the yeast genome. This transformation will proceed through the electroporation of electrocompetent PichiaPink™ Strain 4 cells with linearized pPinkα-HC plasmid containing the insect neuropeptide expression cassettes.

[0430] Firstly, 7500 ng of each DNA sequence verified pPinkα-HC plasmids containing the insecticidal neuropeptide will be linearized in a working volume of 50 µL using restriction enzyme SpeI and by being incubated at 37 °C for an hour. After the hour, the mixture will be heat inactivated at 80 °C for 20 minutes. The DNA will then be ethanol precipitated and finally resuspended in 10 µL of DNAse-free H2O.

[0431] For the electroporation itself, the full 10 µL of the 7500 ng of linearized plasmid DNA construct will first be mixed with 80 µL of the electrocompetent Strain 4 PichiaPinkTM cells in a 1 mL microcentrifuge tube for 5 minutes before being transferred to a 2 mm electroporation cuvette.

[0432] Each cell and DNA mix will then be pulsed at 1500 V using an ECM 399 electroporation system for 5 milliseconds.

[0433] Electroporated Pichia cells will then be revived in 1 mL of ice-cold YPDS media and incubated in a static incubator for 2 hours at 30 °C. After incubation 300 µL of each culture will be spread on individual Pichia Adenine Dropout (PAD) agar plates for morphological adenine autotrophy based red / white selection. In each case, plates containing cells will be incubated in a static incubator for a total of 9 days at 28 °C before red / white selection.

[0434] Individual colonies with the appropriate morphological characteristics’ indicative of ade2 (and therefore neuropeptide expression cassette) integration will be selected for further analysis.

[0435] Firstly, individual morphologically correct yeast cells will be selected and used to inoculate individual 50 mL centrifuge tubes containing 5 mL YPD. These cells will be cultured at 30 °C and 275 RPM for 24 hours before the DNA will be extracted from the cells using a genomic DNA purification method. In this embodiment genomic DNA will be separated and purified using the method and consumables provided by the GeneJET Genomic DNA Purification Kit (ThermoScientific, UK).

[0436] Verification of the successful integration of at least one copy of the expression cassette to the yeast genome will then be performed through the colony PCR on potential integrants’ purified genomic DNA. In this 50 µL total volume PCR based on the OneTaq® Quick-Load® 2X Master Mix (New England Biolabs, UK), 5 µL of each purified genomic DNA sample will be mixed in with 10 µM of expression cassette specific forward and reverse primers and will be subjected to the following programme of PCR conditions. An initial denaturation at 95 °C for 2 mins, followed by 30 cycles of: denaturation at 95 °C for 1 minute, annealing at 54 °C for 1 minute and extension at 72 °C for 1 minute. After the 30 cycles, there will be a final extension step of 72 °C for 7 minutes. PCR products from each genomic DNA sample will then be subject to gel electrophoresis on an agarose gel and the observed sizes of the PCR products compared against the anticipated sizes of a suspected correct expression cassette insert.

[0437] Full DNA sequence confirmation of a suspected correct expression cassette insert into Pichia host cell genomes will then be provided by sending the corresponding PCR product mix for full DNA sequencing.

[0438] In this embodiment, the expression of the DNA sequence-verified constructs in Pichia host cells and production of pre-pro-peptides, precursor peptides or insecticidal neuropeptides will be carried out by culturing the transgenic Pichia host cells in BMGY and BMMY culture media under suitable growth conditions and in a dedicated bioreactor culture vessel.

[0439] The overall bioreactor-scale fermentation will consist of three phases. The first phase post inoculation will be a glycerol batch phase to allow initial growth of the Pichia cell cultures in the sterilized bioreactor environment. The second phase will be a glycerol fed-batch phase for biomass accumulation of the cell population. The third phase will be a methanol fed-batch phase to produce the pre-pro-peptides and insecticidal neuropeptide or precursor peptides from the transgenic Pichia cells.

[0440] Operational growth conditions for all three phases will be as follows: A cultivation temperature of 30°C will be maintained via an integrated temperature control system, a culture pH value of 6.0 ± 0.3 will be maintained by the addition of ~30% (v / v) sterile ammonium hydroxide solution and a dissolved oxygen level of ≥20% will be maintained throughout the aerobic fermentation through a cascade control system which will manage a combination of impeller stirring between 200-1500 RPM, air flow rates of 0.1-1.2 VVM compressed air and supplementary oxygen flow rates of between 0.1-0.3 VVM oxygen. In all phases, foaming will be controlled through the addition of antifoam J 647 (Struktol®) upon the triggering of an integrated antifoam sensor.

[0441] The bioreactor-scale fermentation will begin with the generation of a seed culture of the host Pichia cells to serve as an inoculum for a bioreactor culture. To form the inoculum, a sequence positive Pichia colony from a PAD agar plate or glycerol stock will be used to inoculate a baffled flask containing a volume of BMGY media equivalent to 10% of the starting media volume of the bioreactor. The Pichia cells will then be cultured in a shaking incubator at 30°C and an agitation equating 275 RPM until an OD600 value of between 2-6 can be measured.

[0442] The first phase of growth in the bioreactor will be a batch phase growth using BMGY media containing glycerol as the principal carbon source for growth. The bioreactor will be inoculated with the 10% initial fermentation volume of transgenic Pichia cells and the cells will be grown under the above cultivation conditions for approximately 18-24 hours until the glycerol in the media is exhausted. This glycerol exhaustion will be indicated by an increase in the DO % reading to approximately 100%. This initial exhaustion triggers the start of the glycerol fed-batch phase of growth to increase cell biomass prior to induction of the genetic construct.

[0443] During the glycerol fed-batch phase calibrated peristaltic pumps of the bioreactor control system pump a 50% (v / v) glycerol solution into the Pichia cell culture at a rate of 18.15 mL / hr / L initial fermentation volume. This feed rate will be maintained until a suitable 150 to 220 g / L wet cell weight can be measured from regular bioreactor sampling. At this point, the glycerol fed-batch growth phase of the method completes and can then proceed to a methanol fed-batch induction phase. In the methanol fed-batch induction phase, the AOX1 promoter of the heterologous polynucleotide expression cassette will be induced by the presence of methanol as a primary carbon source (i.e. when glycerol has been fully depleted). During the methanol fed-batch phase, suitable endoproteinases Kex2 and Ste13 will proteolytically cleave the nascent pre-pro-peptide to release the insecticidal neuropeptide / s or precursor peptide / s, which will then be secreted from the cell into the extracellular culture environment. To initiate the methanol fed-batch induction phase, the glycerol solution feed will be terminated and replaced with a 100% methanol feed at an initial rate of 3.6 mL / hr / L initial fermentation volume. Adaptation of the culture to the methanol carbon source will take approximately 2-4 hours. Once adapted, the rate of methanol feed will be increased to 7.3 mL / hr / L initial fermentation volume. Two hours after making this change, the feed rate will be increased to 10.8 mL / hr / L initial fermentation volume for the remainder of the methanol fed-batch induction phase. The methanol fed-batch induction phase is currently estimated to take an approximate total of 70 hours before the fermentation is terminated. Isolation / purification

[0444] Upon termination of the fermentation, isolation of the insecticidal neuropeptide or precursor peptide from the culture will require separation of the peptide products from the Pichia host cells in the culture. This will be performed using centrifugation of the cell culture at 5000 x g for 30 minutes to separate the cells into a cell pellet and leave a supernatant containing the peptide products. This supernatant will first be filtered through a 0.2 µm membrane filter to sterilize the supernatant and remove residual particulates. Isolation of the peptides will then proceed through either through size exclusion chromatography or through separation and concentration using ultrafiltration with size exclusion Macrosep® Advance Centrifugal Devices (PAL Life sciences UK) of either 10 kDa, 5 kDa or 3 kDa followed for an initial ultrafiltration and concentration, followed by a size exclusion Macrosep® Advance Centrifugal Device of less than or equal to 1kDa.

[0445] Lastly, to obtain isolated peptide products, the solvent containing the peptides will be lyophilized at -80 °C and 0.1 mbar pressure for 24 hours to yield a solid peptide powder. 9. Expression of peptides fused to a fluorescent reporter linked via Kex2 / Ste13 recognition sites for in vivo cleavage

[0446] Peptide sequences are fused to the C-terminus of a fluorescent reporter (eYFP) fused to an N-terminal secretion signal. Between the peptide and reporter ORFs are encoded Kex2 and Ste13 protease cleavage sites (Fig. 16). These proteases are endogenous to P. pastoris and are involved in cleavage of the secretion signal peptide (Sleep et al, 1990). This places Kex2 / Ste13 in proximity to recombinant proteins destined for extracellular secretion. This design allows for liberation of peptides from the reporter followed by secretion for recovery from the culture supernatant. The use of a fluorescent reporter allows for rapid screening of candidate strains showing higher target gene expression levels. By reading samples of culture supernatant on a plate reader it is possible to measure the relative fluorescence of samples, allowing rapid identification of strains expressing the highest titres of target recombinant protein (Fig.17). SDS gels demonstrate both eYFP and larger eYFP-peptide fusions in the culture supernatant of strains expressing the fusion constructs (Fig. 18). In eYFP-fusion expressing strains, a doublet corresponding to the expected protein sizes for cleaved and full length eYFP- SB-P-66GC and eYFP-SB-P-44 expressing strains is observed (Fig. 20). This is particularly notable when compared to controls expressing eYFP. The observation of two bands for eYFP-fusion proteins indicates successful but incomplete cutting at the Kex2 / Ste13. 10. Expression of insect neuropeptides as a C-terminal PAM enzyme fusion for in vivo C- terminal amidation.

[0447] Expression of a gene encoding the fusion of a secretion signal and insect neuropeptides to the N- and C- terminal of a PAM enzyme respectively allows for creation of a cell based microbial factory directly producing C-terminally amidated neuropeptides. PAM enzymes amidate the C-terminus of neuropeptides in vivo through the action of two domains, PHM and PAL (Fig. 22). Separating ORFs encoding the PAM enzyme and insect neuropeptides with protease recognition sites permits in vivo cleavage of the fusion proteins (Fig. 21). This is dependent on the cleavage sites being recognized by native proteases found in the host organism. The liberated peptides are exposed at their C-terminus, allowing amidation by the PAM enzyme. We have demonstrated expression of a recombinant modified Rattus norvegicus PAM enzyme fused to a tandem M. persicae peptide construct (MM) through SDS gel electrophoresis (Fig. 23). SDS analysis of purified recombinant PAM shows that 4 bands are identified when running PAM enzyme, these may represent the 2 PAM domains in various breakdown patterns though a similar pattern is found in the culture supernatant of cells expressing a rat PAM-MM fusion. However, the PAM bands in the supernatant samples are larger than in the recombinant rat PAM, suggesting that the PAM fragments are fused to neuropeptide sequence (Fig. 23).

[0448] PAM mediated peptide amidation is a 2-step process controlled by the PHM and PAL domains successively. The PHM domain initiates the amidation by hydroxylating the C- terminal glycine present on the neuropeptide. From this modified α-hydroxyglycine, a glyoxylate is cleaved by the PAL domain, liberating the amidated peptide (Fig. 22). We have shown that PAM enzymes can amidate insect neuropeptides in vitro through LC- MS in an aqueous buffer (Fig. 24). Quantification through HPLC identified complete amidation over a 90-minute incubation at 37 °C with no shaking (Fig. 25). The intermediate peptide has the sequence RQKTVFSSWGG-[OH] (SEQ ID NO: 261), the final amidated peptide of SB-P-181 has the sequence RQKTVFSSWGG-[NH2] (SEQ ID NO: 260).

[0449] PAM activity is inhibited by rich media components. However, we developed minimal media through which PAM activity can be detected with LC-MS (Fig. 26). As the PAM enzyme is expressed with near 1:1 stoichiometry to the insect neuropeptides, it is likely sufficient for neuropeptide amidation in the presence of inhibitors. PAM enzyme acts upon peptides with a C-terminal glycine but is not inhibited by free glycine. We have evidence that PAM is strongly inhibited by the addition of peptone and yeast extract. These components contain a large amount of small peptide fragments which we suspect are inhibiting the PAM enzyme. Subsequently, we propose developing a semi-rich media containing ammonium sulphate, yeast nitrogen base, glucose and casamino acids, a complete hydrolysis of casein consisting of free amino acids. This is unlikely to inhibit PAM but allows for much faster growth when compared to simpler growth media. This has the added advantage of being suited to the maintenance of auxotrophic selection in host organisms using leucine, tryptophan, uracil or adenine synthase genes as a selectable marker. 11. Expression of affinity tagged PAM for pull down, peptide cleavage and elution of rat PAM for post fermentation amidation.

[0450] An alternative strategy for the recombinant production of amidated insect neuropeptides involves encoding the peptide as a fusion protein with a PAM gene containing an affinity tag on the opposing terminus. This allows for affinity pull down of the PAM-peptide fusion. Linkers consisting of a peptide sequence recognized by an external protease between the PAM and peptide allows for ‘on-resin’ cleavage of the peptides. These can be eluted into a buffer suitable for in vitro PAM amidation. Subsequent elution of the PAM enzyme allows for purification of the peptide and the PAM directly into an amidation buffer for incubation at 37 °C, allowing peptide amidation. These can then be recovered through size-exclusion and ion- exchange chromatography. 12. Expression of insect neuropeptides fused to an intein inactivated protease for recombinant production and recovery of target peptides

[0451] Pyrococcus abyssi DNA pol III intein cleaves itself from the translated protein and ligates both exteins together in response to an increase in temperature to 40-70 °C (Mills et al, 2004). This intein can be altered to have a C-terminal alanine / valine, preventing its excision at the C-terminus and blocking intein cleavage post N-S acyl shift. From this position the N-extein can be amidated and cleaved from the intein through addition of thiols and ammonium sulphate / ammonium carbonate.

[0452] Using the unaltered intein to inactivate a protease allows for conditional activation of protease activity by shifting your sample to intein cleaving conditions. Designing a gene construct expressing an affinity tagged intein inactivated protease separated from peptide sequence through a protease cut site and C-terminal alanine / valine modified inteins allows for straightforward recovery, purification and amidation of target peptide.

[0453] The workflow would involve using affinity chromatography to isolate the fusion protein from the culture supernatant. Incubating the sample at 40 °C excises the intein within the protease N- and C- extein, leaving it in an active configuration. Simultaneously, the altered inteins between peptide sequences undergo an N-S acyl shift. The intact protease separates the peptide-altered intein sequences from the protease. The protease can be separated from the peptide-altered intein fusions through size exclusion chromatography. Finally, the peptides can be incubated with a combination of ammonium sulphate / ammonium carbonate and thiols to cleave and amidate the peptides from the inteins (Fig. 27), also shown in Figure 30. 13. Expression of peptides linked to Cysteine and Factor Xa cleavage sites and affinity tagged for pull-down

[0454] Two widely known and commonly used precision protease / peptidase systems are Enterokinase (cleavage site: DDDDK) and Factor Xa (cleavage site: I[E / D]GR). Constructs have been designed incorporating combined cysteine cleavage sites and Factor Xa cleavage sites between repeats of any insect neuropeptide sequence of interest, which in this example is SB-P-80 peptide sequences (see Figure 36). An N-...

Claims

CLAIMS 1. A recombinant expression construct comprising a polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof, operably linked to a polynucleotide comprising a promoter suitable for expression in a microorganism selected from the group consisting of bacteria, yeasts, and fungi, which is heterologous to the polynucleotide encoding the at least one insecticidal neuropeptide or a precursor peptide thereof.

2. The recombinant expression construct of claim 1, wherein said expression construct comprises: (i) a polynucleotide encoding two or more copies of a first insecticidal neuropeptide, or precursor peptide thereof; or (ii) a polynucleotide encoding at least one copy of a first insecticidal neuropeptide, or a precursor peptide, and at least one copy of one or more additional insecticidal neuropeptides, or precursor peptides thereof, wherein the first insecticidal neuropeptide or a precursor peptide, comprises a sequence which is different from the sequence of the or each additional insecticidal neuropeptide or precursor peptide thereof.

3. The recombinant expression construct of claim 2, wherein the first insecticidal neuropeptide, or a precursor peptide, and the one or more additional insecticidal neuropeptides, or precursor peptides thereof are operable to target the same insect.

4. The recombinant expression construct of claim 2 or 3, wherein the polynucleotides encoding each insecticidal neuropeptide or precursor peptides thereof are separated by a polynucleotide sequence encoding an intein, a self-cleaving peptide, a cleavage site for a protease, or a cleavage site for an exogenous or endogenous endopeptidase or exopeptidase, optionally wherein the cleavage site for the protease comprises the sequence: [Arg / Lys-Arg / Lys- (Glu-Ala)n] (SEQ ID NO: 58) wherein n is between 1 to 5, preferably wherein the cleavage site comprises a sequence selected from: KR, RR and KREAEA (SEQ ID NO: 59) or (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269).

5. The recombinant expression construct of claim 2 or 3, wherein the polynucleotides encoding each insecticidal neuropeptide or precursor peptides thereof are separated by apolynucleotide sequence encoding a chemical cleavage site, optionally wherein the chemical cleavage site comprises a Cysteine, and / or a Methionine.

6. The recombinant expression construct of any of claims 1-5, wherein the recombinant expression construct further comprises a polynucleotide encoding one or more of: (a) a targeting peptide; (b) a secretion peptide; and / or (c) a polyadenylation or transcriptional termination signal; wherein the polynucleotides of (i), (ii) and / or (iii) are operably linked to the polynucleotide encoding the at least one insecticidal neuropeptide or a precursor peptide thereof.

7. The recombinant expression construct of any of claims 1-6, wherein the recombinant expression construct further comprises a polynucleotide encoding one or more of: (d) an affinity tag, optionally at the C-terminus or N-terminus, optionally selected from: a His-tag, an Arg-tag, a FLAG-tag, a Strep-tag, Glutathione-S-transferase tag, Twin-Strep Tag, Calmodulin Binding Peptide, Chitin-binding Tag, Maltose-binding Tag, and a Cellulose-binding Tag.

8. The recombinant expression construct of claim 6 or 7, wherein the targeting peptide is selected from: a vacuolar targeting peptide, an endoplasmic reticulum targeting peptide, and / or a plastid targeting peptide.

9. The recombinant expression construct of any of claims 1-8 wherein the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof, encodes a non-naturally occurring pre-pro-peptide, preferably wherein the pre-pro-peptide comprises the sequence: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n wherein (a) S comprises at least one targeting peptide and / or a secretion peptide; (b) [SS] is an optional stuffer sequence; (c) [CC1] and [CC3] are optional consensus cleavage sites, [CC2] is present and is a consensus cleavage site; (d) NP is an insecticidal neuropeptide, a precursor, or a variant thereof;(e) AS is an optional C-terminal amidation signal; (f) n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and (g) i is a numerator indicating that the sequence of each NP unit and / or each: CC unit, AS unit and / or SS unit in the construct is optionally different, optionally wherein the polynucleotide encoding the pre-pro-peptide further comprises a trailer sequence [Tr] at the 3' end thereof.

10. The recombinant expression construct of any of claims 1-8 wherein the polynucleotide encoding at least one insecticidal neuropeptide or a precursor peptide thereof, encodes a non-naturally occurring pre-pro-peptide, preferably wherein the pre-pro-peptide comprises the sequence: S-[CC1i]-([NPi]-[CC2i]-[SSi]-[CC3i])n wherein: (a) S comprises at least one targeting peptide and / or a secretion peptide; (b) [SS] is an optional stuffer sequence; (c) [CC2] is a consensus cleavage site, [CC1] and [CC3] are optional consensus cleavage sites; (d) NP is an insecticidal neuropeptide, a precursor, or a variant thereof; (e) n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and (f) i is a numerator indicating that the sequence of each NP unit and / or each: CC unit, and / or SS unit in the construct can be optionally different, optionally wherein the polynucleotide encoding the pre-pro-peptide further comprises a trailer sequence [Tr] at the 3' end thereof.

11. The recombinant expression construct according to claim 9 or 10, wherein the at least one targeting peptide and / or a secretion peptide comprises an endoplasmic reticulum targeting peptide.

12. The recombinant expression construct according to claims 9, 10 or 11 wherein [CC1], [CC2], when present, and [CC3] each comprise an intein, a self-cleaving peptide, the sequence (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269), or the sequence: [Arg / Lys-Arg / Lys-(Glu-Ala)n](SEQ ID NO: 58) wherein n is between 1 to 5, preferably wherein [CC1], [CC2], when present, and [CC3], each comprise a sequence selected from: KR, RR and KREAEA (SEQ ID NO: 59), or (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269).

13. The recombinant expression construct according to claims 9, 10 or 11 wherein [CC1], [CC2], when present, and [CC3] each comprise a chemical cleavage site, preferably wherein [CC1], [CC2], when present, and [CC3], each comprise a cysteine and / or a methionine.

14. The recombinant expression construct according to any of claims 9-13 wherein each [SS] comprises a nonsense sequence.

15. The recombinant expression construct according to any of claims 9-14 wherein each NP neuropeptide, precursor, or a variant thereof is less than 10 amino acids in length, more preferably between 5 and 10 amino acids in length, and optionally wherein the pre-pro-peptide is less than 200 amino acids in length.

16. The recombinant expression construct according to any of claims 9-15 wherein [Tr] comprises a polyadenylation sequence.

17. The recombinant expression construct according to any of claims 9-16 wherein n is between 1-6, preferably n is at least 2.

18. The recombinant expression construct according to any of claims 9-17 wherein the sequence of each NP neuropeptide, precursor, or variant thereof is identical, or wherein the sequence of each NP neuropeptide, precursor, or variant thereof is different.

19. The recombinant expression construct according to any of claims 9-18 wherein each NP neuropeptide, precursor, or variant thereof is operable to target the same insect.

20. The recombinant expression construct according to any of claims 9-19 wherein the C- terminal amidation signal is a glycine or a cysteine residue.

21. The recombinant expression construct of any of claims 1-8 wherein the polynucleotide encoding at least one insecticidal neuropeptide, or a precursor peptide thereof, is selected from the group consisting of: (i) a complete or partial open reading frame of a wild type gene encoding an insect pre-pro- peptide or a codon optimized variant thereof; (ii) a synthetic gene encoding a pre-pro-peptide according to any of claims 9-20.

22. The recombinant expression construct of any of claims 1 to 21, wherein the polynucleotide encoding the at least one insecticidal neuropeptide, or a precursor peptide thereof, is inserted into a heterologous nuclear or plastid genome of a cell.

23. The recombinant expression construct of any of claims 5-22, wherein the secretion peptide ensures transport through the Golgi vesicle, and is preferably an MFalpha1s secretion peptide.

24. The recombinant expression construct of any of claims 1 to 23, wherein the heterologous promoter is suitable for expression in a yeast, optionally wherein the heterologous promoter is inducible or constitutive, and is preferably selected from the group: AOX1, GAP, ICL1, TEF1, ADH1, PGK1, DAS and FLD1, more preferably the heterologous promoter is AOX1.

25. The recombinant expression construct of any of claims 1 to 24, wherein the precursor peptide, or each of the NP neuropeptides, precursors, or variants thereof comprised in the pre- pro-peptide comprises a sequence selected from: (i) SPPYSPPFSPRL (SEQ ID NO: 175) (ii) Q / EAIMARPQVPRL (SEQ ID NO: 205) (iii) NADEDQQQSVDFTPRL (SEQ ID NO: 182) (iv) GGSMTFSPRL (SEQ ID NO: 183) (v) KVKFSAWG (SEQ ID NO: 82) (vi) RQKTVFSSWG (SEQ ID NO: 77) (vii) PAFSSWG (SEQ ID NO: 76) (viii) Q / ELTFTSSWGG (SEQ ID NO: 143) (ix) QLTFSPDW (SEQ ID NO: 206) (x) Q / ELTFSPDW (SEQ ID NO: 142) (xi) SVPFKPRL (SEQ ID NO: 192) (xii) LRQLQSNGEPAYRVRTPRL (SEQ ID NO: 178) and (xiii) GDTTQSSNGGMWFGPRL (SEQ ID NO: 174) 26. The recombinant expression construct of any of claims 1 to 24 wherein the insecticidal neuropeptide comprises a sequence selected from: i. SPPYSPPFSPRL-[nh2] (SEQ ID NO: 208) ii. [pyr]AIMARPQVPRL-[nh2] (SEQ ID NO: 209) iii. NADEDQQQSVDFTPRL-[nh2] (SEQ ID NO: 210) iv. GGSMTFSPRL-[nh2] (SEQ ID NO: 211) v. KVKFSAWG-[nh2] (SEQ ID NO: 212)vi. RQKTVFSSWG-[nh2] (SEQ ID NO: 213) vii. PAFSSWG-[nh2] (SEQ ID NO: 214) viii. [pyr]-LTFTSSWGG-[nh2] (SEQ ID NO: 215) ix. [palm]-QLTFSPDW-[nh2] (SEQ ID NO: 216) x. [pyr]LTFSPDW-[nh2] (SEQ ID NO: 217) xi. SVPFKPRL-[nh2] (SEQ ID NO: 218) xii. LRQLQSNGEPAYRVRTPRL-[nh2] (SEQ ID NO: 219) xiii. GDTTQSSNGGMWFGPRL-[nh2] (SEQ ID NO: 220) xiv. RQKTVFSSWGG-[nh2] (SEQ ID NO: 260) xv. RQKTVFSSWGG-[OH] (SEQ ID NO: 261) xvi. [Pyr]-LTFSPDW-[OH] (SEQ ID NO:262) xvii. [Pyr]-LTFTSSWGG-[OH] (SEQ ID NO: 263) 27. A eukaryotic nuclear or plastid genome comprising a polynucleotide encoding at least one insecticidal neuropeptide, or a precursor peptide thereof, wherein the polynucleotide is heterologous to the nuclear or plastid genome and wherein the polynucleotide is operably linked to an endogenous promoter of the nuclear or plastid genome.

28. A eukaryotic nuclear or plastid genome according to claim 27, wherein the polynucleotide encoding at least one insecticidal neuropeptide, or a precursor peptide thereof, is further defined according to any of claims 2 to 24.

29. A cell comprising the recombinant expression construct of any one of claims 1 to 26 or the genome of claims 27 or 28, wherein the cell is optionally a bacterial, yeast, fungal, insect, or plant cell.

30. A plant comprising a recombinant expression construct of any of claims 1 to 26, or the genome of claims 27 or 28, or the cell of claim 29.

31. A non-naturally occurring insecticidal [pre-pro-]-peptide comprising the sequence: S-[CC1i]-([SSi]-[CC2i]-[NPi]-[ASi]-[CC3i])n, wherein (a) S comprises at least one targeting peptide and / or a secretion peptide; (b) [SS] is an optional stuffer sequence; (c) [CC1] and [CC3] are optional consensus cleavage sites, [CC2] is present and is a consensus cleavage site; (d) NP is an insecticidal neuropeptide, a precursor, or a variant thereof;(e) AS is an optional C-terminal amidation signal; (f) n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and (g) i is a numerator indicating that the sequence of each NP unit and / or each SS unit in the construct is optionally different.

32. A non-naturally occurring insecticidal [pre-pro-]-peptide comprising the sequence: S-[CC1i]-([NPi]-[CC2i]-[SSi]-[CC3i])n wherein: (a) S comprises at least one targeting peptide and / or a secretion peptide; (b) [SS] is an optional stuffer sequence; (c) [CC2] is a consensus cleavage site, [CC1] and [CC3] are optional consensus cleavage sites; (d) NP is an insecticidal neuropeptide, a precursor, or a variant thereof; (e) n indicates the number of pre-pro-peptide encoding units in the construct and is equal to or larger than 1; and (f) i is a numerator indicating that the sequence of each NP unit and / or each: CC unit, and / or SS unit in the construct can be optionally different, optionally wherein the polynucleotide encoding the pre-pro-peptide further comprises a trailer sequence [Tr] at the 3' end thereof.

33. The non-naturally occurring insecticidal [pre-pro-]-peptide according to claim 31 or 32, wherein the at least one targeting peptide and / or a secretion peptide comprises an endoplasmic reticulum targeting peptide.

34. The non-naturally occurring insecticidal [pre-pro-]-peptide according to claims 31, 32 or 33 wherein [CC1], [CC2], when present, and [CC3] each comprise an intein, a self-cleaving peptide, the sequence (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269), or the sequence: [Arg / Lys- Arg / Lys-(Glu-Ala)n] (SEQ ID NO: 58) wherein n is between 1 to 5, preferably wherein [CC1], [CC2], when present, and [CC3], each comprise a sequence selected from: KR, RR and KREAEA (SEQ ID NO: 59), or (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269).

35. The non-naturally occurring insecticidal [pre-pro-]-peptide according to claims 31, 32 or 33 wherein [CC1], [CC2], when present, and [CC3] each comprise a chemical cleavage site, preferably wherein [CC1], [CC2], when present, and [CC3], each comprise a cysteine and / or a methionine.

36. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-35 wherein each [SS] comprises a nonsense sequence.

37. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-36 wherein each NP neuropeptide, precursor, or a variant thereof is less than 10 amino acids in length, more preferably between 5 and 10 amino acids in length, and optionally wherein the pre-pro-peptide is less than 200 amino acids in length.

38. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-37 wherein [Tr] comprises a polyadenylation sequence.

39. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-38 wherein n is between 1-6, preferably n is at least 2.

40. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-39 wherein the sequence of each NP neuropeptide, precursor, or variant thereof is identical, or wherein the sequence of each NP neuropeptide, precursor, or variant thereof is different.

41. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-40 wherein each NP neuropeptide, precursor, or variant thereof is operable to target the same insect.

42. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-41 wherein the C-terminal amidation signal is a glycine or a cysteine residue.

43. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-42, wherein the secretion peptide ensures transport through the Golgi vesicle, and is preferably an MFalpha1s secretion peptide.

44. The non-naturally occurring insecticidal [pre-pro-]-peptide according to any of claims 31-43, wherein each NP neuropeptide, precursor, or variants thereof comprised in the pre-pro- peptide comprises a sequence selected from: (viii) SPPYSPPFSPRL (SEQ ID NO: 175) (ix) Q / EAIMARPQVPRL (SEQ ID NO: 205) (x) NADEDQQQSVDFTPRL (SEQ ID NO: 182) (xi) GGSMTFSPRL (SEQ ID NO: 183) (xii) KVKFSAWG (SEQ ID NO: 82) (xiii) RQKTVFSSWG (SEQ ID NO: 77) (xiv) PAFSSWG (SEQ ID NO: 76) (xv) Q / ELTFTSSWGG (SEQ ID NO: 143)(xvi) QLTFSPDW (SEQ ID NO: 206) (xvii) Q / ELTFSPDW (SEQ ID NO: 142) (xviii) SVPFKPRL (SEQ ID NO: 192) (xix) LRQLQSNGEPAYRVRTPRL (SEQ ID NO: 178) and (xx) GDTTQSSNGGMWFGPRL (SEQ ID NO: 174).

45. A composition comprising one or more isolated insecticidal neuropeptides or chemically modified variants thereof, and at least 0.1% w / w of a precursor peptide thereof, and / or optionally a pre-pro-peptide thereof according to any of claims 31-44.

46. The composition of claim 45 wherein the composition comprises two or more insecticidal neuropeptides having different sequences, optionally derived from the same pre-pro- peptide.

47. The composition according to claim 45 or 46 wherein the composition comprises between 0.1% and 1% w / w of a precursor peptide thereof, optionally a pre-pro-peptide thereof according to any of claims 31-44.

48. The composition of any of claims 45-47 further comprising an agriculturally, pharmaceutically, or veterinary-practicably acceptable carrier, diluent, or excipient.

49. The composition of any of claims 45-48 wherein the composition is an agricultural composition and / or an insecticidal composition.

50. The composition of any of claims 45-49, wherein the composition further comprises at least one compound selected from the group consisting of wetting agents, surfactants, preservatives, and protease inhibitors.

51. The composition of any of claims 45-50, wherein the composition comprises the one or more insecticidal neuropeptides or chemically modified variants thereof at a concentration of about 0.1, 0.5, 1.0, or 5 pg / ml to about 1, 5, 20, 50, or 100 mg / ml.

52. The composition of any of claims 45-51, wherein the composition comprises a sodium salt at a concentration of at least l00 mM and / or a calcium salt at a concentration of at least 2mM.

53. A process for producing an insecticidal neuropeptide or a precursor thereof comprising the steps of: (i) providing a host cell comprising a recombinant expression construct of any of claims 1-26, the genome of claims 27 or 28, or which is a cell of claim 29;(ii) culturing said host cell under suitable conditions for an insecticidal neuropeptide or precursor peptide or pre-pro-peptide thereof to be produced by the host cell; (iii) optionally further processing any pre-pro-peptide to release an insecticidal neuropeptide or a precursor peptide thereof; and (iv) optionally further chemically and / or enzymatically modifying any precursor peptide to produce an insecticidal neuropeptide.

54. The process according to claim 53 wherein the further processing step (iii) comprises cleavage, optionally proteolytic cleavage of the pre-pro-peptide to release an insecticidal peptide or a precursor peptide thereof, or optionally cleavage of the pre-pro-peptide to release an insecticidal peptide or a precursor peptide thereof via an intein or self-cleaving peptide.

55. The process according to claim 54 wherein the proteolytic cleavage is carried out by an enzyme, optionally by an enzyme selected from a dibasic-processing endoprotease or an exogenous endopeptidase or exopeptidase, optionally wherein the dibasic-processing endoprotease is endogenous to the host cell or caused by a recombinant expression of a dibasic- processing endoprotease within the host cell, optionally wherein the dibasic-processing endoprotease is selected from: Kex2, XPR6, PC2, PC3 / PC1, PC4, furin / PACE, and PACE4; or optionally wherein the exogenous endopeptidase or exopeptidase is selected from adenain or staphylolysin, or optionally wherein the protease is Factor Xa.

56. The process according to claim 53 wherein the further processing step (iii) comprises cleavage, optionally chemical cleavage of the pre-pro-peptide to release an insecticidal peptide or a precursor peptide thereof.

57. The process according to any of claim 53 to 56, wherein: (i) the recombinant expression construct comprises two or more copies of a first insecticidal neuropeptide, or precursor peptide thereof; or a polynucleotide encoding at least one copy of a first insecticidal neuropeptide, or a precursor peptide, and at least one copy of one or more additional insecticidal neuropeptides, or precursor peptides thereof, wherein the first insecticidal neuropeptide or a precursor peptide, comprises a sequence which is different from the sequence of the or each additional insecticidal neuropeptide or precursor peptide thereof; and wherein the insecticidal neuropeptides are separated by a polynucleotide sequence encoding a cleavage site, the cleavage site being selected from: an intein, aself-cleaving peptide, a cleavage site for a protease, or a cleavage site for an exogenous or endogenous endopeptidase or exopeptidase, a Cysteine and / or a Methionine, and wherein the recombinant expression construct further comprises a sequence encoding an affinity tag; ii) the host cell is essentially devoid of proteolytic cleavage activity against any cleavage site present in the recombinant expression construct; iii) the process further comprises a step of isolating the pre-pro-peptide or the precursor r peptide from the host cell or culture, optionally by using the affinity tag; and iv) further processing the isolated pre-pro-peptide or the precursor peptide comprises cleaving the isolated pre-pro-peptide or the precursor peptide by contacting the pre- pro-peptide or the precursor peptide with a protease able to cleave at the or each cleavage site present in the pre-pro-peptide or precursor peptide, thereby producing the insecticidal neuropeptides or the precursor peptides.

58. The process according to claim 57 wherein the cleavage site for the protease comprises the sequence: [Arg / Lys-Arg / Lys-(Glu-Ala)n] (SEQ ID NO: 58) wherein n is between 1 to 5, preferably wherein the cleavage site comprises a sequence selected from: KR, RR and KREAEA (SEQ ID NO: 59) or (Ile-(Glu or Asp)-Gly-Arg) (SEQ ID NO:269).

59. The process according to any of claims 53-58, wherein the step of further processing by cleaving the isolated pre-pro-peptide or the precursor peptide creates a C-terminally amidated insecticidal neuropeptide.

60. The process of claim 55 or 57, wherein the endoprotease is an insect derived endoprotease.

61. The process according to any of claims 53-60 wherein the further chemical and / or enzymatic modification step (iv) comprises amidating the precursor peptide to produce an insecticidal neuropeptide.

62. The process according to claim 61, wherein the precursor peptide is chemically or photochemically amidated, or wherein the precursor peptide is enzymatically amidated by one or more C-terminal [α-]amidation enzymes, optionally wherein the or each enzyme is endogenous to the host cell or caused by a recombinant expression of a C-terminal α-amidating enzyme, optionally wherein the C-terminal α-amidating enzymes comprise PHM and PAL.

63. The process of any of claims 53-62, wherein the host cell is a prokaryotic cell or a eukaryotic cell, optionally wherein the host cell is selected from the group consisting of: Bacillus, Pichia, and filamentous fungi, preferably wherein the host cell is a Bacillus or a Pichia cell, more preferably wherein the host cell is Bacillus subtilis or Pichia pastoris.

64. The process of any of claims 53-63, wherein the process produces an active insecticidal neuropeptide.

65. The process of any of claims 53-64, wherein the process further comprises a step of isolating the insecticidal neuropeptide or a precursor peptide thereof from the culture, optionally prior to step (iv).

66. A composition comprising an isolated insecticidal neuropeptide produced by the process of any of claims 53-65, wherein the composition comprises at least 0.1% w / w of a precursor peptide thereof, and / or optionally a pre-pro-peptide thereof.

67. The composition of claim 66, wherein the insecticidal neuropeptide is amidated.

68. A method for preventing or treating an insect infestation of a plant, the method comprising: contacting a plant, or part of said plant, with an effective amount of the composition of any of claims 45-52 or any of claims 66-67.

69. The method of claim 68 wherein the insect is selected from: M. persicae, Nilaparvata lugens, Drosophila suzukii, Blattella germanica, Plutella xylostella and Spodoptera frugiperda.

70. The method of any of claims 68 or 69 wherein the plant is a crop plant.

71. The use of a Bacillus subtilis strain for the expression of an expression construct according to any of claims 1-26, or as a host cell in a process of any of claims 53-65.

72. The use of a Bacillus subtilis strain according to claim 71, wherein said strain has been modified to delete at least one gene encoding an endogenous endoprotease.

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