Insecticidal combinations

Abstract

The present invention provides novel insecticidal combinations, compositions, and methods of use thereof. The present invention relates to combinations of cysteine rich insecticidal peptides (CRIP) and insecticides (IA). The use of the combination and composition for controlling insects is also described. Here, the present invention describes the following: a gene encoding a CRIP; compositions and combinations comprising CRIP and IA; and methods of using the compositions and combinations useful for controlling pests.

Description

[0001] This application is a divisional application of the invention patent application filed on April 30, 2021, with application number 202180045271.7 (international application number PCT / US2021 / 030277) and entitled "Insect Killing Combination".

[0002] Cross-references to related applications

[0003] This application claims the benefit and priority of U.S. Provisional Application Serial No. 63 / 019,219, filed May 1, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0004] sequence list

[0005] This application is incorporated, by reference, in the sequence list “225312-491452_ST25.txt” (1.41 MB), created on 28 April 2021 at 10:07 p.m., and is hereby submitted electronically. Technical Field

[0006] This invention describes and claims novel insecticidal combinations of cysteine-rich insecticidal proteins (CRIPs) and insecticides (IAs) for the control and / or eradication of pests, said insecticides being, for example, chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, poisons, toxicants, insecticides, pesticides, organic compounds, inorganic compounds, prokaryotes or eukaryotes (and reagents produced by said prokaryotes or eukaryotes). Background Technology

[0007] Many insects are disease vectors. Anopheles mosquitoes are major vectors of Zika virus, chikungunya virus, and malaria, a disease caused by protozoa of the genus Trypanosoma. Aedes aegypti is a major vector of viruses that cause yellow fever and dengue fever. Other viruses, and pathogens of various types of encephalitis, are also carried by Aedes spp. mosquitoes. Wuchereria bancrofti and Brugia malayi are parasitic roundworms that cause filariasis, typically transmitted by mosquitoes of the genera Culex, Mansonia, and Anopheles.

[0008] Horseflies and deer flies can transmit bacterial pathogens of tularemia (Pasteurella tularensis) and anthrax (Bacillus anthracis), as well as the parasitic roundworm (Loa filaria) that causes loa filariasis in tropical Africa.

[0009] Eye leafminers (Hippelates) can carry the spirochetal pathogen that causes yaws (Treponema pertenue) and can also transmit conjunctivitis (pink eye). Tsetse flies (Glossina) transmit the protozoan pathogens that cause African sleeping sickness (Trypanosoma gambiense and T. rhodesiense). Sandflies (Phlebotomus) are vectors for the bacteria (Bartonella bacilliformis) that causes Oroa fever in South America. In parts of Asia and North Africa, they transmit viral agents that cause sandfly fever (three-day fever) and the protozoan pathogen (Leishmania) that causes leishmaniasis.

[0010] Therefore, effective insect control is needed to protect the crops we depend on for survival and to safeguard the health of humans and animals.

[0011] Here, we describe a combination of an insecticide (IA) and a cysteine-rich insecticidal peptide (CRIP). An IA is one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, poisons, toxicants, insecticides, pesticides, organic compounds, inorganic compounds, prokaryotes and / or their products (such as bacterial toxins) or eukaryotes and / or their products (such as fungal toxins). IAs can be combined to provide a greater insecticidal effect than the sum of the effects of any IA used alone.

[0012] CRIPs are peptides, polypeptides, and / or proteins containing cysteine ​​residues that, in some embodiments, are capable of forming disulfide bonds. These disulfide bonds form scaffold motifs that are observed in a variety of unrelated protein families. An example of a peptide belonging to the CRIP family is the inhibitory cystine knot (ICK) peptide. ICK peptides include many molecules with insecticidal activity. Such ICK peptides are typically toxic to naturally occurring biological target species, usually insects or some type of arachnid. ICK peptides may often possess venom of arthropod origin, such as scorpions or spiders.

[0013] Here, we describe novel combinations of insecticides, including IAs and CRIPs. For example, we particularly describe effective insecticide combinations comprising (1) one or more CRIPs or pharmaceutically acceptable salts thereof; one or more CRIP-insecticide proteins or pharmaceutically acceptable salts thereof; or combinations thereof; and (2) one or more insecticides (IAs), and methods of using them to protect the crops on which we depend for survival and to safeguard the health of humans and animals. Summary of the Invention

[0014] This invention describes how to combine CRIP and IA such that they provide an insecticidal effect greater than the sum of the insecticidal effects of either IA or CRIP used alone. This disclosure describes how to prepare and use combinations of CRIP and IA to kill and control insects, even insecticide-resistant insects, and even at low doses. Free from theoretical constraints, our understanding of CRIP and IA enables us to teach those skilled in the art to create novel methods, compositions, compounds (proteins and peptides), and procedures to protect plants and control insects.

[0015] This disclosure describes a combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA).

[0016] Furthermore, this disclosure describes a combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA), wherein the IA is a bacterial toxin, a fungal toxin, a lectin, an Azadirachta indica compound, a boron compound, a virus, or a combination thereof; and wherein the CRIP is a U1-funnel-web spider toxin-Ta1b peptide, a U1-funnel-web spider toxin-Ta1b variant peptide (TVP), an anemone toxin, an Av3 variant peptide (AVP), a Brazilian wandering spider (Phoneutria) toxin, or an atracotoxin (ACTX).

[0017] Furthermore, this disclosure describes a composition comprising a combination of a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA), and also comprising an excipient.

[0018] In addition, this disclosure describes a combination comprising one or more fermentation solids, spores or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and a U1-funnel web spider toxin-Ta1b peptide having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:1.

[0019] In addition, this disclosure describes a combination comprising one or more fermentation solids, spores or toxins isolated from Bacillus thuringiensis Goldsde subspecies EVB-113-19, and a U1-funnel web spider toxin-Ta1b variant polypeptide (TVP) having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:2.

[0020] In addition, this disclosure describes a combination comprising one or more fermentation solids, spores or toxins isolated from Bacillus thuringiensis Goldsde subspecies EVB-113-19, and an Av3-variant polypeptide (AVP) having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:67.

[0021] Furthermore, this disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis Goldsde subspecies EVB-113-19, and a Γ-CNTX-Pn1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:65.

[0022] In addition, this disclosure describes a combination comprising Beauveria bassiana strain ANT-03 spores and U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:61.

[0023] Furthermore, this disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. tenebrionis strain NB-176, and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:61.

[0024] In addition, this disclosure describes a combination comprising one or more fermentation solids, spores or toxins isolated from Bacillus thuringiensis Goldsde subspecies EVB-113-19, and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:61.

[0025] Furthermore, this disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. israelensis strain BMP 144, and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:61.

[0026] Furthermore, this disclosure describes a combination comprising a photorhabdus luminescens toxin and ACTX; wherein the photorhabdus luminescens toxin is a photorhabdus luminescens toxin complex (Tca), comprising TcaA (SEQ ID NO:616), TcaB (SEQ ID NO:617), TcaC (SEQ ID NO:618), and TcaZ (SEQ ID NO:619); and wherein the ACTX peptide is U+2-ACTX-Hv1a toxin (SEQ ID NO:61).

[0027] Furthermore, this disclosure describes a combination comprising snowdrop (Galanthus nivalis) lectin (GNA) and ACTX; wherein the GNA has the amino acid sequence shown in SEQ ID NO:35; and wherein the ACTX peptide is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:61.

[0028] Furthermore, this disclosure describes a combination comprising azadirachtin and ACTX; wherein the azadirachtin is an azadirachtin having the following chemical formula: C 35 H 44 O 16 ; and wherein ACTX is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:61.

[0029] Furthermore, this disclosure describes a combination comprising a boric acid compound and ACTX; wherein the boric acid compound has the chemical formula H3BO3; and wherein the ACTX peptide is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:61.

[0030] Furthermore, this disclosure describes a combination comprising codling moth (Cydia pomonella) granulovirus (CpGV) and ACTX; wherein CpGV is a codling moth granulovirus isolate V22 virus; and wherein the ACTX peptide is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:61.

[0031] Furthermore, this disclosure describes a method for controlling insects using a combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA), the method comprising providing a combination of at least one CRIP and at least one IA, and applying the combination comprising the cysteine-rich insecticidal peptide (CRIP) and the insecticide (IA) to the location of the insects.

[0032] Furthermore, this disclosure describes a method for controlling Bacillus thuringiensis toxin-resistant insects using a combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA), the method comprising providing a combination of at least one CRIP and at least one IA; and then applying the combination to the location of the insects.

[0033] Furthermore, this disclosure describes a method for combating, preventing, or suppressing pests, comprising applying an insecticidally effective amount of a combination of an insecticidal peptide (CRIP) rich in cysteine ​​and an insecticide (IA) to the location of the pest, or to plants or animals susceptible to pest infestation. Attached Figure Description

[0034] Figure 1 A graph depicting the 24-hour mortality rate of Aedes aegypti (mosquito) larvae after dietary incorporation assays using (1) U+2-ACTX-Hv1a with Bti, (2) Bti toxin alone, (3) U+2-ACTX-Hv1a alone, and (4) control (water) is shown.

[0035] Figure 2 A graph depicting the 3-day mortality rate of the lepidopteran species *Spodoptera exigua* following a foliar spray assay using a combination of *Bacillus thuringiensis* var. *kurstaki* toxin (Btk) and Γ-CNTX-Pn1a. Here, the treatments were (1) Γ-CNTX-Pn1a alone; (2) Btk toxin alone; (3) a combination of Γ-CNTX-Pn1a and Btk toxin; or (4) a control (0.125% Vintre, surfactant).

[0036] Figure 3A graph depicting the 3-day mortality rate of the lepidopteran species *Betula spp.* after foliar spraying with a Btk-binding Av3 variant peptide (AVP) is presented. Here, tests were performed on (1) AVP alone; (2) Btk toxin alone; (3) a combination of both AVP and Btk toxin; or (4) a control (0.125% Vintre, a surfactant). The AVP tested here was AVPb.

[0037] Figure 4 Chromatograms evaluating WT-Ta1b degradation in Helicoverpa zea gut extract (HGE), a simulated lepidopteran insect gut environment, are shown. Boxes indicate the main and secondary peaks, thus representing WT-Ta1b degradation. Nested insets show magnified and reduced views of the chromatograms. Boxes highlight peaks demonstrating proteolytic events, evidenced by the presence of two shoulder peaks: the smaller "shoulder" to the right of the main peak indicates partial proteolytic events.

[0038] Figure 5 Chromatograms evaluating the degradation of TVP-R9Q in *Helicoverpa armigera* gut extract (HGE), a simulated lepidopteran insect gut environment, are shown. Boxes indicate the presence of a single peak, thus representing the stability of TVP-R9Q. Nested insets show magnified and reduced views of the chromatograms. Here, the presence of a single main peak (shown in the box) indicates the stability of the TVP-R9Q peptide.

[0039] Figure 6 A graph depicting the results of defoliation assays on the lepidopteran species *Bollworm simonii* (corn earworm) is shown. Treatments were as follows: (1) WT-Ta1b alone; (2) Btk toxin alone; (3) a combination of both WT-Ta1b and Btk toxin; or (4) a control (0.125% Vintre, surfactant). Here, Btk toxin is shown as "Btk".

[0040] Figure 7 A graph depicting the results of defoliation assays on the lepidopteran species *Bollworm simonii* (corn earworm) was presented. Treatments were as follows: (1) TVP-R9Q alone; (2) Btk toxin alone; (3) a combination of both TVP-R9Q and Btk toxin; or (4) a control (0.125% Vintre, surfactant). Here, Btk toxin is shown as "Btk".

[0041] Figure 8A graph depicting the mortality results of the lepidopteran species *Bollworm simonii* (corn earworm) when tested with WT-Ta1b, Btk toxin, and combinations thereof. Treatments were as follows: (1) WT-Ta1b alone; (2) Btk toxin alone; (3) a combination of both WT-Ta1b and Btk toxin; or (4) a control (0.125% Vintre, surfactant). Here, Btk toxin is shown as "Btk".

[0042] Figure 9 A graph depicting the mortality results of the lepidopteran species *Bollworm simonii* (corn worm) when tested with TVP-R9Q, Btk toxin, and combinations thereof. Treatments were as follows: (1) TVP-R9Q alone; (2) Btk toxin alone; (3) a combination of both TVP-R9Q and Btk toxin; or (4) a control (0.125% Vintre, surfactant). Here, Btk toxin is shown as "Btk".

[0043] Figure 10 A graph depicting the 4-day mortality rate of the Coleopteran species *Alphitobius diaperinus* after dietary incorporation assays using (1) U+2-ACTX-Hv1a alone; (2) Btt toxin alone; (3) a combination of U+2-ACTX-Hv1a and Btt toxin; or (4) an untreated control (water).

[0044] Figure 11 A graph depicting the 4-day mortality rate of Colorado potato beetle (Leptinotarsadecemlineata) after spraying (1) U+2-ACTX-Hv1a alone; (2) Btt toxin alone; (3) a combination of U+2-ACTX-Hv1a and Btt toxin; or (4) an untreated control (water).

[0045] Figure 12 A graph depicting the 4-day mortality rates of corn earworm larvae treated with the following substances is presented: (a) water; (b) a luminescent bacillus toxin complex extract alone (4.75% v / v); (c) 10 mg / mL U+2-ACTX-Hv1a (1% w / v); and (d) a luminescent bacillus toxin complex extract (4.75% w / v) with 10 mg / mL U+2-ACTX-Hv1a (1% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water.

[0046] Figure 13A graph depicts the mortality rates of newborn corn earworms on day three after treatment with (a) 0 mg / mL GNA (0% w / v) and 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control); (b) 2.5 mg / mL GNA (0.25% w / v) and 0 mg / mL U+2-ACTX-Hv1a (0% w / v); (c) 0 mg / mL GNA (0% w / v) and 5 mg / mL U+2-ACTX-Hv1a (0.5% w / v); and (d) 2.5 mg / mL GNA (0.25% w / v) and 5 mg / mL U+2-ACTX-Hv1a (0.5% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water. Proportional mortality refers to the proportion of individual insects killed during the experiment, i.e., the proportion of dead individuals to the total number of individuals.

[0047] Figure 14 The image depicts the effects of treatments on the Spodoptera exigua (grass armyworm) on day three following treatment: (a) chitinase 0 μL / L (0% w / v), U+2-ACTX-Hv1a 0 mg / mL (0% w / v), sucrose (10% w / v); (b) chitinase 100 μL / L (0.01% w / v), U+2-ACTX-Hv1a 0 mg / mL (0% w / v), sucrose (10% w / v); (c) chitinase 0 μL / L (0% w / v), U+2-ACTX-Hv1a 5 mg / mL (0.5% w / v), sucrose (10% w / v); and (d) chitinase 100 μL / L (0.01% w / v), U+2-ACTX-Hv1a 5 mg / mL (0.5% w / v), sucrose (10% w / v). A graph showing the mortality rate of frugiperda larvae. Here, %w / v is the percentage w / v of the total volume of the composition, the remainder being water.

[0048] Figure 15 The chemical structure of azadirachtin, an insect growth regulator, was described.

[0049] Figure 16A graph depicts the mortality rates of newborn corn bollworms (Helicoverpa armigera) on day three after treatment with (a) 0 μL / L azadirachtin (0% v / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control); (b) 80 μL / L azadirachtin (0.008% v / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v); (c) 0 μL / L azadirachtin (0% v / v), 10 mg / mL U+2-ACTX-Hv1a (1% w / v); and (d) 80 μL / L azadirachtin (0.008% v / v), 10 mg / mL U+2-ACTX-Hv1a (1% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water.

[0050] Figure 17 A graph depicting the mortality rates of newborn larvae of *Acer micranthum* (small mealybug) on ​​day three after treatment with the following substances is presented: (a) 0 mg / mL U+2-ACTX-Hv1a (0% w / v), 0 mg / mL boric acid (0% w / v) (control); (b) 0 mg / mL U+2-ACTX-Hv1a (0% w / v), 2.5 mg / mL boric acid (0.25% w / v); (c) 1 mg / mL U+2-ACTX-Hv1a (0.1% w / v), 0 mg / mL boric acid (0% w / v); and (d) 1 mg / mL U+2-ACTX-Hv1a (0.1% w / v), 2.5 mg / mL boric acid (0.25% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water.

[0051] Figure 18 A graph depicting the mortality rates of newborn apple leafrollers (codling moths) on day seven after treatment with the following substances is presented: (a) 0 mg / mL Beauveria bassiana toxin (0% w / v); 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control); (b) 1.2 mg / mL Beauveria bassiana toxin (0.12% w / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v); (c) 0 mg / mL Beauveria bassiana toxin, 2 mg / mL U+2-ACTX-Hv1a (0.2% w / v); and (d) 1.2 mg / mL Beauveria bassiana toxin (0.12% w / v), 2 mg / mL U+2-ACTX-Hv1a (0.2% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water.

[0052] Figure 19The graph depicts the mortality rates of newborn apple leafrollers (codling moths) the day after treatment with (a) 0 μL / L CpGV (0% w / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control); (b) 58.5 μL / L CpGV (0.00585% w / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v); (c) 0 μL / L CpGV (0% w / v), 2 mg / mL U+2-ACTX-Hv1a (0.2% w / v); and (d) 58.5 μL / L CpGV (0.00585% w / v), 2 mg / mL U+2-ACTX-Hv1a (where 0.2% w / v is the percentage w / v of the total volume of the composition, the remainder being water).

[0053] Figure 20 A graph depicting the results of a dietary incorporation assay of diphenylfluorouracil with U+2-ACTX-Hv1a and the mortality rate of the corn ear borer (Bollworm) after 3 days is presented. As shown here, in the dietary incorporation assay of the corn ear borer (Bollworm), there is no evidence that the effect of diphenylfluorouracil combined with U+2-ACTX-Hv1a is greater than the sum of its effects. Here, U+2 refers to U+2-ACTX-Hv1a. The concentrations of diphenylfluorouracil were as follows: (a) 80 μL / L diphenylfluorouracil (0.008% w / v); (b) 8 μL / L diphenylfluorouracil (0.0008% w / v); (c) 0.8 μL / L diphenylfluorouracil (0.00008% w / v); and (d) 0 μL / L diphenylfluorouracil (0% w / v). 10 ppt Spear corresponds to 1 mg / mL (1% w / v) U+2-ACTX-Hv1a.

[0054] Figure 21 A graph depicting the results of a dietary incorporation assay of nanoparticles with U+2-ACTX-Hv1a and the mortality rate of the corn earworm (Bollworm) after 3 days is presented. As shown here, in the dietary incorporation assay of the corn earworm (Bollworm), there is no evidence that the effect of combining nanoparticles with U+2-ACTX-Hv1a is greater than the additive effect. Here, "Spear" refers to U+2-ACTX-Hv1a. The concentrations of the nanoparticles are as follows: (a) 50 nm aminated silica (2700 ppm); (b) 50 nm silica (2575 ppm); (c) 20 nm silica (1177 ppm); and (d) 10 nm silica (12500 ppm). Here, "U+2" corresponds to 5 ppt U+2-ACTX-Hv1a, i.e., 0.5 mg / mL (0.5% w / v of the total volume of the composition) of U+2-ACTX-Hv1a. Proportional mortality rate = number of dead insects divided by total number of insects. "UTC" refers to the untreated control (water).

[0055] Figure 22 A graph depicting the dose-response of mortality rates in a diet incorporation assay of corn earworm (Bollworm) after 3 days is presented. As shown here, in the diet incorporation assay of corn earworm (Bollworm), there is no evidence that the effect of combining cryolite with U+2-ACTX-Hv1a is greater than the summative effect. Here, U+2 refers to U+2-ACTX-Hv1a. The concentrations of the nanoparticles are as follows: (a) 10000 ppm; (b) 2000 ppm; (c) 400 ppm; and (d) 0 ppm. Here, 10 ppt "U+2" (i.e., U+2-ACTX-Hv1a) corresponds to 1 mg / mL (1% w / v of the total volume of the composition) of U+2-ACTX-Hv1a. Proportional mortality rate = number of dead insects divided by the total number of insects. Detailed Implementation

[0056] Definitions

[0057] "5'-end" and "3'-end" refer to directionality, that is, the end-to-end orientation of a nucleotide polymer (such as DNA). The 5'-end of a polynucleotide is the end of a polynucleotide that has a fifth carbon.

[0058] "5'- and 3'- homologous arms" or "5' and 3' arms" or "left arm and right arm" refer to polynucleotide sequences in vectors and / or targeting vectors that undergo homologous recombination with target genome sequences and / or endogenous genes of interest in the host organism in order to achieve successful genetic modification of chromosomal loci in the host organism.

[0059] "Γ-CNTX-Pn1a" or "γ-CNTX-Pn1a" or "γ-CNTX-Pn1a" or "γ" refers to an insecticidal neurotoxin derived from the Brazilian armed spider (Phoneutria nigriventer). Γ-CNTX-Pn1a targets the N-methyl-D-aspartate (NMDA) isoform and sodium channel of the ionotropic glutamate receptor (GRIN).

[0060] "ω / κ-HXTX-Hv1a" or "Omega / Kappa-HXTX-Hv1a" refers to an insecticidal toxin derived from the Australian Blue Mountain funnel-web spider (Hadronyche versuta). ω / κ-HXTX-Hv1a is an ACTX peptide, belonging to the insecticidal ICK peptide family isolated from spiders belonging to the Atracinae family. ω / κ-HXTX-Hv1a is a positive allosteric regulator of nicotinic acetylcholine receptors and can also be an insect voltage-gated Ca2+ receptor.2+ Channel and voltage gating K + Dual antagonists of the channel. See Chambers et al., “Insecticidal spider toxins are highaffinity positive allosteric modulators of the nicotinic acetylcholine receptor,” FEBS Lett., June 2019, Vol. 593, No. 12: 1336-1350; and Windley et al., “Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors,” Neuropharmacology, December 2017, Vol. 127: 224-242. The full text of these publications is incorporated herein by reference.

[0061] "ACTX," or "ACTX peptide," or "atracotoxin," refers to the insecticidal ICK peptide family isolated from spiders belonging to the subfamily Atracinae. One such spider is known as the Australian Blue Mountain Funnel-web Spider, scientifically named *Atracotoxinus australis*. Two examples of ACTX peptides from this species are Omega peptide and U peptide.

[0062] The “ADN1 promoter” refers to a DNA fragment consisting of the promoter sequence of the adhesion-deficient protein 1 gene from the yeast Schizosaccharomyces pombe.

[0063] "α-MF signaling" or "αMF secretion signaling" refers to proteins that direct newly formed recombinant polypeptides to the secretion pathway.

[0064] "Agriculturally acceptable carriers" encompass all adjuvants, inert components, dispersants, surfactants, thickeners, binders, etc., commonly used in pesticide formulation techniques; these are well-known to those skilled in the field of pesticide formulation.

[0065] “Agriculturally acceptable salt” is used in this document as a synonym for the term “pharmaceutically acceptable salt”.

[0066] "Agrobacterial infection" refers to a plant transformation method that uses Agrobacterium tumefaciens or Agrobacterium rhizogenes to introduce DNA into plant cells.

[0067] "Alignment" refers to the process of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide, or protein sequences) to determine the relationship between them. Alignment is typically performed by computer programs that apply various algorithms; however, it can also be performed manually. Alignment programs usually iterate through potential sequence alignments and score alignments using permutation tables, employing multiple strategies to achieve a potentially optimal alignment score. Commonly used alignment algorithms include, but are not limited to, CLUSTALW (see Thompson JD, Higgins DG, Gibson TJ, “CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, Nucleic Acids Research, Vol. 22: pp. 4673-4680, 1994), CLUSTALV (see Larkin MA et al., CLUSTALW2, ClustalW and ClustalX version 2, Bioinformatics, Vol. 23, No. 21: pp. 2947-2948, 2007), Mafft, Kalign, ProbCons, and T-Coffee (see Notredame et al., “T-Coffee: A novel method for multiple sequence alignments”, Journal of Molecular Biology, Vol. 302: pp. 205-217, 2000). Exemplary programs implementing one or more of the aforementioned algorithms include, but are not limited to, MegAlign (DNAStar, Inc., 3801 Regent St. Madison, Wis., 53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio (Accelrys, Inc., 10188 Teleesis Ct, Suite 100, San Diego, Calif., 92121), from DNAStar. In some implementations, alignment introduces “phase shift” and / or “vacancy” into one or both of the sequences being compared to maximize the similarity between the two sequences, and scoring refers to a process that quantitatively expresses the relevance of the aligned sequences.

[0068] "α-MF signaling" or "αMF secretion signaling" refers to proteins that direct newly formed recombinant polypeptides to the secretion pathway.

[0069] "Arachnida" refers to the class of arthropods. For example, in some implementation schemes, Arachnida may refer to spiders, scorpions, ticks, mites, harmless spiders, or sun spiders (solifuges).

[0070] “Av2” or “ATX-II” or “neurotoxin 2” or “Anemonia viridis toxin 2” or “δ-AITX-Avd1c” refers to a toxin isolated from the venom of the sea anemone *Anemonia sulcata*. An example of the Av2 polypeptide is a polypeptide having the amino acid sequence SEQ ID NO: 588.

[0071] “Av3” refers to a polypeptide isolated from sea anemones (Anemone scutellarioides) that targets receptor site 3 on the α-subunit III of a voltage-gated sodium channel. An example of an Av3 polypeptide is the Av3 polypeptide (NCBI accession number P01535.1) having the amino acid sequence SEQ ID NO:44.

[0072] “AVP” or “Av3 variant peptide” refers to an Av3 peptide sequence and / or a peptide encoded by a variant Av3 polynucleotide sequence that has been altered to produce a non-naturally occurring peptide and / or polynucleotide sequence.

[0073] “BAAS” refers to barley α-amylase signal peptide and is an example of ERSP. An example of BAAS is BAAS with the amino acid sequence SEQ ID NO:37 (NCBI accession number AAA32925.1).

[0074] "Biomass" refers to any measured plant product.

[0075] "Binary vector" or "binary expression vector" refers to an expression vector that can replicate itself in both E. coli and Agrobacterium strains. Furthermore, this vector contains a DNA region (often called t-DNA) enclosed by left and right boundary sequences, which is recognized by virulence genes, thereby being replicated by Agrobacterium and delivered into plant cells.

[0076] A "bp" or "base pair" refers to a molecule containing two chemical bases bonded together. For example, a DNA molecule consists of two intertwined strands, each with a backbone composed of alternating deoxyribose sugars and phosphate groups. Attached to each deoxyribose sugar is one of four bases: adenine (A), cytosine (C), guanine (G), or thymine (T), where adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.

[0077] "Bt toxin" refers to the fermentation solids, spores, and toxins produced by Bacillus thuringiensis (Bt), a Gram-positive sporoforming bacterium, such as Bacillus thuringiensis Goldsteine ​​(Btk), Bacillus thuringiensis Tenebrionoides (Btt), and Bacillus thuringiensis Israel (Bti). During sporulation, Bacillus thuringiensis produces crystalline proteins (i.e., protein inclusions) with insecticidal activity, called δ-endotoxins. In some embodiments, Bt toxins can be crystalline (Cry) proteins, cytolytic (Cyt) proteins, plant insecticidal proteins (Vip), or other toxins produced by Bacillus thuringiensis.

[0078] "Bt resistance" or "Bt resistance" or "Bt-resistant insects" or "Bacillus thuringiensis toxin-resistant insects" refers to a heritable change in the susceptibility of a pest population that is reflected in the repeated failure of products (such as Bt) to achieve the expected control levels when used against that pest species.

[0079] "C-terminus" refers to the free carboxyl group (i.e., -COOH) located at the end of a polypeptide.

[0080] “cDNA” or “copy DNA” or “complementary DNA” refers to a molecule complementary to an RNA molecule. In some embodiments, cDNA can be single-stranded or double-stranded. In some embodiments, cDNA can be double-stranded DNA synthesized from a single-stranded RNA template in a reverse transcriptase-catalyzed reaction. In other embodiments, “cDNA” refers to all nucleic acids sharing an arrangement of sequence elements found in naturally mature mRNA species, wherein the sequence elements are exons and 3' and 5' non-coding regions. Typically, mRNA species have consecutive exons, with intercalated introns removed by splicing of nuclear RNA to form consecutive open reading frames encoding proteins. In some embodiments, “cDNA” refers to DNA complementary to and derived from an mRNA template.

[0081] “CEW” refers to the corn ear worm.

[0082] See "Cutable connector" for connector.

[0083] "Cloning" refers to the process and / or method involving the insertion of a DNA fragment from one source (e.g., typically a gene of interest, such as tvp) and its recombination with a DNA fragment from another source (e.g., typically a vector, such as a plasmid), and guiding the replication of that recombinant DNA, or "recombinant DNA," typically by transforming that recombinant DNA into a bacterial or yeast host.

[0084] "Chimeric gene" refers to a DNA sequence that encodes a gene derived from a portion of one or more coding sequences to produce a new gene.

[0085] A “coding sequence” or “CDS” is a polynucleotide or nucleic acid sequence that, when placed under the control of appropriate regulatory sequences and in the presence of necessary transcriptional and / or translational molecules, can be transcribed (e.g., in the case of DNA) or translated (e.g., in the case of mRNA) into a peptide, polypeptide, or protein. The boundaries of a coding sequence are defined by a translation start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. The transcription termination sequence is typically located at the 3' end of the coding sequence. In some embodiments, the coding sequence may have untranslated regions flanking the 5' and / or 3' ends. In some embodiments, the coding sequence can be used to produce peptide, polypeptide, or protein products. In some embodiments, the coding sequence may or may not be fused with another coding sequence or a localization signal such as a nuclear localization signal. In some embodiments, the coding sequence may be cloned into a vector or expression construct, integrated into the genome, or exist as a DNA fragment.

[0086] "Codon optimization" refers to the process by which one or more endogenous, natural, and / or wild-type codons are replaced by codons that still encode the same amino acid but are preferred in the corresponding host.

[0087] "Combination" means any association between two or more items. The association may be spatial, temporal, and / or indicate the use of the two or more items for a common purpose. For example, a combination may be any spatiotemporal association, mixture, or arrangement of the following: (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; CRIP-insecticides, or pharmaceutically acceptable salts thereof; or combinations thereof; and (2) one or more insecticides (IA) as described herein, wherein (1) and (2) are used for the common purpose of controlling or combating insect pests, causing the insect pests to die, stop or slow their movement; stop or slow their feeding; stop or slow their growth; become confused (e.g., regarding navigation, food location, sleep behavior, and / or mating); be unable to pupate; interfere with reproduction; and / or prevent the insect from producing offspring and / or prevent the insect from producing fertile offspring.

[0088] Unless the context otherwise indicates, the term “combination” can include simultaneous, separate or sequential application: application in any order of (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticides, or pharmaceutically acceptable salts thereof; or combinations thereof; with (2) one or more insecticides (IA).

[0089] It should be understood that in some embodiments, (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; CRIP-insecticide protein, or pharmaceutically acceptable salts thereof; or combinations thereof; and (2) one or more insecticides (IAs) are considered to be applied as a “combination” or “combination” when the pest or the location of the pest is exposed to both (1) and (2), or when the location to be protected from pests (e.g., plants) is treated with simultaneous exposure to (1) and (2). In some embodiments, (1) the one or more CRIPs, or pharmaceutically acceptable salts thereof; CRIP-insecticide protein, or pharmaceutically acceptable salts thereof; or combinations thereof; and (2) each of the one or more insecticides (IAs) may be applied sequentially or at different times—in fact, it is not necessary to apply single doses of different agents simultaneously or in the same composition. Rather, they are considered to be applied in combination as long as (1) the one or more CRIPs, or pharmaceutically acceptable salts thereof; CRIP-insecticide protein, or pharmaceutically acceptable salts thereof; or combinations thereof; and (2) the one or more insecticides (IA) both retain insecticidal effect (i.e. have insecticidal activity).

[0090] In some implementations, “combination” means the simultaneous application of: (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticides, or pharmaceutically acceptable salts thereof; or combinations thereof; with (2) one or more insecticides (IA).

[0091] In some implementations, “combination” means the separate application of: (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticides, or pharmaceutically acceptable salts thereof; or combinations thereof; with (2) one or more insecticides (IA).

[0092] In other embodiments, “combination” means the sequential application of (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticides, or pharmaceutically acceptable salts thereof; or combinations thereof; with (2) one or more insecticides (IA).

[0093] In some embodiments of the invention, where the combination is applied sequentially or separately, the delay in application of the second component should not result in the complete loss of the beneficial effects of the combination (i.e., (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticides, or pharmaceutically acceptable salts thereof; or combinations thereof; or (2) a combination with one or more insecticides (IA)). When the combination of two or more components is applied separately or sequentially, it should be understood that the dosage regimen for each component may be different and independent of the other components.

[0094] In some embodiments, the one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticide proteins, or pharmaceutically acceptable salts thereof; or combinations thereof, may be applied on the same day as the one or more insecticides (IA). In other embodiments, the one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticide proteins, or pharmaceutically acceptable salts thereof; or combinations thereof, may be applied in the same week or month as the one or more insecticides (IA).

[0095] In some implementations, the combination may be a “mixture”. As used herein, a “mixture” means a combination of two or more agents, such as (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticides, or pharmaceutically acceptable salts thereof; or a combination thereof; with (2) one or more insecticides (IAs) that are in physical and / or chemical contact with each other.

[0096] "Complementarity" refers to the topological compatibility or matching of the interaction surfaces of two polynucleotides as understood by those skilled in the art. Therefore, two sequences are "complementary" if they can hybridize to form a stable antiparallel double-stranded nucleic acid structure. The first polynucleotide is complementary to the second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide-binding partner of the second polynucleotide, or if the first polynucleotide hybridizes with the second polynucleotide under strict hybridization conditions. Thus, a polynucleotide with the sequence 5'-TATAC-3' is complementary to a polynucleotide with the sequence 5'-GTATA-3'.

[0097] "Conditioned culture medium" refers to a cell culture medium that has been used by cells and is rich in cell derivatives but does not contain cells.

[0098] "Cone snail" or "cone snail" refers to an organism belonging to the genus *Conus*, a predatory marine gastropod. For example, in some implementations, a cone snail can be one of the following species: *Conus amadis*; *Conus catus*; *Conus ermineus*; *Conus geographus*; *Conus gloriamaris*; *Conus kinoshitai*; *Conus magus*; *Conus marmoreus*; *Conus purpurascens*; *Conus stercusmuscarum*; *Conus striatus*; *Conus textile*; or *Conus tulipa*.

[0099] "Conotoxin" refers to a toxin isolated from the cone snail that works by interfering with neuronal communication. For example, in some embodiments, conotoxin can be α-, ω-, μ-, δ-, or κ-conotoxin. In short, α-conotoxin (and αA-conotoxin and φ-conotoxin) targets nicotinic ligand-gated channels; ω-conotoxin targets voltage-gated calcium channels; μ-conotoxin targets voltage-gated sodium channels; δ-conotoxin targets voltage-gated sodium channels; and κ-conotoxin targets voltage-gated potassium channels.

[0100] "Copy number" refers to the same number of copies of a vector, expression cassette, amplification unit, gene, or any defined nucleotide sequence present in the host cell at any given time. For example, in some embodiments, a gene or another defined chromosomal nucleotide sequence may exist in one, two, or more copies on the chromosome. Autonomously replicating vectors may exist in one or several hundred copies per host cell.

[0101] "CRIP" refers to a cysteine-rich insecticidal peptide. A CRIP is a peptide rich in cysteine ​​residues, which in some embodiments are operable to form disulfide bonds between such cysteine ​​residues. In some embodiments, in a protein or peptide having at least 10 amino acids, a CRIP contains at least four (4), sometimes six (6), and sometimes eight (8) cysteine ​​amino acids, wherein the cysteines form two (2), three (3), or four (4) disulfide bonds. In some embodiments, the disulfide bonds contribute to the folding, three-dimensional structure, and activity of the insecticidal peptide. Cysteine-cysteine ​​disulfide bonds and the three-dimensional structure they form play an important role in the insecticidal properties of these insecticidal peptides. In some embodiments, a CRIP may or may not contain an inhibitory cystine knot (ICK) motif. For example, in some embodiments, CRIPs with an ICK motif can be ACTX peptides derived from spiders; in other embodiments, CRIPs without an ICK motif, i.e., non-ICK CRIPs, can be peptides such as Av2 and Av3, which are peptides isolated from sea anemones. Non-ICK CRIPs may have 4 to 8 cysteine ​​residues that form 2 to 4 disulfide bonds. These cysteine-cysteine ​​disulfide bond-stabilized toxic peptides (CRIPs) can exhibit significant stability upon exposure to the environment. Many CRIPs are isolated from venomous animals such as spiders, scorpions, snakes, and snails, as well as sea anemones, and they are toxic to insects.

[0102] "CRIP construct" refers to the three-dimensional arrangement / orientation of a motif (e.g., CRIP-insecticide protein) of a peptide, polypeptide, and / or operatively linked polypeptide fragment. For example, a CRIP expression ORF may include one or more of the following components or motifs: CRIP; endoplasmic reticulum signal peptide (ERSP); adaptor peptide (L); translationally stable protein (STA); or any combination thereof. Furthermore, as used herein, the term "CRIP construct" is used to describe the name and / or orientation of the structural motif. In other words, a CRIP construct describes the arrangement and orientation of the components or motifs contained in a given CRIP expression ORF. For example, in some embodiments, a CRIP construct describes, but is not limited to, the orientation of one of the following CRIP-insecticide proteins: ERSP-CRIP, ERSP-(CRIP). N ERSP-CRIP-L, ERSP-(CRIP) N -L, ERSP-(CRIP-L) N ,ERSP-L-CRIP,ERSP-L-(CRIP) N ERSP-(L-CRIP) N ,ERSP-STA-CRIP,ERSP-STA-(CRIP) N, ERSP-CRIP-STA, ERSP-(CRIP) N -STA, ERSP-(STA-CRIP) N , ERSP-(CRIP-STA) N , ERSP-L-CRIP-STA, ERSP-L-STA-CRIP, ERSP-L-(CRIP-STA) N , ERSP-L-(STA-CRIP) N , ERSP-L-(CRIP) N -STA, ERSP-(L-CRIP) N -STA, ERSP-(L-STA-CRIP) N , ERSP-(L-CRIP-STA) N , ERSP-(L-STA) N -CRIP, ERSP-(L-CRIP) N -STA, ERSP-STA-L-CRIP, ERSP-STA-CRIP-L, ERSP-STA-L-(CRIP) N , ERSP-(STA-L) N -CRIP, ERSP-STA-(L-CRIP) N , ERSP-(STA-L-CRIP) N , ERSP-STA-(CRIP) N -L, ERSP-STA-(CRIP-L) N , ERSP-(STA-CRIP) N -L, ERSP-(STA-CRIP-L) N , ERSP-CRIP-L-STA, ERSP-CRIP-STA-L, ERSP-(CRIP) N -STA-L ERSP-(CRIP-L) N -STA, ERSP-(CRIP-STA) N -L, ERSP-(CRIP-L-STA) N or ERSP-(CRIP-STA-L) N ; where N is an integer from 1 to 200. See also "structural motif".

[0103] A “CRIP ORF diagram” refers to the composition of one or more CRIP ORFs, as shown in a diagram or equation. For example, a “CRIP ORF diagram” can be written using acronyms or shorthand to represent the DNA fragments contained within the ORF. Thus, in one example, a “CRIP ORF diagram” could describe polynucleotide fragments encoding ERSP, L, STA, and CRIP, illustrated in equation form as “ersp” (i.e., the polynucleotide sequence encoding the ERSP polypeptide); “linker” or “L” (i.e., the polynucleotide sequence encoding the linker polypeptide); “sta” (i.e., the polynucleotide sequence encoding the STA polypeptide); and “crip” (i.e., the polynucleotide sequence encoding CRIP). An example of a CRIP ORF diagram is “ersp-sta-(linker)”. i -crip j ) N "or "ersp-(crip j -linker i ) N -sta and / or any combination of their DNA fragments.

[0104] "CRIP polynucleotide" refers to a polynucleotide or polynucleotide group operable to express and / or encode an insecticidal protein that contains one or more CRIPs in addition to one or more non-CRIP polypeptides or proteins.

[0105] "CRIP-insecticide protein" refers to any protein, peptide, polypeptide, amino acid sequence, conformation, or arrangement consisting of: (1) at least one CRIP, or two or more CRIPs; and (2) additional peptides, polypeptides, or proteins, wherein said additional peptides, polypeptides, or proteins have the ability to: (a) increase insect mortality and / or inhibit insect growth when insects are exposed to CRIP-insecticide protein relative to CRIP alone; (b) increase the expression of said CRIP-insecticide protein, for example, in a host cell or expression system; and / or (c) affect the post-translational processing of CRIP-insecticide protein.

[0106] In some embodiments, the insecticidal protein may comprise one or more CRIPs disclosed herein. In some embodiments, the CRIP-insectic protein may be a polymer comprising two or more CRIPs. In some embodiments, the insecticidal protein may comprise a CRIP homopolymer, such as two or more CRIP monomers that are the same CRIP. In some embodiments, the insecticidal protein may comprise a CRIP heteropolymer, such as two or more CRIP monomers, wherein the CRIP monomers are different.

[0107] In some implementations, CRIP-insecticide protein can be a polymer of amino acids that, when properly folded or in its most natural thermodynamic state, exerts insecticidal activity against one or more insects.

[0108] In some embodiments, the CRIP-insectic protein may be a polymer comprising two or more CRIPs, wherein the CRIPs are operatively linked via a linker peptide (e.g., a cleavable and / or non-cleavable linker). In some embodiments, the CRIP-insectic protein may refer to one or more CRIPs operatively linked to one or more proteins, such as a stabilizing domain (STA), an endoplasmic reticulum signaling protein (ERSP), an insect-cleavable or insect-non-cleavable linker (L), and / or any other combination thereof. In some embodiments, the CRIP-insectic protein may be a non-naturally occurring protein, including (1) a wild-type CRIP protein; and (2) additional peptides, polypeptides, or proteins, such as ERSPs, linkers, STAs, UBIs, or histidine tags or similar markers.

[0109] "Cultivation" or "cell culture" refers to maintaining cells in an artificial in vitro environment.

[0110] “Cultivation” refers to the propagation of an organism on or in a variety of culture media. For example, the term “cultivation” can mean the growth of a cell population in a liquid or solid culture medium under suitable conditions. In some embodiments, cultivation refers to the fermentation (typically in a vessel or reactor) of recombinantly producing heterologous peptides of interest and / or other desired end products.

[0111] "Cysteine" refers to the oxidized cysteine ​​dimer. Cysteine ​​is a sulfur-containing amino acid obtained by oxidizing two cysteine ​​molecules and linking them together by disulfide bonds.

[0112] "Defined composition medium" refers to a medium composed of known chemical components but without crude protein extracts or byproducts such as yeast extracts or peptones.

[0113] “Degeneracy” or “codon degeneracy” refers to the phenomenon that an amino acid can be encoded by different nucleotide codons. Therefore, the nucleic acid sequence of a nucleic acid molecule encoding a protein or polypeptide can vary due to degeneracy. Because of the degeneracy of the genetic code, many nucleic acid sequences can encode a given polypeptide with specific activities; this article considers such functionally equivalent variants.

[0114] "Desmethylrimomycin B" refers to [(5R,7R,8R,9R,10R,13S,17S)-17-[(3R)-5-hydroxyoxacyclopenten-3-yl]-4,4,8,10,13-pentamethyl-3,16-dioxo-6,7,9,11,12,17-hexahydro-5H-cyclopenta[a]phenanthrene-7-yl]acetate.

[0115] A "disulfide bond" refers to a covalent bond between two cysteine ​​residues derived by coupling two thiol groups on their side chains.

[0116] "DNA" refers to deoxyribonucleic acid, a polymer containing one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form. For example, one or more nucleotides produce polynucleotides.

[0117] “dNTP” refers to nucleoside triphosphates that make up DNA and RNA.

[0118] "Dual expression cassette" refers to two heterologous polypeptide expression cassettes contained on the same vector.

[0119] "Double transgenic peptide expression vector" or "double transgenic expression vector" refers to a yeast expression vector containing two copies of a heterologous peptide expression cassette.

[0120] "Endogenous" refers to processes that occur and / or exist naturally in an organism, such as molecules or activities that are already present in the host cell prior to a specific genetic manipulation.

[0121] An "enhancer element" is a DNA sequence that is operatively linked to a promoter and can enhance the transcriptional activity of the promoter relative to the transcriptional activity produced by the promoter in the absence of an enhancer element.

[0122] The ER, or endoplasmic reticulum, is a common subcellular organelle in all eukaryotes, where some post-translational modification processes occur.

[0123] "ERSP," or "Endoplasmic Reticulum Signal Peptide," is an N-terminal sequence of amino acids that is recognized and bound by host cell signaling granules during the translation of the mRNA molecule encoding CRIP. These granules move the protein-translation ribosome / mRNA complex to the ER in the cytoplasm. As a result, protein translation is paused until it docks with the ER, where translation resumes and the resulting protein is injected into the ER.

[0124] “ersp” refers to the polynucleotide that encodes the peptide ERSP.

[0125] "ER transport" refers to the transport of proteins expressed by cells to the ER for post-translational modification, sorting, and transport.

[0126] "Excipient" refers to any pharmacologically inactive, natural or synthetic component or substance formulated together (e.g., simultaneously) or subsequently with the active ingredient of the present invention (i.e., CRIP or CRIP-insecticide protein). In some embodiments, the excipient can be any additive, adjuvant, binder, thickener, carrier, coating, diluent, disintegrant, filler, flow aid, lubricant, preservative, solvent, or combination thereof, with which the CRIP or CRIP-insecticide protein of the present invention can be administered, and / or which can be used to prepare the compositions of the present invention. Excipients include any such substances known in the art that are non-toxic and do not interact with other components of the composition. In some embodiments, when preparing the composition, the excipient may be formulated together with CRIP or CRIP-insecticide protein for the purpose of swelling the composition (therefore commonly referred to as a swelling agent, filler, or diluent). In other embodiments, the excipient may be used to impart enhancement to the active ingredient in the final dosage form, such as promoting absorption and / or dissolution. In other embodiments, the excipient may be used to provide stability or prevent contamination (e.g., microbial contamination). In other embodiments, excipients may be used to impart physical properties to the composition (e.g., a composition in the physical form of dried granules or dried flowable powder). References to excipients include one or more such excipients. Suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, EWMartin, the entire disclosure of which is incorporated herein by reference.

[0127] An “expression cassette” refers to (1) a DNA sequence of interest, such as a polynucleotide operable to encode CRIP; and one or more of the following: (2) a promoter, terminator, and / or enhancer element; (3) a suitable mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) an intron; and / or (6) a posttranscriptional regulatory element. A combination of (1) and at least one of (2) through (6) is called an “expression cassette.” In some embodiments, a plurality of expression cassettes cloned into a vector may be present. For example, in some embodiments, a first expression cassette may be present, which contains a polynucleotide operable to encode CRIP. In an alternative embodiment, two expression cassettes are present, each containing a polynucleotide operable to encode CRIP (i.e., a dual expression cassette). In other embodiments, three expression cassettes operable to encode CRIP are present (i.e., a triple expression cassette). In some embodiments, a dual expression cassette can be generated by subcloning a second expression cassette into a vector containing a first expression cassette. In some implementations, a three-expression cassette can be generated by subcloning a third expression cassette into a vector containing the first and second expression cassettes. Methods involving expression cassettes and cloning techniques are well known in the art and are described herein. See also CRIP expression cassette.

[0128] "Expression ORF" refers to the nucleotide that encodes a protein complex and is defined as the nucleotide in the ORF.

[0129] “FECT” refers to a transient plant expression system using a foxtail mosaic virus with the outer coat protein gene and three gene frames removed.

[0130] "Fermented beer" refers to used fermentation medium, i.e., the supernatant of the fermentation medium after the removal of organisms, which has been inoculated with and consumed by transformed host cells (e.g., yeast cells operable to express the CRIP of the present invention). In some embodiments, fermented beer refers to the solution recovered after fermentation of the transformed host cells. The term "fermentation" is broadly defined as the enzymatic and anaerobic or aerobic decomposition of organic matter (e.g., carbon substrate) by microorganisms under controlled conditions (e.g., temperature, oxygen, pH, nutrients, etc.) to produce fermentation products (e.g., one or more peptides of the present invention). Although fermentation is generally described as a process that occurs under anaerobic conditions, as used herein, this does not mean that the term is limited to strictly anaerobic conditions, as the term "fermentation" as used herein can also occur in the presence of oxygen.

[0131] "Fermentation solids" refers to the solids (including dissolved solids) retained from fermented beer during the yeast-based fermentation process, and consists primarily of salts, complex protein sources, vitamins, and other yeast byproducts, with a molecular weight cutoff of approximately 200 kDa to approximately 1 kDa.

[0132] "GFP" refers to the green fluorescent protein of the jellyfish (Aequorea victoria).

[0133] "HIS" or "His" refers to histidine. For example, in some embodiments, "HIS" or "His" may refer to a histidine tag, such as a histidine tag having the amino acid sequence shown in SEQ ID NO:591.

[0134] "Homologous" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if a position in each of two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of shared matching or homologous positions divided by the number of compared positions × 100. Therefore, in some embodiments, the term "homologous" refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if a position in each of two DNA molecules is occupied by adenine—then the molecules are homologous at that position. Homology between two sequences is a function of the number of shared matching or homologous positions. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. For example, DNA sequences ATTGCC and TATGGC share 50% homology.

[0135] When used in relation to nucleic acids, the term "homology" refers to the degree of complementarity. Partial homology or complete homology can exist, and therefore they are identical. "Sequence identity" is a measure of the correlation between two or more nucleic acids and is given as a percentage of the total comparison length. Identity calculations take into account those nucleotide residues that are identical in their respective larger sequences and those that are in the same relative positions.

[0136] Homologous recombination refers to the event in which a DNA fragment is replaced by another DNA fragment having the same (homologous) or nearly identical regions. For example, in some implementations, homologous recombination refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. In short, homologous recombination is most widely used by cells to precisely repair harmful breaks that occur on the two strands of DNA, called double-strand breaks. Although homologous recombination varies considerably between different organisms and cell types, most forms involve the same basic steps: after a double-strand break occurs, a portion of DNA around the 5' end of the break is removed during a process called excision. In the subsequent strand invasion step, the protruding 3' end of the broken DNA molecule "invades" an unbroken, similar or identical DNA molecule. After strand invasion, the further sequence of events can follow either of two main pathways: the double-strand break repair pathway or the synthesis-dependent strand annealing pathway. Homologous recombination is conserved in all three domains of life as well as viruses, suggesting it is a virtually universal biological mechanism. For example, in some embodiments, homologous recombination can occur using site-specific integration (SSI) sequences, thereby establishing a strand exchange event between nucleic acid sequences with substantially similar nucleotide compositions. These exchange events can occur between sequences contained in the targeted construct of the invention (i.e., SSI sequences) and endogenous genomic nucleic acid sequences (e.g., polynucleotides encoding peptide subunits). Furthermore, in some embodiments, more than one site-specific homologous recombination event may occur, resulting in a substitution event in which a nucleic acid sequence contained in the targeted construct has replaced a specific sequence present in the endogenous genomic sequence.

[0137] "ICK motif," "ICK motif protein," "inhibitory cystine knot motif," "ICK peptide," "cystine knot motif," or "cystine knot peptide" refers to a peptide of 16 to 60 amino acids having at least six cysteine ​​core amino acids. These cysteine ​​core amino acids have three disulfide bonds, three of which are covalent bonds, and among the six cysteine ​​residues, the covalent disulfide bonds are located between the first and fourth, second and fifth, and third and sixth cysteine ​​residues of the six core cysteine ​​amino acids, starting from the N-terminal amino acid. Typically, this type of peptide contains a β-hairpin secondary structure, usually composed of residues located between the fourth and sixth core cysteine ​​residues of the motif. The hairpin is stabilized by structural crosslinks provided by the three disulfide bonds of the motif. Note that additional cysteine / cystine or cysteine ​​amino acids may be present in the inhibitory cystine knot motif.

[0138] “ick” refers to the nucleotide that encodes the ICK motif protein.

[0139] "ICK motif protein expression ORF" or "expression ORF" refers to the nucleotides that encode the ICK motif protein complex and is defined as nucleotides in the ORF.

[0140] "ICK motif protein expression vector" or "ICK expression vector" refers to a binary vector containing an expression ORF. The binary vector also contains the necessary transcription promoter and terminator sequences surrounding the ORF to promote the expression of the ORF and its encoded protein.

[0141] "Identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. The term "identity" also means the degree of sequence correlation between polypeptide or polynucleotide sequences, as determined by matching strings of such sequences. "Identity" and "similarity" can be readily calculated by any of the numerous methods known to those skilled in the art, including but not limited to those described below: Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., Vol. 48: p. 1073, 1988. The full text of these publications is incorporated herein by reference. Furthermore, methods for determining identity and similarity are encoded in publicly available computer programs. For example, in some implementations, methods for determining identity and similarity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., Nucleic Acids Research, Vol. 12, No. 1: p. 387, 1984), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol., Vol. 215: pp. 403–410 (1990)).The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md., 20894; Altschul, S. et al., J.Mol.Biol., Vol. 215: pp. 403-410, 1990), the full text of which is incorporated herein by reference.

[0142] "IGER" refers to the name of a short peptide based on its actual sequence, which is determined by its single-letter code. It is an example of an intercalation linker.

[0143] "In vivo" refers to the natural environment (e.g., an animal or a cell) and the processes or reactions that occur in the natural environment.

[0144] "Inactivation" refers to a state in which something is not in use, such as being in a dormant and / or non-functional state. For example, when used in the context of a gene or when referring to a gene, the term inactivation means that the gene is no longer actively synthesizing gene products, translating gene products into proteins, or otherwise enabling the gene to perform its normal functions. For example, in some embodiments, the term inactivation may refer to the failure of gene transcription of RNA, failure of RNA processing (e.g., pre-mRNA processing, RNA splicing, or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and / or inhibition and / or interference with any of the following molecular polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors involved in any of the above processes.

[0145] "Inoperable" refers to a situation where something is not functioning, malfunctioning, or no longer able to function. For example, when used in the context of a gene or when referring to a gene, the term "inoperable" means that the gene is no longer able to function permanently or temporarily as it normally would. For example, in some embodiments, "inoperable" means that a gene is no longer able to synthesize a gene product, translate the gene product into a protein, or otherwise fail to perform its normal function. For example, in some embodiments, the term inoperable may refer to failure of gene transcription of RNA, failure of RNA processing (e.g., pre-mRNA processing, RNA splicing, or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and / or inhibition and / or interference with any of the following molecular polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors involved in any of the above processes.

[0146] The term "insect" includes all organisms in the class Insecta. The term "pre-adult" insect refers to any form of organism prior to the adult stage, including, for example, eggs, larvae, and nymphs. As used herein, the term "insect" refers to any arthropod and nematode, including mites, and insects known to infest all crops, vegetables, and trees, including insects considered pests in forestry, horticulture, and agriculture. Examples of specific crops that can be protected using the methods disclosed herein are soybeans, corn, cotton, alfalfa, and vegetable crops. A list of specific crops and insects is included herein.

[0147] The term "insect gut environment" or "gut environment" refers to the specific pH and protease conditions found in the foregut, midgut, or hindgut of an insect or insect larva.

[0148] "Insect hemolymphatic environment" refers to specific pH and protease conditions found in insects or insect larvae.

[0149] "Insecticidal activity" means that when an insect is exposed to a compound, reagent, or peptide, or afterward, the insect dies, stops or slows its movement; stops or slows its feeding; stops or slows its growth; becomes confused (e.g. about navigation, locating food, sleep behavior, and / or mating); is unable to pupate; interferes with reproduction; and / or prevents the insect from producing offspring and / or prevents the insect from producing fertile offspring.

[0150] "Insecticide" or "IA" or "reagent" means one or more chemical substances, molecules, nucleotides, polynucleotides, RNA, DNA, peptides, polypeptides, proteins, lipids, glycolipids, enzymes, toxins, poisons, toxicants, insecticides, pesticides, organic compounds, inorganic compounds, viruses, prokaryotes or eukaryotes (and reagents produced by said prokaryotes or eukaryotes). In some embodiments, IA includes, but is not limited to, members selected from the following categories: RNAi; stomach poisons; type O chitin biosynthesis inhibitors; type I chitin biosynthesis inhibitors; insect viruses; compounds isolated from neem (Azadirachta taindica); compounds having an unknown MOA; bacteria (and their products); fungi (and their products); nematodes (and their products); plant extracts; mechanical interferences; fluorescent whitening agents; silica nanospheres; chitinases; lectins; membrane attack complex / perforin (MACPF) proteins; plant virus coat protein-toxin fusions; glycan-binding domain / toxin fusion proteins; acetylcholinesterase (AChE) inhibitors; GABA-gated chloride channel blockers; sodium channel modulators; nicotinic acetylcholine receptor (nAChR) competitive modulators; nicotinic acetylcholine receptor (nAC) competitive modulators. hR) allosteric modulators - site I; glutamate-gated chloride channel (GluCl) allosteric modulators; juvenile hormone mimics; other non-specific (multi-site) inhibitors; stringer TRPV channel modulators; mite growth inhibitors; mitochondrial ATP synthase inhibitors; uncoupling agents by disrupting proton gradient oxidative phosphorylation; nicotinic acetylcholine receptor (nAChR) channel blockers; molting disruptors (Diptera); molting hormone receptor agonists; octopamine receptor agonists; mitochondrial complex III electron transport inhibitors; mitochondrial complex I electron transport inhibitors; voltage-dependent sodium channel blockers; acetyl-CoA carboxylase inhibitors; mitochondrial complex IV electron transport inhibitors; mitochondrial complex II electron transport inhibitors; reniform base receptor modulators; stringer modulators - undefined target sites; or GABA-gated chloride channel allosteric modulators. In some embodiments, the insecticide may be a polymer of amino acids, peptides, polypeptides, or proteins; such peptides-IA may be prepared and / or used according to any of the methods described herein related to peptides and / or proteins.

[0151] "Integrative expression vector" or "integrative vector" refers to a yeast expression vector that can insert itself into a specific locus in the yeast cell genome and stably become part of the yeast genome.

[0152] "Insecticide resistance" or "insecticide-resistant insects" refers to a genetic variation in the sensitivity of a pest population to an insecticide, which is reflected in the repeated failure of the insecticide to achieve the expected level of control when used against a pest species.

[0153] "Intercalation linkers" are short peptide sequences that separate different parts of a protein, or short DNA sequences placed in the reading frame of an ORF to separate upstream and downstream DNA sequences. For example, in some embodiments, intercalation linkers can be used to allow proteins to achieve their independent secondary and tertiary structure formation during translation. In some embodiments, intercalation linkers may be resistant or readily cleaved in plant cell environments, insect and / or lepidopteran gut environments, and insect and lepidopteran hemolymph environments.

[0154] "Separated" means a substance and / or component separated from its natural environment. For example, a toxin separated from a given genus or species means a toxin separated from its natural environment, such as a toxin removed from a WT organism.

[0155] "Kappa-ACTX peptide" refers to an excitoxin that inhibits calcium-activated potassium (KCa) channels (Slo type) in insects. As used herein, "Kappa-ACTX peptide" may refer to a peptide isolated from the Australian Blue Mountain funnel-web spider (Australasian funnel-web spider) or its variants.

[0156] “kb” stands for kilobase, or 1000 bases. As used herein, the term “kb” refers to the length of a nucleic acid molecule. For example, 1kb refers to a nucleic acid molecule that is 1000 nucleotides long. A 1kb length of double-stranded DNA contains two thousand nucleotides (i.e., one thousand nucleotides per strand). Alternatively, a 1kb length of single-stranded RNA contains one thousand nucleotides.

[0157] “kDa” refers to kilodaltons, which is equal to 1,000 Daltons; “Dalton” or “Da” is the unit of molecular weight (MW).

[0158] "Knock-in" (or "knock-in", "knocks-in", or "knocking-in") refers to the replacement of an endogenous gene with a foreign or heterologous gene or a portion thereof. For example, in some embodiments, the term "knock-in" refers to the introduction of a nucleic acid sequence encoding a desired protein into a target locus via homologous recombination, thereby causing the expression of the desired protein. In some embodiments, a "knock-in" mutation may modify a gene sequence to produce a loss-of-function or gain-of-function mutation. The term "knock-in" may also refer to a procedure that introduces a foreign or heterologous polynucleotide sequence or fragment thereof into a genome (e.g., "they were knocked in" or "they knocked in a heterologous gene") or the resulting cell and / or organism (e.g., "the cell is knocked in" or "the animal is knocked in").

[0159] "Knockout" (or "knock-out", "knocks-out", or "knocking-out") refers to the partial or complete suppression of the expression of a gene product (e.g., mRNA) encoding a protein from an endogenous DNA sequence in a cell. In some embodiments, "knockout" can be achieved by targeting and deleting the entire gene or a portion of a gene that encodes a peptide, polypeptide, or protein. As a result, the deletion can inactivate, partially inactivate, render inoperable, partially inoperable, or otherwise reduce the expression of the gene or its product in any cell throughout the organism and / or in the cells where it is normally expressed. The term "knockout" can refer to a procedure that completely or partially inactivates or renders an endogenous gene (e.g., "they were knocked out" or "they knocked out an endogenous gene") or the resulting cell and / or organism (e.g., "the cell is knocked out" or "the animal is knocked out"), or renders it completely or partially inoperable.

[0160] "Knockout Dose 50" or "KD" 50 "This refers to the median dose required to cause paralysis or cessation of movement in 50% of populations, such as those of houseflies (Musca domestica) and / or Aedes aegypti (mosquitoes)."

[0161] “l” or “linker” refers to the nucleotide that encodes the linker peptide.

[0162] In the appropriate context, "L" refers to a linker peptide that connects a translationally stable protein (STA) to another polypeptide, such as a heteropeptide and / or multiple heteropeptides. When referring to amino acids, "L" can also mean leucine.

[0163] The "LAC4 promoter" or "Lac4 promoter" refers to a DNA fragment consisting of a promoter sequence derived from the β-galactosidase gene of Lactobacillus (K. lactis). The LAC4 promoter is a strong and inducible reporter gene used to drive the expression of foreign genes transformed into yeast.

[0164] The “LAC4 terminator” or “Lac4 terminator” refers to a DNA fragment consisting of a transcription terminator sequence from the Lactobacillus β-galactosidase gene.

[0165] “LD 20 "This refers to the dose required to kill 20% of the population."

[0166] “LD 50 "50" refers to a lethal dose, meaning the dose required to kill 50% of the population.

[0167] "Lepidoptera gut environment" refers to the specific pH and protease conditions found in the foregut, midgut, or hindgut of lepidopteran insects or larvae.

[0168] "Lepidoptera hemolymphatic environment" refers to specific pH and protease conditions found in lepidopteran insects or larvae.

[0169] A “linker” (or “LINKER”), “peptide linker”, “L”, or “intercalation linker” refers to a short peptide sequence operable to link two peptides together. A linker can also refer to a short DNA sequence placed within the reading frame of an ORF to separate upstream and downstream DNA sequences. In some embodiments, the linker may be cleaved by insect proteases. In some embodiments, the linker may allow proteins to achieve their independent secondary and tertiary structure formation during translation. In some embodiments, the linker may be resistant or readily cleaved in plant cell environments, insect and / or lepidopteran gut environments, and / or insect and lepidopteran hemolymph environments. In some embodiments, the linker may be cleaved by proteases, for example, in some embodiments, the linker may be cleaved by plant proteases (e.g., papain, bromelain, fig proteases, kiwi proteases, ginger proteases, and / or cardosin), insect proteases, fungal proteases, vertebrate proteases, invertebrate proteases, bacterial proteases, mammalian proteases, reptile proteases, or avian proteases. In some embodiments, the linker may be cleavable or non-cleavable. In some embodiments, the adapter comprises binary or ternary regions, each of which can be cleaved by at least two types of proteases: one being an insect and / or nematode protease, and the other a human protease. In some embodiments, the adapter may have one of at least three functions: cleavage in the insect gut environment, cleavage in plant cells, or designed not to cleave intentionally.

[0170] "Culture medium" refers to the nutrient solution used to culture cells in cell culture.

[0171] "MOA" refers to the mechanism of action.

[0172] Molecular weight (MW) refers to the mass or weight of a molecule and is typically measured in Daltons (Da) or kilodaltons (kDa). In some embodiments, MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical ultracentrifugation, or light scattering. In some embodiments, the SDS-PAGE method is as follows: The sample of interest is separated on a gel using a set of molecular weight standards. The sample is run, and the gel is then treated with the desired staining agent, followed by destaining for approximately 2 to 14 hours. The next step is to determine the relative migration distance (Rf) between the standards and the protein of interest. The migration distance can be determined using the following equation:

[0173]

[0174] Next, the logarithm of the molecular weight (MW) can be determined based on the values ​​obtained for the bands in the standard; for example, in some embodiments, the logarithm of the molecular weight of the SDS-denatured peptide and its relative migration distance (Rf) are plotted. After plotting, interpolating the obtained values ​​will provide the molecular weight of the unknown protein band.

[0175] A "motif" refers to a polynucleotide or polypeptide sequence that has some biological significance and / or plays some role or participates in some biological process.

[0176] "Multiple cloning sites" or "MCS" refer to DNA fragments found on vectors that contain many restriction sites into which DNA sequences of interest can be inserted.

[0177] A “mutant” refers to an organism, DNA sequence, amino acid sequence, peptide, polypeptide, or protein that has an alteration or variation (e.g., in its nucleotide or amino acid sequence) that results in the organism and / or sequence differing from a naturally occurring or wild-type organism, wild-type sequence, and / or reference sequence compared to the mutant. In some embodiments, the alteration or variation may be one or more nucleotide and / or amino acid substitutions or modifications (e.g., deletions or additions). In some embodiments, the one or more amino acid substitutions or modifications may be conserved; here, such conserved amino acid substitutions and / or modifications in the “mutant” do not substantially reduce the activity of the mutant relative to its non-mutant form. For example, in some embodiments, the “mutant” has one or more conserved amino acid substitutions, as shown in SEQ ID NO, compared to a peptide having a disclosed and / or claimed sequence.

[0178] "N-terminus" refers to the free amino group (i.e., -NH2) located at the beginning or starting point of a polypeptide.

[0179] "NCBI" refers to the National Center for Biotechnology Information.

[0180] “nm” refers to nanometers.

[0181] "Non-ICK CRIP" refers to peptides having 4 to 8 cysteine ​​residues that form 2 to 4 disulfide bonds. Non-ICK peptides include cysteine-bound peptides that are not ICK peptides. Non-ICK peptides may have a different disulfide bond linkage pattern than ICK peptides. Examples of non-ICK CRIPs are peptides such as Av2 and Av3 isolated from sea anemones; these anemone peptides are examples of a class of compounds that regulate sodium channels in the insect peripheral nervous system (PNS).

[0182] "Nonpolar amino acids" are weakly hydrophobic amino acids, and include glycine, alanine, proline, valine, leucine, isoleucine, phenylalanine, and methionine. Glycine or Gly is the most preferred nonpolar amino acid for the dipeptide of this invention.

[0183] "Standardized peptide yield" refers to the peptide yield in conditioned medium divided by the corresponding cell density at which the peptide yield was measured. Peptide yield can be expressed as the mass of peptide produced per unit volume, for example, mg / L or mg / L, or as the UV absorption peak area of ​​the peptide produced in HPLC chromatography, for example, mAu.sec. Cell density can be expressed as the visible light absorbance of the culture at a wavelength of 600 nm (OD600).

[0184] "OD" refers to optical density. OD is typically measured using a spectrophotometer. When measuring the growth of cell populations over time, OD600 is superior to the UV spectrum; this is because at a wavelength of 600 nm, cells are not as damaged as they are under much UV light.

[0185] “OD660nm” or “OD” 660nm "" refers to the optical density at 660 nanometers (nm).

[0186] "Omega peptide," "Omega toxin," "Omega-ACTX-Hv1a," or "natural OmegaACTX-Hv1a" all refer to the ACTX peptide first isolated from a spider called the Australian Blue Mountain funnel-web spider (Australian funnel-web spider). Omega peptides are allosteric regulators of nicotinic acetylcholine receptors and can also be voltage-gated Ca2+ receptors in insects. 2+ Channel and voltage gating K +Dual antagonists of the channel. See Chambers et al., “Insecticidal spider toxins are highaffinity positive allosteric modulators of the nicotinic acetylcholine receptor,” FEBS Lett., June 2019, Vol. 593, No. 12: 1336-1350; and Windley et al., “Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors,” Neuropharmacology, December 2017, Vol. 127: 224-242. The full text of these publications is incorporated herein by reference.

[0187] "Single-letter codes" refer to peptide sequences listed with single-letter codes to distinguish various amino acids in the primary structure of a protein: alanine = A, arginine = R, asparagine = N, aspartic acid = D, asparagine or aspartic acid = B, cysteine ​​= C, glutamic acid = E, glutamine = Q, glutamine or glutamic acid = Z, glycine = G, histidine = H, isoleucine = I, leucine = L, lysine = K, methionine = M, phenylalanine = F, proline = P, serine = S, threonine = T, tryptophan = W, tyrosine = Y, and valine = V.

[0188] "Operable" refers to the ability to be used, the ability to do something, and / or the ability to achieve a certain function or result. For example, in some embodiments, "operable" refers to the ability of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence or gene to encode a peptide, polypeptide, and / or protein. For example, in some embodiments, a polynucleotide may be operable to encode a protein, meaning that the polynucleotide contains information that confers upon it the ability to produce a protein (e.g., by transcribing mRNA, which is then translated into a protein).

[0189] "Operationally linked" refers to juxtaposition, where the components described so far are in a relationship that allows them to function in their intended manner. For example, in some embodiments, operationally linked may refer to two or more DNA, peptide, or polypeptide sequences. In other embodiments, operationally linked may refer to two adjacent DNA sequences placed together such that transcriptional activation of one DNA sequence can act on the other DNA sequence. In other embodiments, the term "operationally linked" may refer to two or more peptides and / or polypeptides linked in a manner that produces a single polypeptide chain; alternatively, the term operationally linked may refer to two or more peptides linked in a manner in which one peptide exerts some effect on the other. In other embodiments, operationally linked may refer to two adjacent DNA sequences placed together such that transcriptional activation of one sequence can act on the other sequence.

[0190] An “ORF” or “open reading frame” refers to the length of an RNA or DNA sequence between one or more of the translation initiation signal (e.g., AUG or ATG, respectively) and a known stop codon encoding one or more polypeptide sequences. In other words, the ORF describes a reference frame from the perspective of the ribosome encoding the translating RNA, as the ribosome is able to maintain reading (i.e., add amino acids to the nascent protein) due to the absence of a stop codon. Therefore, an “open reading frame” or “ORF” refers to the amino acid sequence encoded between the translation initiation and stop codons of the coding sequence. Here, the terms “start codon” and “stop codon” refer to the units (i.e., codons) of three adjacent nucleotides in the coding sequence that respectively designate the start and termination of protein synthesis (mRNA translation).

[0191] In some embodiments, an ORF is a continuous extension of a codon that begins with a start codon (typically ATG for DNA and AUG for RNA) and ends with a stop codon (typically UAA, UAG, or UGA). In other embodiments, an ORF can be the length of an RNA or DNA sequence between a translation start signal (e.g., AUG or ATG) and one or more known stop codons, wherein the length of the RNA or DNA sequence encodes one or more polypeptide sequences. In some other embodiments, an ORF can be a DNA sequence encoding a protein that begins with an ATG start codon and ends with a TGA, TAA, or TAG stop codon. ORF can also refer to a DNA-encoded translated protein. Generally, those skilled in the art distinguish the terms "open reading frame" and "ORF" from the term "coding sequence" simply by considering the fact that it refers to a series of codons that do not contain a stop codon, based on the broadest definition of "open reading frame." Therefore, although an ORF may contain introns, coding sequences are distinguished by reference to nucleotides that can be broken down into codons (e.g., linked exons), which are actually translated into amino acids by ribosomal translation mechanisms (i.e., coding sequences do not contain introns); however, as used herein, the terms “coding sequence,” “CDS,” “open reading frame,” and “ORF” are used interchangeably.

[0192] “Out-recombined” or “out-recombination” refers to the removal of genes and / or polynucleotide sequences (e.g., endogenous genes) flanked by two site-specific recombination sites (e.g., the 5'- and 3'-nucleotide sequences of a target gene homologous to the homologous arm of the target vector) during in vivo homologous recombination. See “knockout”.

[0193] "Pasporon crystal toxin" refers to any of the peptides, polypeptides, and / or proteins that are part of a parasporium or parasporium crystal, and is a bipyramidal crystal containing one or more peptides, polypeptides, and / or proteins. When the parasporium or parasporium crystal is ingested by an insect, this toxin-containing parasporium crystal dissolves in alkaline intestinal fluid and is subsequently cleaved by midgut proteases of the protoxin to produce an active peptide toxin, such as δ-endotoxin.

[0194] "Peptide expression cassette" or "expression cassette" refers to a DNA sequence consisting of all the DNA elements necessary for the transcription of an insecticidal protein in a biological expression system. In the methods described herein, it includes a transcription promoter, a DNA sequence encoding an α-mating factor signal sequence, a cleavage site, an insecticidal protein transgene, a stop codon, and a transcription terminator.

[0195] "Peptide expression vector" refers to a host organism expression vector containing a heterologous peptide transgene.

[0196] "Peptide-expressing yeast strains," "peptide-expressing strains," or "peptide-producing strains" refer to yeast strains that are capable of producing heteropeptides.

[0197] "Peptide-IA" refers to insecticides that are amino acids, peptides, polypeptides, and / or proteins.

[0198] "Peptide transgenes," "insecticide peptide transgenes," or "insecticide protein transgenes" refer to DNA sequences that encode peptides of interest and can be translated in biological expression systems.

[0199] "Peptide yield" refers to the concentration of insecticidal peptides in a conditioned medium produced by cells of peptide-expressing yeast strains. It can be expressed as the mass of peptides produced per unit volume, for example, mg / L or mg / L, or as the UV absorption peak area of ​​the peptides produced in HPLC chromatography, for example, mAu.sec.

[0200] The term "peritoneum" refers to the lining inside an insect's gut that traps large food particles. It helps these particles move through the gut, allows for digestion, and protects the intestinal wall.

[0201] "Pests" include, but are not limited to: insects, fungi, bacteria, nematodes, mites, ticks, etc.

[0202] "Insecticidal effective dose" refers to the amount of insecticide that can kill or significantly reduce the growth, feeding, or normal physiological development of at least one pest. This amount will vary depending on factors such as the specific target pest to be controlled, the specific environment, location, plant, crop, or agricultural site to be treated, environmental conditions, and the method of application, ratio, concentration, stability, and amount of the insecticidal effective polypeptide composition. The formulation may also vary depending on climatic conditions, environmental considerations, and / or application frequency and / or the severity of pest infestation.

[0203] "Pharmaceutically acceptable salt" is synonymous with "agriculturally acceptable salt" and, as used herein, refers to a compound modified by preparing its acid or base salt.

[0204] "Plant" should mean the whole plant, plant tissue, plant organ (e.g., leaf, stem, root, etc.), seed, plant cell, propagule, embryo and its offspring. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplast, leaf cells, root cells, phloem cells and pollen).

[0205] "Plant genetically modified protein" refers to a protein expressed in a plant after one or more of its DNA or RNA encoding a protein from a heterologous species have been delivered into the plant cell.

[0206] "Plant cleavable connectors" refers to cleavable connector peptides or nucleotides encoding cleavable connector peptides that contain plant protease recognition sites and can be cleaved during protein expression in plant cells.

[0207] "Plant embedded protectant" or "PIP" refers to an insecticidal protein produced by genetically modified plants, along with the genetic material necessary for the plant to produce that protein.

[0208] A plasmid is a DNA fragment that serves as a vector for a gene of interest. When transformed or transfected into an organism, it can replicate and express the DNA sequence contained within the plasmid independently of the host organism. A plasmid is a type of vector and can be a "cloning vector" (i.e., a simple plasmid used to clone DNA fragments and / or select host populations carrying the plasmid via some selection indicator) or an "expression plasmid" (i.e., a plasmid used to produce large amounts of polynucleotides and / or polypeptides).

[0209] "Polar amino acids" are polar amino acids, including serine, threonine, cysteine, asparagine, glutamine, histidine, tryptophan and tyrosine; preferred polar amino acids are serine, threonine, cysteine, asparagine and glutamine; among which serine is the most preferred.

[0210] "Polynucleotide" refers to a polymeric form of nucleotides of any length (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof); for example, a sequence of two or more ribonucleotides or deoxyribonucleotides. As used herein, the term "polynucleotide" includes double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA; it also includes modified and unmodified forms of polynucleotides (modifications to polynucleotides and modifications as polynucleotides may include, for example, methylation, phosphorylation, and / or capping). In some embodiments, a polynucleotide can be one of: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag); genomic DNA; a fragment of genomic DNA; an exon; an intron; messenger RNA (mRNA); transfer RNA; ribosomal RNA; a ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probe; primer; or an amplified copy of any of the foregoing.

[0211] In other embodiments, a polynucleotide may refer to a polymeric form of a nucleotide that is operable to encode an open reading frame of a gene.

[0212] In some implementations, polynucleotides may refer to cDNA.

[0213] In some embodiments, polynucleotides can have any three-dimensional structure and perform any known or unknown function. The structure of a polynucleotide can also be referred to by its 5'- or 3'-terminus, indicating its orientation. Adjacent nucleotides in a polynucleotide single strand are typically linked by a phosphodiester bond between their 3' and 5' carbons. However, different internucleotide bonds, such as those including methylene, aminophosphate, etc., can also be used. This means that the corresponding 5' and 3' carbons can be exposed at either end of the polynucleotide, which can be referred to as 5' and 3' ends or ends. The 5' and 3' ends can also be referred to as phosphoryl (PO4) and hydroxyl (OH) ends, respectively, because chemical groups are attached to those ends. The term polynucleotide also refers to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment preparing or using a polynucleotide includes both double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0214] In some embodiments, the polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with non-natural bases, nucleotides with modified natural bases, such as aza- or denitro-purines, etc.). If present, modifications to the nucleotide structure may be imparted before or after polynucleotide assembly.

[0215] In some implementations, the polynucleotide may be further modified after polymerization, such as by conjugation with a labeled component. Additionally, the nucleotide sequence in the polynucleotide may be interrupted by non-nucleotide components. One or more ends of the polynucleotide may be protected or otherwise modified to prevent that end from interacting with other polynucleotides in a specific manner (e.g., forming covalent bonds).

[0216] In some implementations, the polynucleotide may consist of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T). Uracil (U) may also be present, for example, as a natural substitute for thymine when the polynucleotide is RNA. Uracil may also be used in DNA. Therefore, the term "sequence" refers to the letter representation of a polynucleotide or any nucleic acid molecule, including both natural and non-natural bases.

[0217] The term "RNA molecule" or ribonucleic acid molecule refers to a polynucleotide having ribose instead of deoxyribose and typically uracil instead of thymine as one of its pyrimidine bases. The RNA molecules of this invention are typically single-stranded, but can also be double-stranded. In the context of RNA molecules originating from an RNA sample, RNA molecules may include single-stranded molecules transcribed from DNA in the cell nucleus, mitochondria, or chloroplasts, having a linear sequence of nucleotide bases complementary to the DNA strand that transcribed them.

[0218] In some embodiments, the polynucleotide may further comprise one or more heterologous regulatory elements. For example, in some embodiments, the regulatory element is one or more promoters, enhancers, silencers, operators, splicing signals, polyadenylation signals, termination signals, RNA export elements, internal ribosome entry sites (IRES), poly-U sequences, or combinations thereof.

[0219] "Posttranscriptional gene silencing" or "PTGS" refers to the cellular process of suppressing gene expression within living cells.

[0220] "Posttranscriptional regulatory elements" are DNA segments and / or mechanisms that affect mRNA after transcription. Posttranscriptional mechanisms include splicing events, capping, addition of a poly(A) tail, and other mechanisms known to those skilled in the art.

[0221] A "promoter" is a DNA region that RNA polymerase binds to and initiates gene transcription.

[0222] In this article, "protein" has the same meaning as "peptide" and / or "polypeptide".

[0223] A "ratio" refers to a quantitative relationship between two quantities, indicating how many times one value is contained within or included in another.

[0224] A "reading frame" refers to one of the six possible reading frames in a double-stranded DNA molecule, three in each direction. The reading frame used determines which codons in the coding sequence of the DNA molecule are used to encode amino acids. In some embodiments, a reading frame is a way of dividing a nucleotide sequence in a polynucleotide and / or nucleic acid (e.g., DNA or RNA) into a set of consecutive, non-overlapping triplets.

[0225] "Recombinant DNA" or "rDNA" refers to DNA composed of two or more different DNA fragments.

[0226] "Recombinant vector" refers to a DNA plasmid vector into which foreign DNA has been inserted.

[0227] "Regulatory element" refers to a genetic element that controls aspects of nucleic acid sequence expression and / or processing. For example, in some embodiments, regulatory elements can be found at the transcriptional and post-transcriptional levels. Regulatory elements can be cis-regulatory elements (CREs) or trans-regulatory elements (TREs). In some embodiments, regulatory elements can be one or more promoters, enhancers, silencers, operators, splicing signals, polyadenylation signals, termination signals, RNA export elements, internal ribosome entry sites (IRES), poly-U sequences, and / or other elements that affect gene expression, such as by increasing or decreasing expression in a tissue-specific or time-related manner and / or by inducing constitutive expression.

[0228] "Restriction enzymes" or "restriction endonucleases" are enzymes that cut DNA at specific restriction sites. For example, restriction enzymes can cut plasmids at EcoRI, SacII, or BstXI restriction sites, thereby linearizing the plasmid and ligating the DNA of interest.

[0229] A "restriction site" is a location on DNA containing a sequence of 4 to 8 nucleotides that is recognized by a specific restriction enzyme.

[0230] "Salannin" refers to an insecticidal compound isolated from neem trees. In some embodiments, Salannin has the molecular formula C2. 34 H 44 O9, with a molecular weight of 596.7 g / mol.

[0231] "Sea anemone" refers to a group of marine animals belonging to the order Actiniaria. Named after the anemone, sea anemones are terrestrial flowering plants, many of which have colorful appearances. For example, in some implementations, sea anemones are one of the following species: red sea anemone (Actinia equine); Anemonia erythraea; grooved wind-facing sea anemone; snake-locked sea anemone; elegant yellow sea anemone (Anthopleura elegantissima); rust-green lateral-flowered sea anemone (Anthopleura fuscoviridis); yellow sea anemone (Anthopleura xanthogrammica); Bunodosoma caissarum; Bunodosoma cangicum; warty sea anemone (Bunodosoma granulifera); purple-spotted sea anemone (Heteractis crispa); Parascyonisactinostoloides; Radianthus paumotensis; or sunflower sea anemone (Stoichactishelianthus).

[0232] "Selective genes" refer to genes that give genetically modified organisms an advantage in growth under selection pressure.

[0233] "Serotype" or "serological variant" refers to a group of closely related microorganisms distinguished by a set of characteristic antigens. In some implementations, a serological variant is an antigenic and serologically distinct microbial variant.

[0234] “sp.” refers to a species.

[0235] “ssp.” or “subsp.” refers to a subspecies.

[0236] "Subcloning" or "subcloned" refers to the process of transferring DNA from one vector to another (usually a preferred vector). For example, after selecting yeast cells transformed with the pKLAC1 plasmid, a polynucleotide encoding a mutant or peptide can be subcloned into the pKlac1 plasmid.

[0237] "SSI" is a context-dependent acronym. In some contexts, it can refer to "site-specific integration," which refers to sequences that allow homologous recombination to occur at specific sites within the host organism's genome. Therefore, in some implementations, the term "site-specific integration" refers to the process of directing a transgene to a target site in the host organism's genome, thereby allowing the integration of the gene of interest into a pre-selected genomic location within the host organism. However, in other contexts, SSI can refer to "indoor surface spraying," a technique for applying a variable volume of sprayable insecticide to surfaces inhabited by disease vectors, such as walls, windows, floors, and ceilings.

[0238] The term "STA," or "translationally stable protein," "stable domain," or "stable protein" (used interchangeably in this text) refers to a peptide or protein with sufficient tertiary structure to accumulate in the cell without being targeted by cellular protein degradation processes. The protein's length can range from 5 to 50 amino acids. Translationally stable proteins are encoded by a DNA sequence in the ORF that is operatively linked to a sequence encoding an insecticidal protein or CRIP. The operatively linked STA can be upstream or downstream of the CRIP, and any intercalation sequence can exist between the two sequences (STA and CRIP), as long as the intercalation does not cause a frameshift in either DNA sequence. Translationally stable proteins may also have activity that enhances the delivery of CRIP across the intestinal wall and into the insect's hemolymph.

[0239] “sta” refers to the nucleotide that encodes proteins that are stable for translation.

[0240] A “structural motif” refers to the three-dimensional arrangement of peptides and / or polypeptides, and / or the arrangement of operatively linked polypeptide segments. For example, a polypeptide having an ERSP motif, a STA motif, a LINKER motif, and a CRIP polypeptide motif has an overall “structural motif” of ERSP-STA-L-CRIP. See also “CRIP constructs”.

[0241] “Ta1b” or “U1-funnel-web spider venom-Ta1b” or “Ta1bWT” or “wild-type U1-funnel-web spider venom-Ta1b” refers to a polypeptide isolated from the hobo spider (Eratigena agrestis). An example of U1-funnel-web spider venom-Ta1b is a polypeptide having the amino acid sequence of SEQ ID NO:1 (NCBI accession number O46167.1).

[0242] "Ta1b variant polynucleotide" or "U1-funnel-web spider toxin-Ta1b variant polynucleotide" refers to a polynucleotide or polynucleotide group operable to express and / or encode an insecticidal protein containing one or more TVPs. The term "U1-funnel-web spider toxin-Ta1b variant polynucleotide" is described as "tvp" and / or "Tvp" when used to describe the U1-funnel-web spider toxin-Ta1b variant polynucleotide sequence contained in a TVP expression ORF, its inclusion in a vector, and / or when describing a polynucleotide encoding an insecticidal protein.

[0243] "Toxin" refers to venom and / or poisons, particularly proteins or conjugated proteins produced by certain animals, higher plants, and pathogens. Generally, the term "toxin" is reserved for naturally occurring products, such as molecules and peptides found in scorpions, spiders, snakes, poisonous mushrooms, etc., while the term "poison" is reserved for man-made and / or artificial products, such as synthetic chemical pesticides. However, as used herein, the terms "toxin" and "poison" are used synonymously.

[0244] Both “transfection” and “transformation” refer to the process of introducing exogenous and / or heterologous DNA or RNA (e.g., a vector containing a polynucleotide encoding CRIP) into a host organism (e.g., a prokaryote or eukaryote). Generally, those skilled in the art sometimes retain the term “transformation” to describe the process of introducing exogenous and / or heterologous DNA or RNA into bacterial cells; and retain the term “transfection” to describe the process of introducing exogenous and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the terms “transformation” and “transfection” are used synonymously, regardless of whether the method describes the introduction of exogenous and / or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plant, or animal).

[0245] "Genetically modified" refers to a heterologous DNA sequence that encodes a protein that has been transformed into a plant.

[0246] "Transgenic host cell" refers to a cell that has been transformed with a gene and whose transgenic state has been selected by another selection gene.

[0247] "Genetically modified plant" refers to a plant derived from a single cell transformed with foreign DNA, so that every cell in the plant contains that genetic material.

[0248] "Transient expression system" refers to a system based on Agrobacterium tumefaciens that delivers DNA encoding a plant virus into plant cells for expression. The plant virus has been engineered to express the protein of interest at high concentrations of up to 40% TSP.

[0249] "Three expression boxes" refers to three CRIP expression boxes contained on the same carrier.

[0250] "TRBO" refers to a transient plant expression system that uses tobacco mosaic virus and removes the viral coat protein gene.

[0251] "TSP" or "Total Soluble Protein" refers to the total amount of protein that can be extracted from plant tissue samples and dissolved in the extraction buffer.

[0252] "TVP" or "U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP)" or "Ta1b variant polypeptide (TVP)" refers to a mutant or variant of the wild-type U1-funnel-web spider toxin-Ta1b polypeptide sequence and / or a polynucleotide sequence encoding the wild-type U1-funnel-web spider toxin-Ta1b polypeptide, which has been altered to produce a non-naturally occurring polypeptide and / or polynucleotide sequence. An exemplary wild-type U1-funnel-web spider toxin-Ta1b polypeptide sequence having SEQ ID NO:1 is provided herein. An exemplary wild-type U1-funnel-web spider toxin-Ta1b precursor polypeptide sequence having SEQ ID NO:48 (NCBI accession number O46167.1) is provided herein, which includes the signal sequence "MKLQLMICLVLLPCFFC" (SEQ ID NO:59). In some embodiments, the TVP may have an amino acid sequence according to any of the amino acid sequences listed in Table 1. Therefore, the term "TVP" refers to a polypeptide having one or more mutants relative to the amino acid sequence shown in SEQ ID NO:1. In some implementations, the TVP may have an amino acid sequence according to formula (I):

[0253] EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7

[0254] Formula (I)

[0255] The polypeptide contains at least one amino acid substitution relative to the wild-type sequence of U1-funnel web spider toxin-Ta1b shown in SEQ ID NO:1, and wherein X1 is A, S or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E or R; X6 is K or absent; and X7 is G or absent.

[0256] In some embodiments, the TVP may have an amino acid sequence according to formula (II): EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG

[0257] Equation (II)

[0258] The polypeptide contains at least one amino acid substitution relative to the wild-type sequence of U1-funnel web spider toxin-Ta1b shown in SEQ ID NO:1, and wherein X1 is R or Q; and Z1 is T or A; or a pharmaceutically acceptable salt thereof.

[0259] "U-ACTX-Hv1a," "heterogeneous peptide," "heterogeneous toxin," "heterogeneous-ACTX-Hv1a," "naturally occurring heterogeneous ACTX-Hv1a," "U peptide," "U toxin," "natural U," or "natural U-ACTX-Hv1a" all refer to an ACTX peptide found in a spider called the Australian Blue Mountain funnel-web spider (Australian funnel-web spider). U-ACTX-Hv1a is a positive allosteric regulator of nicotinic acetylcholine receptors and can also be an insect voltage-gated Ca2+ receptor. 2+ Channel and voltage gating K + A dual antagonist of the channel. See Chambers et al., “Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor,” FEBS Lett., June 2019, Vol. 593, No. 12: pp. 1336-1350; and Windley et al., “Lethal effects of aninsecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors,” Neuropharmacology, December 2017, Vol. 127: pp. 224-242, the full text of which is incorporated herein by reference. An exemplary U-ACTX-Hv1a peptide is provided in SEQ ID NO:60.

[0260] The terms “U+2 peptide,” “U+2 protein,” “U+2 toxin,” “U+2,” “U+2-ACTX-Hv1a,” or “Spear” all refer to U-ACTX-Hv1a having an additional dipeptide operatively linked to the native peptide. The additional dipeptide operatively linked to the U peptide, indicated by “+2” or “plus 2,” can be selected from several peptides, any of which can produce a “U+2 peptide” with the unique properties discussed herein. In some preferred embodiments, the dipeptide is “GS”; an exemplary U+2-ACTX-Hv1a peptide is shown in SEQ ID NO:61.

[0261] “UBI” refers to ubiquitin. For example, in some implementations, UBI may refer to ubiquitin monomers isolated from maize (Zea mays).

[0262] “var.” refers to a variety or variant. The term “var.” is used to indicate a taxonomic category that is listed below the species level and / or subspecies (when present). In some implementations, the term “var.” represents a member that differs from other members of the same subspecies or species in minor but permanent or heritable characteristics.

[0263] A “variant” or “variant sequence” or “variant peptide” refers to an amino acid sequence having one or more conserved amino acid substitutions or modifications. The conserved amino acid substitutions in a “variant” do not substantially reduce the activity of the variant relative to its non-variant form. For example, in some embodiments, a “variant” has one or more conserved amino acid substitutions, as shown in SEQ ID NO, compared to a peptide having a disclosed and / or claimed sequence.

[0264] A "vector" is a DNA segment that receives a foreign gene of interest (e.g., crip). The gene of interest is called an "insertion" or "transgenic gene."

[0265] "Vip," "VIP," or "vegetative-stage insecticidal protein" refers to proteins with potential insecticidal activity found in the supernatant of vegetatively grown Bt strains. Vip shares little or no similarity with Cry proteins. Proteins called VIP3 or Vip3, which exhibit Lepidoptera activity, are particularly useful and preferred in this study. Vip is considered to have a similar mode of action to Bt cry peptides.

[0266] "Vitrification" refers to the process of transforming a material into a glassy amorphous material. Glassy amorphous solids may contain no crystalline structure. The solidification of glassy solids occurs at the glass transition temperature (Tg).

[0267] "Wild type" or "WT" refers to the phenotype and / or genotype (i.e., appearance or sequence) of an organism, polynucleotide sequence and / or polypeptide sequence found and / or observed in its naturally occurring state or conditions.

[0268] "Yeast expression vector" or "expression vector" or "vector" refers to a plasmid that can introduce a heterologous gene and / or expression cassette into yeast cells for transcription and translation.

[0269] "Yield" refers to the production of peptides, and an increase in yield can mean an increase in production volume, an increase in productivity, an increase in average or median yield, and an increase in the frequency of higher yields. When used in relation to plant crop growth and / or production, such as in "plant yield," the term "yield" refers to the quality and / or quantity of biomass produced by the plant.

[0270] Throughout the specification, unless otherwise specified or required by context, references to a single step, composition of substance, group of steps, or group of composition of substance shall include one or more of those steps, compositions of substance, groups of steps, or groups of composition of substance (i.e., one or more).

[0271] Unless otherwise stated, this disclosure does not require extensive experimentation, but rather utilizes conventional techniques from molecular biology, microbiology, virology, recombinant DNA technology, solid-phase and liquid nucleic acid synthesis, peptide synthesis in solution, solid-phase peptide synthesis, immunology, cell culture, and formulation. Such procedures are described in, for example, Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratories, New York, 2nd edition, 1989, Volumes 1, 2, and 3; *DNA Cloning: A Practical Approach*, Volumes 1 and 2, edited by DNGlover, 1985, IRL Press, Oxford, full text; *Oligonucleotide Synthesis: A Practical Approach*, edited by MJ Gait, 1984, IRL Press, Oxford, full text, especially Gait's paper, pp. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151; 4. *Nucleic Acid Hybridization: A Practical Approach*, edited by B.D. Hames and S.J. Higgins, 1985, IRL Press, Oxford, full text; *Immobilized Cells and Enzymes: A Practical Approach, 1986, IRL Press, Oxford, full text; Perbal, B., A Practical Guide to Molecular Cloning, 1984; Methods In Enzymology, edited by S. Colowick and N. Kaplan, Academic Press, Inc., series; J.F. Ramalho Ortigao, “The Chemistry of Peptide Synthesis”, in: Knowledge Database for Accessing Virtual Labs, Interactiva, Germany; Sakakibara, D., Teichman, J., Lien, EL., and Fenichel, RL., 1976, Biochem. Biophys. Res. Commun., Vol. 73, pp. 336-342; Merrifield, RB., 1963, J. Am. Chem. Soc.Volume 85, pp. 2149-2154; Barany, G. and Merrifield, RB, 1979, “The Peptides”, Gross, E. and Meienhofer, 3 eds., Volume 2, pp. 1-284, Academic Press, New York, 12. Wiinsch, E. ed., 1974, Synthese von Peptiden in Houben-Weyls Metoden der Organicischen Chemie, Muler, E. ed., Volume 15, 4th edition, Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M., 1984, Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. and Bodanszky, A., 1984, The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M., 1985, Int. J. Peptide Protein Res., Vol. 25, pp. 449–474; Handbook of Experimental Immunology, Vols. I–IV, edited by D.M. Weir and C.C. Blackwell, 1986, Blackwell Scientific Publications; and Animal Cell Culture: Practical Approach, 3rd edition, edited by John R.W. Masters, 2000; the full text of each of these references is incorporated herein by reference.

[0272] Throughout this specification, unless the context otherwise requires, the word “comprising” or variations thereof such as “including” or “containing” shall be understood to imply inclusion of the said step or element or integer or group of steps or elements or integers, but not to exclude any other step or element or integer or group of elements or integers.

[0273] All patent applications, patents, and print publications mentioned herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application were expressly and individually indicated to be incorporated by reference. Furthermore, all patent applications, patents, and print publications cited herein are incorporated herein by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and except where the incorporated material differs from the express disclosure herein to the extent that the language of this disclosure shall prevail.

[0274] Cysteine-rich insecticidal proteins (CRIPs)

[0275] This invention provides combinations comprising (1) one or more CRIPs, or pharmaceutically acceptable salts thereof; one or more CRIP-insecticide proteins, or pharmaceutically acceptable salts thereof; or combinations thereof; and (2) one or more insecticides (IA). Several types of CRIPs are contemplated and taught herein. The CRIPs of this invention, which can be used in combination with the insecticides (IA) of this invention, are described in detail below. All CRIPs suitable for the combinations of this invention and contemplated below include CRIP-insecticide proteins.

[0276] Spider peptides and toxins

[0277] In some implementations, CRIP can be a spider venom peptide or protein isolated from one of the following: Brazilian wandering spider; Allagelena opulenta; Cupiennius salei; Plectreurys tristis; Coremiocnemis valida; Haplopelma huwenum; Agelena orientalis; Allagelena opulenta; Segestria florentina; Apomastus schlingeri; Phoneutria keyserlingi; Macrothele gigas; Macrothele raveni; Missulena bradleyi; Pireneitega luctuosa; Phoneutria reidyi; Illawara wisharti; Eucratoscelus constrictus; Agelenopsis aperta; Hololena curta; Oxyopes lineatus; Mexican golden-backed red-tailed spider (Brachypelma albiceps); or Mexican red-kneed spider (Brachypelma smithi).

[0278] In some implementations, CRIP can be isolated from the Australian funnel-web spider, Hadronyche venenata, Sydney funnel-web spider (Atrax robustus), Atrax formidabilis, or Atrax infensus.

[0279] In some embodiments, CRIP can be any of the following spider peptides, polypeptides, and / or toxins: U+2-ACTX-Hv1a, Γ-CNTX-Pn1a, U13-ctenitoxin-Pn1a, U13-ctenitoxin-Pn1b, U13-ctenitoxin-Pn1c, U1-funnel web spider toxin-Aop1a, U1-ctenitoxin-Cs1a, U1-nemetoxin-Csp1a, U1-nemetoxin-Csp1b, U1-nemetoxin-Csp1c, U1-plectoxin-Pt1a, U1-plectoxin-Pt1b , U1-plectoxin-Pt1c, U1-plectoxin-Pt1d, U1-plectoxin-Pt1f, U1-theraphotoxin-Cv1a, U1-theraphotoxin-Hh1a_1, U1-theraphotoxin-Hh1a_2 , U1-theraphotoxin-Hh1a_3, U1-theraphotoxin-Hh1b, U1-theraphotoxin-Hh1c_1, U1-theraphotoxin-Hh1c_2, U1-theraphotoxin-Hh1d, U1-ther aphotoxin-Hh1e, U1-theraphotoxin-Hh1f_1, U1-theraphotoxin-Hh1f_2, U1-theraphotoxin-Hh1f_3, U1-theraphotoxin-Hh1f_4, U1-theraphotoxin-Hh1g, U2-funnel-web spider toxin-Ao1a, U2-funnel-web spider toxin-Aop1a, U2-ctenitoxin-Cs1a, U2-ctenitoxin-Pn1a, U2-cyrtautoxin-As1a, U2-segestritoxin-Sf1a, U2-s egestritoxin-Sf1b, U2-segestritoxin-Sf1c, U2-segestritoxin-Sf1d, U2-segestritoxin-Sf1e, U2-segestritoxin-Sf1f, U2-segestritoxin-S f1g, U2-segestritoxin-Sf1h, U2-theraphotoxin-Hh1a, U3-cyrtautoxin-As1a, U3-plectoxin-Pt1a, U5-ctenitoxin-Pn1a, U7-ctenitoxin-Pk1a,β-hexatoxin-Mg1a, β-hexatoxin-Mr1a, Γ-ctenitoxin-Pn1a, δ-actinopoditoxin-Mb1a, δ-Amaurobitoxin-Pl1a, δ-Amaurobitoxin-Pl1b, δ-Amau robitoxin-Pl1c, δ-Amaurobitoxin-Pl1d, δ-ctenitoxin-Asp2e, δ-ctenitoxin-Pn1a_1, δ-ctenitoxin-Pn1a_2, δ-ctenitoxin-Pn1b, δ-ctenitox in-Pn2a, δ-ctenitoxin-Pn2b, δ-ctenitoxin-Pn2cδ-ctenitoxin-Pr2d, δ-hexatoxin-Ar1a, δ-hexatoxin-Hv1a, δ-hexatoxin-Hv1b, δ-hexatoxin -Iw1a, δ-hexatoxin-Mg1a, δ-hexatoxin-Mg1b, κ-hexatoxin-Hf1a, κ-hexatoxin-Hv1a, κ-hexatoxin-Hv1b, κ-hexatoxin-Hv1c_1, κ-hexatoxin-Hv 1c_2, κ-hexatoxin-Hv1c_3, κ-hexatoxin-Hv1c_4, κ-hexatoxin-Hv1d, κ-hexatoxin-Hv1e, κ-theraphotoxin-Ec2a, κ-theraphotoxin-Ec2b, μ-funnel-web spider toxin-Aa1a, μ-funnel-web spider toxin-Aa1b, μ-funnel-web spider toxin-Aa1c, μ-funnel-web spider toxin-Aa1d, μ-funnel-web spider toxin-Aa1e, μ-funnel-web spider toxin-Aa1f, μ-funnel-web spider toxin-Hc1a, μ-funnel-web spider toxin-Hc1b, μ-funnel-web spider toxin-Hc1c, μ- hexatoxin-Mg1a, μ-hexatoxin-Mg1b, μ-hexatoxin-Mg1c, μ-hexatoxin-Mg2a, μ-theraphotoxin-Hh1a, ω-actinopoditoxin-Mb1a, ω-funnel web spider toxin-Aa4a , ω-Funnel Web Spider Toxin-Aa4b, ω-Funnel Web Spider Toxin-Aa4c, ω-hexatoxin-Ar1a_1, ω-hexatoxin-Ar1a_3, ω-hexatoxin-Ar1b_1, ω-hexatoxin-Ar1d_1, ω-hexatoxin-Ar1d_4,ω-hexatoxin-Ar1e_1、ω-hexatoxin-Ar1f、ω-hexatoxin-Ar1g_1、ω-hexatoxin-Ar1h、ω-hexatoxin-Ar2a、ω-hexatoxin-Ar2b、ω-hexatoxin-Ar2c、ω-hexatoxin-Ar2d、ω-hexatoxin-Ar2e_1、ω-hexatoxin-Ar2e_2、ω-atracotoxin-Asp2a、ω-hexatoxin-Asp2b、ω-hexatoxin-Hf1a、ω-hexatoxin- Hi1a_1, ω-hexatoxin-Hi1a_2, ω-hexatoxin-Hi1a_3, ω-hexatoxin-Hi1b_1, ω-hexatoxin-Hi1b_10, ω-hexatoxin-Hi1b_2, ω-hexatoxin-Hi1b_5, ω-hexatoxin-Hi1b_8, ω-hexatoxin-Hi1c_1, ω-hexatoxin-Hi1c_2, ω-hexatoxin-Hv1a, ω-hexatoxin-Hv1b, ω-hexatoxin-Hv1c, ω-hexatoxin-Hv1d ω-hexatoxin-Hv1e ω-hexatoxin-Hv1f ω-hexatoxin-Hv1g_1 ω-hexatoxin-Hv1g_5 ω-hexatoxin-Hv1g_6 ω-hexatoxin-Hv2a ω-hexatoxin-Hv2b_1 ω-hexatoxin-Hv2b_2 ω-hexatoxin-Hv2b_3 ω-hexatoxin-Hv2b_4 ω-hexatoxin-Hv2b_5 ω-hexatoxin-Hv2b_6 ω-hexatoxin-Hv2b_7 ω-hexa toxin-Hv2c、ω-hexatoxin-Hv2d_1、ω-hexatoxin-Hv2d_2、ω-hexatoxin-Hv2d_3、ω-hexatoxin-Hv2e、ω-hexatoxin-Hv2f、ω-hexatoxin-Hv2g、ω-hexatoxin-Hv2h_1、ω-hexatoxin-Hv2h_2、ω-hexatoxin-Hv2i、ω-hexatoxin-Hv2j_1、ω-hexatoxin-Hv2j_2、ω-hexatoxin-Hv2k、ω-hexatoxin-Hv2l、ω-hexatoxin-Hv2m_1, ω-hexatoxin-Hv2m_2, ω-hexatoxin-Hv2m_3, ω-hexatoxin-Hv2n, ω-hexatoxin-Hv2o, ω-hexatoxin-Hvn1 a, ω-hexatoxin-Hvn1b_1, ω-hexatoxin-Hvn1b_2, ω-hexatoxin-Hvn1b_3, ω-hexatoxin-Hvn1b_4, ω-hexatoxin-Hvn1b_6, ω-hexa toxin-Iw2a, ω-oxotoxin-Ol1b, ω-plectoxin-Pt1a, ω-theraphotoxin-Asp1a, ω-theraphotoxin-Asp1f, ω-theraphotoxin-Asp 1g, ω-theraphotoxin-Ba1a, ω-theraphotoxin-Ba1b, ω-theraphotoxin-Bs1a, ω-theraphotoxin-Bs2a or ω-theraphotoxin-Hh2a. ,

[0280] In some embodiments, CRIP may be a spider venom or a peptide having an amino acid sequence shown in any one of SEQ ID NO:192-278 and 281-370.

[0281] In some embodiments, the polynucleotide encoding CRIP may encode an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least 89% identity with the amino acid sequences shown in SEQ ID NO:192-278 and 281-370. CRIPs of amino acid sequences with at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity.

[0282] ACTX peptides

[0283] In some implementations, CRIP can be an ACTX peptide.

[0284] In some implementations, CRIP can be one or more of the following ACTX peptides: U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, rκ-ACTX-Hv1c, ω-ACTX-Hv1a, and / or ω-ACTX-Hv1a+2.

[0285] Exemplary ACTX peptides include: U-ACTX-Hv1a, having the amino acid sequence “QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO:60); U+2-ACTX-Hv1a, having the amino acid sequence “GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO:61); Omega-ACTX-Hv1a, having the amino acid sequence “SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO:62); and “ω+2-ACTX-Hv1a+2” (or Omega+2-ACTX-Hv1a), having the amino acid sequence “GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO:62). NO:63); and Kappa+2-ACTX-Hv1a (or κ+2-ACTX-Hv1a, which has the amino acid sequence “GSAICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP” (SEQ ID NO:64).

[0286] In some implementations, the CRIP may be “Kappa-ACTX-Hv1a” (or κ+2-ACTX-Hv1a) having the amino acid sequence “AICTGADRPCAACCPCCPGTS CKAESNGVSYCRKDEP” (SEQ ID NO:594).

[0287] In some embodiments, the ACTX peptide may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with SEQ ID NO:60-64, 192-370, and 594.

[0288] In some embodiments, the polynucleotide encoding the ACTX peptide may encode an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, or at least 89% identity with the amino acid sequence shown in SEQ ID NO:60-64 and 594. ACTX peptides with amino acid sequences of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity.

[0289] Gamma-CNTX-Pn1a peptides

[0290] In some preferred embodiments, the CRIP can be a Γ-CNTX-Pn1a or γ-CNTX-Pn1a toxin. The Γ-CNTX-Pn1a peptide is an insecticidal neurotoxin derived from the Brazilian tarantula (Brazilian wandering spider). Γ-CNTX-Pn1a targets the N-methyl-D-aspartate (NMDA) isoform and sodium channel of the ionotropic glutamate receptor (GRIN). An exemplary wild-type full-length Γ-CNTX-Pn1a peptide has the following amino acid sequence: MKVAIVFLSLLVLAFASESIEENREEFPVEESARCADING ACKSDCDCCGDSVTCDCYWSDSCKCRESNFKIGMAIRKKFC (SEQ ID NO:689) (NCBI Registry No. P59367). A recombinant mature Γ-CNTX-Pn1A peptide is provided, having the amino acid sequence “GSCADINGACKSDCDCCGDSVTCDCYWSDSCKCRESNFKIGMAIRKKF C” (SEQ ID NO:65).

[0291] In some embodiments, the Γ-CNTX-Pn1a peptide may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with SEQ ID NO:65.

[0292] In some embodiments, the polynucleotide encoding the Γ-CNTX-Pn1a peptide may encode an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, or at least 90% identity with the amino acid sequence shown in SEQ ID NO:65. A Γ-CNTX-Pn1a peptide with an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identity.

[0293] Wild-type U1-agatoxin and TVP

[0294] The "hobo spider" (formerly known as the wanderer spider (Tegenaria agrestis)) is a member of the family Agelenidae or funnel-web spiders. See Ingale A, "Antigenic epitopesprediction and MHC binder of a paralytic insecticidal toxin (ITX-1) of Tegenaria agrestis (hobo spider)", August 4, 2010, Vol. 2010, No. 2: pp. 97-103. The venom of the hobo spider is believed to have insecticidal activity. See Johnson et al., “Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system,” Arch Insect Biochem Physiol., 1998, Vol. 38, No. 1: pp. 19-31; Klint et al., Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli, PLoS One, 2013, Vol. 8, No. 5: p. e63865.

[0295] Wandering spiders—along with several other spiders in the family Lymnaeidae—produce venom containing funnel-web spider toxins—which exhibit insecticidal activity. Funnel-web spider toxins are a diverse group of toxins that induce varying insecticidal effects depending on the target species; for example, they cause slow-onset spastic paralysis in Coleoptera, Lepidoptera, and Diptera; increase the neuronal firing rate in the central nervous system (CNS) of the housefly (common housefly); and are lethal to other insects (e.g., the green glass fly (Lucilia cuprina)). Therefore, funnel-web spider toxins involve targeting the CNS. See Undheim et al., “Weaponization of a hormone:convergent recruitment of hyperglycemichormone into the venom of arthropod predators,” Structure, Vol. 23, pp. 1283-1292; and Johnson et al., “Novel insecticidal peptides from Tegenaria agrestisspider venom may have a direct effect on the insect central nervous system,” Arch. Insect Biochem. Physiol., Vol. 38, pp. 19-31 (1998).

[0296] Two types of funnel-web spider venoms include U1-funnel-web spider venom-Ta1a and U1-funnel-web spider venom-Ta1b, both members of the helical arthropod-neuropeptide-derived (HAND) venom family. Besides spiders, these venoms are also found in the venom of centipedes. Funnel-web spider venoms belong to an evolutionary branch of the ancient ecdysone family, namely the ion transport peptide / crustacean hyperglycemic hormone (ITP / CHH) family. See Undheim et al., “Weaponization of ahormone:convergent recruitment of hyperglycemic hormone into the venom of arthropod predators,” Structure, Vol. 23: pp. 1283–1292; and Johnson et al., “Novelinsecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system,” Arch. Insect Biochem. Physiol., Vol. 38: pp. 19–31 (1998).

[0297] The U1-funnel-web spider venom-Ta1b toxin derived from the wandering spider has the complete amino acid sequence “MKLQLMICLVLLPCFFCEPDEICRARMTNKEFTYKSNVCNNCGDQVA ACEAECFRNDVYTACHEAQKG (SEQ ID NO:48)”, which includes a signal peptide from amino acid positions 1-17 and a mature toxin from positions 18-68, as described in the aforementioned literature. The protein contains four tightly packed α-helices, lacks a β-chain, and the mature toxin has a molecular weight of 5700.39 Daltons (Da), as described in the aforementioned literature.

[0298] An exemplary mature wild-type U1-funnelweb spider venom-Ta1b polypeptide from the vagrant spider is provided, having the amino acid sequence: “EPDEICRARMTNKEFTYKSNVCNNCGD QVAACEAECFRNDVYTACHEAQKG” (SEQ ID NO:1).

[0299] During protein processing, mature wild-type U1-funnel-web spider toxin-Ta1b toxin undergoes a C-terminal glycine cleavage event, resulting in the following amino acid sequence: EPDEECRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHE AQK (SEQ ID NO: 60). Subsequent post-translational events lead to C-terminal amidation of mature wild-type U1-funnel-web spider toxin-Ta1b toxin.

[0300] A U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) is a mutant or variant that differs in some respects from wild-type U1-funnel-web spider toxin-Ta1b (SEQ ID NO:1). For example, in some embodiments, this change may be an amino acid substitution, deletion, or addition; or an alteration of the polynucleotide encoding wild-type U1-funnel-web spider toxin-Ta1b, resulting in an amino acid substitution, deletion, or addition. The result of this variation is a non-naturally occurring polypeptide and / or a polynucleotide sequence encoding that polypeptide that exhibits enhanced insecticidal activity against one or more insect species relative to wild-type U1-funnel-web spider toxin-Ta1b.

[0301] In some embodiments, the TVP may have an amino acid sequence according to SEQ ID NO:2-15, 49-53, 621-622, 624-628, 631-640, 642-651 or 653-654, as shown in Table 1.

[0302] Table 1. TVP of the present invention.

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] In some embodiments, a polynucleotide sequence having a sequence according to SEQ ID NO: 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654 is operable to encode a TVP. For example, in some embodiments, the polynucleotides shown in Table 2 are operable to encode a TVP.

[0310] Table 2. Polynucleotides of the present invention.

[0311]

[0312]

[0313]

[0314]

[0315] Exemplary TVPs

[0316] An exemplary description of TVP and polynucleotides operable to encode TVP is provided in international application PCT / US21 / 28254, the entire disclosure of which is incorporated herein by reference.

[0317] In some embodiments, the TVP contains one or more mutations relative to the wild-type sequence of U1-funnelweb spider toxin-Ta1b shown in SEQ ID NO:1. For example, in some embodiments, the TVP may have a first, second, or third mutation relative to the wild-type sequence of U1-funnelweb spider toxin-Ta1b shown in SEQ ID NO:1.

[0318] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent, or a pharmaceutically acceptable salt thereof.

[0319] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; and wherein TVP has an amino acid substitution at X1, X2, X3, X4, or X5, or a pharmaceutically acceptable salt thereof.

[0320] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; wherein TVP has an amino acid substitution at X1, X2, X3, X4, or X5; and wherein X7 is glycine, or a pharmaceutically acceptable salt thereof.

[0321] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; wherein TVP has an amino acid substitution at X1, X2, X3, X4, or X5; and wherein X7 is absent, or a pharmaceutically acceptable salt thereof.

[0322] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; and wherein TVP has an amino acid substitution at X1, X2, X3, X4, or X5; and wherein X6 and X7 are absent, or are pharmaceutically acceptable salts of them.

[0323] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; and TVP contains the amino acid sequence shown in any one of SEQ ID NO:2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654, or a pharmaceutically acceptable salt thereof.

[0324] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; and wherein TVP is encoded by the polynucleotide sequence or their complementary nucleotide sequence shown in any one of SEQ ID NO:17-30, 54-58, or 655-688.

[0325] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; and wherein the TVP further comprises homopolymers or heteropolymers of two or more TVPs, wherein the amino acid sequences of each TVP are the same or different.

[0326] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnelweb spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; and wherein the TVP is a fusion protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each TVP may be the same or different.

[0327] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnelweb spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; and wherein the TVP is a fusion protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each TVP may be the same or different, and wherein the linker is cleavable in the insect's gut or hemolymph.

[0328] In some embodiments, the adapter has an amino acid sequence shown in any one of SEQ ID NO:61-70.

[0329] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% identity with the amino acid sequence according to formula (I). TVPs with amino acid sequences of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity: EPDEECR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is relative to SEQ The wild-type sequence of U1-funnel web spider toxin-Ta1b shown in ID NO:1 contains at least one amino acid substitution, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent; and wherein if Z1 is T or S, then TVP is glycosylated or a pharmaceutically acceptable salt thereof.

[0330] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, and so on, with the amino acid sequence “EPDEICRAQMTNKEFTYKSNVCNNCGD QVAACEAECFRNDVYAACHEAQKG” (SEQ ID NO: 51). TVPs with amino acid sequences of at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity.

[0331] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, or at least TVPs with amino acid sequences of 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity: EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG; wherein the polypeptide contains at least one amino acid substitution relative to the wild-type sequence of U1-funnel-web spider toxin-Ta1b shown in SEQ ID NO:1, and wherein X1 is R or Q; and Z1 is T or A; or a pharmaceutically acceptable salt thereof.

[0332] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, or at least TVPs with amino acid sequences of 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity: EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG; wherein the polypeptide contains at least one amino acid substitution relative to the wild-type sequence of U1-funnel web spider toxin-Ta1b shown in SEQ ID NO:1, and wherein X1 is R or Q; and Z1 is T or A; or a pharmaceutically acceptable salt thereof; wherein if Z1 is T, the TVP is glycosylated.

[0333] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, or at least TVPs with amino acid sequences of 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity: EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG; wherein the polypeptide contains at least one amino acid substitution relative to the wild-type sequence of U1-funnel-web spider toxin-Ta1b shown in SEQ ID NO:1, and wherein X1 is R or Q; and Z1 is T or A; or a pharmaceutically acceptable salt thereof, wherein X1 is Q; and Z1 is A.

[0334] In some embodiments, the insecticidal U1-funnel-web spider toxin-Ta1b variant polypeptide (TVP) may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identity with the amino acid sequence shown in any one of SEQ ID NO:2, 49, or 51. TVPs of amino acid sequences with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity, or pharmaceutically acceptable salts thereof.

[0335] In some embodiments, TVP may comprise an amino acid sequence “EPDEICRAQMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYAACH EAQKG” (SEQ ID NO: 1). NO:51) has an amino acid sequence with at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity.

[0336] In some preferred embodiments, the TVP may be TVP-R9Q / T43A (SEQ ID NO: 51).

[0337] In various embodiments, polynucleotides encoding TVPs can be used to transform plant cells, yeast cells, or bacterial cells. In some embodiments, insecticidal TVP transgenic proteins can be formulated into compositions that can be sprayed or otherwise applied to the surface of plants or parts thereof in any manner known to those skilled in the art. Therefore, DNA constructs are provided herein operable to encode one or more TVPs in host cells (e.g., plant cells) under suitable conditions. Methods for controlling insect infestations of plant cells by parasitic insects include applying or introducing polynucleotides encoding TVPs as described herein into plants, plant tissues, or plant cells via recombinant technology, and allowing the recombinantly altered plants, plant tissues, or plant cells to grow in fields exposed to pests. Alternatively, TVPs can be formulated into sprayable compositions consisting of TVPs and excipients and applied directly to susceptible plants by direct application, causing harmful effects upon ingestion of the TVP by infectious insects.

[0338] Scorpion peptides and toxins

[0339] In some implementations, CRIP can be the following scorpion ( scorpionAny of the following peptides, polypeptides, and / or toxins: Imperatoxin-A (IpTxa), potassium channel toxin α-KTx 10.2 (Cobatoxin-2), potassium channel toxin α-KTx 11.1 (Parabutoxin-1), potassium channel toxin α-KTx 11.2 (Parabutoxin-2), potassium channel toxin α-KTx 11.3 (Parabutoxin-10), potassium channel toxin α-KTx 12.1 (Butantoxin), potassium channel toxin α-KTx 12.2 (Butantoxin), potassium channel toxin α-KTx 12.3 (Butantoxin-like peptide), potassium channel toxin α-KTx 15.1 (peptide Aa1), potassium channel toxin α-KTx 15.3 (toxin AmmTX3), potassium channel toxin α-KTx 15.6 (Discrepin), potassium channel toxin α-KTx 16.1 (Tamulotoxin), Potassium channel toxin α-KTx 19.1 (Neurotoxin BmBKTx1), Potassium channel toxin α-KTx 1.3 (African scorpion venom), Potassium channel toxin α-KTx 1.4 (Limbatotoxin), Potassium channel toxin α-KTx 1.7 (Lqh15-1), Potassium channel toxin α-KTx 1.9 (Hongotoxin-2), Potassium channel toxin α-KTx 1.10 (Parabutoxin-3), Potassium channel toxin α-KTx 1.11 (Slotoxin), Potassium channel toxin α-KTx 1.13 (Kalyu (Bud) scorpion venom c), Potassium channel toxin α-KTx 2.1 (Noxiustoxin), Potassium channel toxin α-KTx 2.2 (Magetoxin), Potassium channel toxin α-KTx 2.3 (CllTx1), Potassium channel toxin α-KTx 2.4 (Noxiustoxin-2), Potassium channel toxin α-KTx 2.5 (Hongotoxin-1), Potassium channel toxin α-KTx 2.6 (Hongotoxin-3), Potassium channel toxin α-KTx 2.7 (CllTx2), Potassium channel toxin α-KTx 2.8 (Toxin Ce1), Potassium channel toxin α-KTx 2.9 (Toxin Ce2), Potassium channel toxin α-KTx 2.10 (Toxin Ce3), Potassium channel toxin α-KTx 2.11 (Toxin Ce4), Potassium channel toxin α-KTx 2.12 (Toxin Ce5), Potassium channel toxin α-KTx 3.1 (Shortskin scorpion venom-1), Potassium channel toxin α-KTx 3.2 (Agitoxin-2), Potassium channel toxin α-KTx 3.3 (Agitoxin-3), Potassium channel toxin α-KTx 3.4 (Agitoxin-1), potassium channel toxin α-KTx 3.7 (OsK-1), potassium channel toxin α-KTx 3.8 (Carlyle (Budd) scorpion venom-like peptide Bs 6), potassium channel toxin α-KTx 3.9 (Short-skinned scorpion venom-3), potassium channel toxin α-KTx 4.1 (East Asian scorpion venom K-α), potassium channel toxin α-KTx 4.3 (toxin TdK1), potassium channel toxin α-KTx 4.4 (toxin Tc30), potassium channel toxin α-KTx 5.1 (Leiurotoxin-1), potassium channel toxin α-KTx 5.2 (Leiurotoxin I-like toxin P05), potassium channel toxin α-KTx 5.4 (Tamapin), potassium channel toxin α-KTx 5.5 (Tamapin-2), potassium channel toxin α-KTx 6.1 (potassium channel blocking toxin 1), potassium channel toxin α-KTx 6.2 (Maurotoxin), potassium channel toxin α-KTx 6.3 (neurotoxin HsTX1), potassium channel toxin α-KTx 6.12 (Anuroctoxin), Potassium channel toxin α-KTx 6.13 (Spinoxin), Potassium channel toxin α-KTx 6.14 (HgeTx1), Potassium channel toxin α-KTx 7.2 (toxin PiTX-K-β), Potassium channel toxin γ-KTx 1.2 (Ergtoxin-like protein 1), Potassium channel toxin γ-KTx 1.3 (Ergtoxin-like protein 1), Potassium channel toxin γ-KTx 1.4 (Ergtoxin-like protein 1), Potassium channel toxin γ-KTx 1.5 (Ergtoxin-like protein 1), Potassium channel toxin γ-KTx 1.6 (Ergtoxin-like protein 1), Potassium channel toxin γ-KTx 4.2 (Ergtoxin-like protein 5), Insectotoxin-I1. Small toxins (peptide I), Insectotoxin-I3 (BeI3), Insectotoxin-I4 (BeI4), Insectotoxin-I5A, neurotoxin 8 (neurotoxin VIII), possible toxins Lqh 8 / 6, neurotoxin 9 (neurotoxin IX), Maurocalcin (MCa), chloramphenicol-like peptide Bs14 (Bs14), chloramphenicol (CTX), neurotoxin P2, Insectotoxin-I5 (BeI5), potassium channel toxin α-KTx 6.15 (semi-toxin), toxin GaTx1, AahIT1, Phaiodotoxin, BaIT2, BotIT1, BotIT2, BmK M1, BmK-M2, BmK-M4, BmK-M7, BmK IT-AP, Bom3, Bom4, BjaIT, Bj-xtrIT, BjIT2, LqhaIT, Lqhb1, LqhIT2, LqhdprIT3a, Lgh-xtrIT, Lqh3, Lqh6, Lqh7, LqqIT1, LqqIT2, Lqq3, OD1, Ts1 or Tz1. .

[0340] In some embodiments, CRIP may be a scorpion peptide having an amino acid sequence shown in any of SEQ ID NO:88-191.

[0341] In some implementations, CRIP can be imperatoxin. Imperatoxin is a peptide toxin derived from the venom of the African scorpion (Pandinus imperator).

[0342] In some embodiments, the CRIP may be imperatoxin, wherein the imperatoxin is imperatoxin A (IpTx-a) or a variant thereof. In some embodiments, IpTx-a has the amino acid sequence GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR (SEQ ID NO:66).

[0343] In some embodiments, the CRIP can be the AaIT1 toxin. The protein toxin AaIT1 is a sodium channel site 4 toxin from the North African desert scorpion (Androctonus australis). An exemplary AaIT1 toxin is a peptide having the amino acid sequence according to SEQ ID NO:88 (NCBI accession number P01497.2). AaIT1 is a site 4 toxin that forces insect sodium channels to open by lowering the energy barrier of the activation response.

[0344] In some embodiments, the scorpion peptide may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with SEQ ID NO:66, 88-191.

[0345] In some embodiments, the polynucleotide encoding the scorpion peptide or toxin may encode an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identity with the amino acid sequence shown in SEQ ID NO:66, 88-191. Scorpion peptides or toxins with amino acid sequences of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity.

[0346] Sea anemone peptides and toxins

[0347] In some embodiments, CRIP can be isolated from the sea anemone. For example, in some embodiments, the sea anemone may be *Actinia equina*; *Anemonia erythraea*; *Anemonia grosvenorii*; *Anemonia serpentina*; *Anemonia glabra*; *Anemonia rust-green*; *Anemonia jasminoides*; *Anemonia jasminoides*; *Bunodosoma caissarum*; *Bunodosoma cangicum*; *Anemonia verrucosum*; *Anemonia purpurea*; *Parasicyonis actinostoloides*; *Radianthus paumotensis*; or *Anemonia semperflorens*. In other embodiments, the anemone toxin may be Av2; Av3; or variants thereof.

[0348] In some implementations, CRIP can be one of the following anemone toxins: toxin AETX-1 (AETX I), toxin APETx1, toxin APETx2, antihypertensive protein BDS-1 (blood inhibitor I), antihypertensive protein BDS-2 (blood inhibitor II), neurotoxin Bg-2 (BgII), neurotoxin Bg-3 (BgIII), toxin APE 1-1, toxin APE 1-2, neurotoxin-1 (toxin ATX-I), neurotoxin-1 (neurotoxin I), neurotoxin 1 (toxin RTX-I), neurotoxin 1 (toxin SHP-I), toxin APE 2-1, toxin APE 2-2, neurotoxin-2 (toxin ATX-II), (aka Neurotoxin-2 (AFT-II), Neurotoxin 2 (RTX-II), Neurotoxin 2 (Neurotoxin II), Neurotoxin 3 homolog (Neurotoxin III homolog), Neurotoxin 3 (RTX-III), Neurotoxin 3 (Neurotoxin-III), Neurotoxin 4 (RTX-IV), Neurotoxin-5 (ATX-V), Neurotoxin 5 (RTX-V), Anemone Cardiotonic Peptide-A (AP-A), Anemone Cardiotonic Peptide-B (AP-B), Anemone Cardiotonic Peptide-C (AP-C), Potassium Channel Toxin Aek, Potassium Channel Toxin Bgk, Major Neurotoxin BcIII, Neurotoxin BcIV, Cangitoxin (CGTX), Potassium Channel Toxin ShK, Toxin PCR1 (PCR1-2), Toxin PCR2 (PCR2-5), Toxin PCR3 (PCR2-1), Toxin PCR4 (PCR2-10), Toxin PCR6 (PCR3-7), Cangitoxin-2 (Cangitoxin II) or Cangitoxin-3 (Cangitoxin III).

[0349] In some embodiments, CRIP may be anemone peptide having the amino acid sequence shown in SEQ ID NO:371-411.

[0350] In some embodiments, the CRIP of the present invention can be one or more polypeptides derived from sea anemones (Anemone scabra), which possess a variety of toxins for self-defense. One of the toxins derived from Anemone scabra is the neurotoxin "Av3". Av3 is a type III anemone toxin that inhibits voltage-gated sodium (Na+) at receptor site 3. +Inactivation of the sodium channel leads to contractile paralysis. The binding of the Av3 toxin to site 3 destabilizes the inactive state of the sodium channel, which in turn causes the channel to remain open (see Blumenthal et al., “Voltage-gated sodium channel toxins: poisons, probes, and future promise,” Cell Biochem Biophys., 2003, Vol. 38, No. 2: pp. 215-238). Av3 exhibits high selectivity for crustacean and insect sodium channels and low selectivity for mammalian sodium channels (see Moran et al., “Sea anemone toxins affecting voltage-gated sodium channels—molecular and evolutionary features,” Toxicon, December 15, 2009, Vol. 54, No. 8: pp. 1089-1101). An exemplary Av3 polypeptide from the sea anemone *Syngonium scabra* is provided, having the amino acid sequence of SEQ ID NO: 44.

[0351] In some embodiments, the CRIP of the present invention may be an Av3 variant polypeptide (AVP). In some embodiments, the AVP may have the following amino acid variations from SEQ ID NO:44: an N-terminal amino acid substitution of R1K relative to SEQ ID NO:44, thereby changing the polypeptide sequence from wild-type “RSCCPCYWGGCPWGQNCYPEGCSGPKV” to “KSCCPCYWGGCPWGQNCYPEGCSGPKV” (SEQ ID NO:45); a C-terminal amino acid deletion relative to SEQ ID NO:44, thereby changing the polypeptide sequence from wild-type “RSCCPCYWGGCPWGQNCYPEGCSGPKV” to “RSCCPCYWGGCPWGQNCYPEGCSGPK” (SEQ ID NO:46); and / or an N-terminal mutation and a C-terminal mutation, wherein the N-terminal amino acid may have a substitution of R1K relative to SEQ ID NO:44, and relative to SEQ ID NO:44, the C-terminal amino acid may have a substitution of R1K, and relative to SEQ ID NO:44, the C-terminal amino acid may have a substitution of R1K. NO:44, the C-terminal amino acid can be deleted, thereby changing the polypeptide sequence from wild-type "RSCCPCYWGGCPWGQNCYPEGCSGPKV" to "KSCCPCYWGGCPWGQNCYPEGCSGPK" (SEQ ID NO:47).

[0352] In some embodiments, exemplary Av3 peptides or variants thereof are described in the applicant’s PCT application (application number PCT / US19 / 51093) filed on September 13, 2019, entitled “Av3 Mutant Insecticidal Polypeptides and Methods for Producing and Using Same”, the disclosure of which and the disclosure of Av3 peptides or variants thereof are described herein and are incorporated herein by reference in their entirety.

[0353] In some embodiments, the sea anemone peptide may comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with SEQ ID NO:44-47 and 371-411.

[0354] In some embodiments, the polynucleotide encoding the sea anemone peptide may encode an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identity with the amino acid sequences shown in SEQ ID NO:44-47 and 371-411. Sea anemone peptides with amino acid sequences of at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, at least 99.9% identity, or 100% identity.

[0355] Conus peptides and conotoxins

[0356] Conotoxins are toxins isolated from the cone snail; these toxins act by interfering with neuronal communication. Examples of conotoxins include α-, ω-, μ-, δ-, and κ-conotoxins. In short, α-conotoxins (and αA- and φ-conotoxins) target nicotinic ligand-gated channels; ω-conotoxins target voltage-gated calcium channels; μ-conotoxins target voltage-gated sodium channels; δ-conotoxins target voltage-gated sodium channels; and κ-conotoxins target voltage-gated potassium channels.

[0357] In some implementations, CRIP can be isolated from organisms belonging to the genus Conodontia, wherein the isolated peptide is conodontoxin.

[0358] In some implementations, CRIP can be isolated from: Amady cone snail; cat cone snail; turtle cone snail; killer cone snail; sea glory cone snail; wood-shining cone snail; monk's robe cone snail; marble cone snail; purple gold cone snail; fly cone snail; thread cone snail; brocade cone snail; or tulip cone snail.

[0359] Other CRIPs

[0360] In some implementations, CRIP can be a toxin, peptide, or protein (also referred to as venom, toxic peptide, or protein) produced and / or isolated from arthropods, spiders, scorpions, insects, bees, wasps, centipedes, crustaceans, reptiles, snakes, lizards, amphibians, frogs, salamanders, mollusks, conch shells, cnidarians, sea anemones, jellyfish, hydroids, cephalopods, octopuses, squid, cuttlefish, fish, or mammals.

[0361] In some implementations, CRIP can be snake venom or a toxin derived from snake venom.

[0362] CRIP - insecticidal proteins

[0363] CRIP-insecticide protein is any protein, peptide, polypeptide, amino acid sequence, conformation, or arrangement that consists of: (1) at least one CRIP, or two or more CRIPs; and (2) additional non-CRIP peptides, polypeptides, or proteins, for example, in some embodiments, having the ability to: increase insect mortality and / or inhibit insect growth when insects are exposed to CRIP-insecticide protein relative to CRIP alone; increase the expression of said CRIP-insecticide protein, for example, in a host cell or expression system; and / or affect the post-translational processing of CRIP-insecticide protein.

[0364] In some embodiments, the CRIP-insecticide protein may be a polymer comprising two or more CRIPs. In some embodiments, the CRIP-insecticide protein may be a polymer comprising two or more CRIPs, wherein the CRIPs are operatively linked via linker peptides (e.g., cleavable and / or uncleavable linkers).

[0365] In some embodiments, CRIP-insectic protein may refer to one or more CRIPs operatively linked to one or more proteins, such as stabilization domains (STA), endoplasmic reticulum signaling proteins (ERSP), insect-cleavable or insect-inoperable linkers (L), and / or any other combination thereof.

[0366] In some implementations, the CRIP-insecticide protein may be a non-naturally occurring protein, including (1) wild-type CRIP; and (2) other peptides, polypeptides or proteins, such as ERSP, adapters, STA, UBI or histidine tags or similar markers.

[0367] In some implementations, the CRIP-insecticide protein may be a non-naturally occurring protein, including (1) wild-type CRIP; and (2) non-naturally occurring CRIP.

[0368] In some implementations, the CRIP-insecticide protein may be a non-naturally occurring protein, including (1) wild-type CRIP; and (2) non-naturally occurring CRIP; and (3) other peptides, polypeptides or proteins, such as ERSP, adapters, STA, UBI or histidine tags or similar markers.

[0369] In some implementations, CRIP-insecticide protein may include any of the CRIPs described herein.

[0370] In some embodiments, the insecticidal protein may comprise one or more CRIPs disclosed herein. In some embodiments, the insecticidal protein may comprise CRIP homopolymers, such as two or more CRIP monomers that are the same CRIP. In some embodiments, the insecticidal protein may comprise CRIP heteropolymers, such as two or more CRIP monomers, wherein the CRIP monomers are different.

[0371] In some embodiments, the insecticidal protein may comprise a fusion protein comprising two or more CRIPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRIP may be the same or different.

[0372] In some embodiments, the insecticidal protein may comprise a fusion protein comprising two or more CRIPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRIP may be the same or different, and wherein the linker is cleavable in the insect's gut or hemolymph.

[0373] In some embodiments, the insecticidal protein may comprise a fusion protein comprising two or more CRIPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRIP may be the same or different, and wherein the linker is cleavable in the mammalian gut.

[0374] Exemplary methods for producing cuttable and non-cuttable joints can be found in U.S. Patent Application No. 15 / 727,277 and PCT Application No. PCT / US2013 / 030042, the disclosures of which are incorporated herein by reference in their entirety.

[0375] Methods of producing CRIPs or peptides-IA

[0376] Methods for producing proteins are well known in the art, and various techniques are available. For example, in some embodiments, proteins may be produced using recombinant methods or chemical synthesis. This disclosure provides methods for producing CRIP, CRIP-insecticide protein, and other peptide insecticides (peptide-IA). These methods will be described in detail below.

[0377] In some embodiments, the CRIP of the present invention can be produced using any known method for producing proteins. For example, in some embodiments, and without limitation, the CRIP can be produced using a recombinant expression system, such as a yeast expression system or a bacterial expression system. However, those skilled in the art will recognize that other protein production methods can be used.

[0378] In some embodiments, the present invention provides a method for producing CRIPs using a recombinant expression system.

[0379] In some embodiments, the present invention comprises, is substantially composed of, or is composed of a method for producing CRIP, said method comprising: (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising, is substantially composed of, or is composed of a polynucleotide or a complementary nucleotide sequence operable to encode CRIP; (b) introducing the vector into a host cell, such as bacteria or yeast, or insect, or plant cell, or animal cell; and (c) culturing a yeast strain in a growth medium under conditions operable to enable CRIP expression and secretion into a growth medium. In some related embodiments, the host cell is a yeast cell.

[0380] This invention is feasible in a variety of host cells (see the Host Cell section below). In fact, the end user of this invention can practice its teachings in any host cell of his or her choice. Therefore, in some embodiments, the host cell can be any host cell that meets the end user's requirements; that is, in some embodiments, CRIP expression can be performed using a variety of host cells and in accordance with the teachings herein. For example, in some embodiments, the user may expect to use a particular type of host cell (e.g., yeast cells or bacterial cells) instead of another; the preferred range of a given host cell can range from availability to cost.

[0381] For example, in some embodiments, the invention comprises, is substantially composed of, or is composed of a method for producing CRIP, said method comprising: (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising, is substantially composed of, or is composed of a polynucleotide or complementary nucleotide sequence operable to encode CRIP; (b) introducing the vector into a host cell, such as bacteria or yeast, or insect, or plant cell, or animal cell; and (c) culturing a yeast strain in a growth medium under conditions operable to enable CRIP expression and secretion into a growth medium. In some related embodiments, the host cell is a yeast cell.

[0382] Isolation and mutation of wild-type CRIPs

[0383] CRIP or peptide-insecticidal agents (peptide-IA) can be obtained directly from the source (e.g., isolated from animals). Mutant CRIP or peptide-IA can be produced by: generating a mutation in the wild-type CRIP or peptide-IA polynucleotide sequence; inserting the CRIP or peptide-IA polynucleotide sequence into a suitable vector; transforming a host organism in a manner that expresses the polynucleotide encoding the CRIP or peptide-IA; culturing the host organism to produce the desired amount of CRIP or peptide-IA; and then purifying the CRIP or peptide-IA from the host organism and / or surrounding environment.

[0384] Inducing mutations in wild-type CRIP or peptide-IA polynucleotide sequences can be achieved by a variety of methods well known to those skilled in the art. Mutagenesis methods include the Kunkel method; cassette mutagenesis; site-directed PCR mutagenesis; the "perfect murder" technique ("perfect crime"); direct gene deletion and site-directed mutagenesis using PCR and a cyclic marker; direct gene deletion and site-directed mutagenesis using PCR and a cyclic marker using a long homologous region; the shift "bounce-in-bounce" method; and CRISPR-Cas 9. Exemplary methods of site-directed mutagenesis can be found in Ruvkun and Ausubel, “A general method for site-directed mutagenesis in prokaryotes,” Nature, January 1, 1981, Vol. 289, No. 5793: pp. 85-88; Wallace et al., “Oligonucleotide-directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA,” Nucleic Acids Res., August 11, 1981, Vol. 9, No. 15: pp. 3647-3656; Dalbadie-McFarland et al., “Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function,” Proc Natl Acad Sci USA, November 1982, Vol. 79, No. 21: pp. 6409-6413; and Bachman, “Site-directed mutagenesis.” Methods Enzymol., 2013, Vol. 529: pp. 241-248; Carey et al., “PCR-mediated site-directed mutagenesis”, Cold Spring Harb Protoc., August 1, 2013, Vol. 8: pp. 738-742; and Cong et al., “Multiplex genome engineering using CRISPR / Cassystems”, Science, February 15, 2013, Vol. 339, No. 6121: pp. 819-823; The full text of these publications is incorporated herein by reference.

[0385] Wild-type CRIPs, such as spider, scorpion, and / or other toxins, can be isolated from venom. For example, spider venom can be isolated from the venom glands of spiders (e.g., spiders such as hobo spiders) using any technique known to those skilled in the art. For example, in some embodiments, venom can be isolated from spiders according to the method described in U.S. Patent No. 5,688,764, the entire disclosure of which is incorporated herein by reference.

[0386] Wild-type CRIP or peptide-IA polynucleotide sequences can be obtained by screening genomic libraries using primers and probes targeting the CRIP or peptide-IA polynucleotide sequence. Alternatively, wild-type CRIP or peptide-IA polynucleotide sequences and / or mutant CRIP or peptide-IA polynucleotide sequences can be chemically synthesized. For example, CRIP or peptide-IA polynucleotide sequences and / or mutant CRIP or peptide-IA polynucleotide sequences can be generated using oligonucleotide synthesis methods, such as phosphoramide; triester, phosphite, or H-phosphonate methods. See Engels, JW and Uhlmann, E., Gene Synthesis (New Synthetic Methods (77)), Angew. Chem. Int. Ed. Engl., Vol. 28: pp. 716-734, the full text of which is incorporated herein by reference.

[0387] Chemical synthesis of CRIP or peptide-IA polynucleotides

[0388] In some implementations, the polynucleotide sequence encoding CRIP or peptide-IA can be obtained using commercially available polynucleotide synthesis services (such as those provided by [Provider Name]). (For example, TurboGENE) TM PriorityGENE and FragmentGENE) or (For example, custom DNA and RNA oligomer design and custom DNA oligomers) provided by chemical synthesis. Exemplary methods for producing DNA and / or custom chemically synthesized polynucleotides are well known in the art and are illustratively provided in U.S. Patent No. 5,736,135, Serial No. 08 / 389,615, filed February 13, 1995, the disclosure of which is incorporated herein by reference in its entirety. See also Agarwal et al., “Chemical synthesis of polynucleotides,” Angew Chem Int Ed Engl., June 1972, Vol. 11, No. 6: pp. 451-459; Ohtsuka et al., “Recent developments in the chemical synthesis of polynucleotides,” Nucleic Acids Res., November 11, 1982, Vol. 10, No. 21: pp. 6553-6570; Sondek and Shortle, “A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites,” Proc Natl Acad Sci USA, April 15, 1992, Vol. 89, No. 8: pp. 3581-3585; Beaucage S.L. et al., “Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach,” Tetrahedron, Elsevier Science. Publishers, Amsterdam, NL, Vol. 48, No. 12, 1992, pp. 2223-2311; Agrawal, 1993, “Protocols for Oligonucleotides and Analogs: Synthesis and Properties”, Methods in Molecular Biology, Vol. 20. The full text of these publications is incorporated herein by reference.

[0389] Chemically synthesized polynucleotides allow the production of DNA sequences tailored to produce desired polypeptides based on the arrangement of nucleotides within the sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A], or thymine [T] molecules); the mRNA sequence transcribed from the chemically synthesized DNA polynucleotide can be translated into an amino acid sequence, each amino acid corresponding to a codon in the mRNA sequence. Therefore, the amino acid composition of the polypeptide chain translated from the mRNA sequence can be altered by changing the base codon that determines which of the 20 amino acids will be added to the growing polypeptide; thus, mutations in DNA such as insertions, substitutions, deletions, and frameshifts can cause amino acid insertions, substitutions, or deletions, depending on the base codon.

[0390] Obtaining CRIPs or peptide-IAs from chemically synthesized DNA polynucleotide sequences and / or wild-type DNA polynucleotide sequences altered via mutagenesis can be achieved by cloning the DNA sequence into a suitable vector. A variety of available expression vectors, host organisms, and cloning strategies are known to those skilled in the art. For example, a vector can be a plasmid that can introduce a heterologous gene and / or expression cassette into yeast cells for transcription and translation. The term "vector" is used to refer to a vector nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell, where the nucleic acid sequence can be replicated. A vector may contain "vector elements" such as an origin of replication (ORI); genes that confer antibiotic resistance to allow selection; multiple cloning sites; promoter regions; selection markers for non-bacterial transfection; and primer binding sites. The nucleic acid sequence can be "exogenous," meaning it is foreign to the cell into which the vector is introduced, or the sequence is homologous to a sequence in the cell but is not typically found in the host cell's nucleic acid. Vectors include plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will readily construct vectors using standard recombination techniques, as described in Sambrook et al. (1989) and Ausubel et al. (1996), both of which are incorporated herein by reference. In addition to encoding CRIPs or peptide-IA polynucleotides, vectors may encode target molecules. Target molecules are molecules that guide the desired nucleic acid to a specific tissue, cell, or other location.

[0391] Vectors and transformation

[0392] In some implementations, CRIP or peptide-IA polynucleotides can be cloned into vectors using various cloning strategies and commercially available cloning kits and materials readily available to those skilled in the art. For example, CRIP or peptide-IA polynucleotides can be cloned into vectors using strategies such as SnapFast, Gateway, TOPO, Gibson, LIC, InFusionHD, or Electra. Many commercially available vectors exist for the production of CRIP or peptide-IA. For example, CRIP or peptide-IA polynucleotides can be generated using polymerase chain reaction (PCR) and coupled with pCR... TM II-TOPO vector or PCR TM 2.1- carrier (as) TA The kit (obtained commercially from Invitrogen) can be mixed at room temperature for 5 minutes; then... The reactants are converted into competent cells, which can then be selected based on color changes (see Janke et al., “Aversatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes”, Yeast, August 2004, Vol. 21, No. 11: pp. 947-962; see also Adams et al., Methods in YeastGenetics., Cold Spring Harbor, NY, 1997, the full text of which is incorporated herein by reference).

[0393] In some implementations, the polynucleotide encoding CRIP or peptide-IA can be cloned into vectors such as plasmids, kinases, viruses (bacteriophages, animal viruses, and plant viruses) and / or artificial chromosomes (e.g., YAC).

[0394] In some embodiments, the polynucleotide encoding CRIP or peptide-IA can be inserted into a vector (e.g., a plasmid vector using *E. coli* as the host) by digesting approximately 2 to 5 μg of the vector DNA with a restriction enzyme necessary to allow insertion of the DNA fragment of interest, followed by overnight incubation to complete digestion (alkaline phosphatase can be used for 5' dephosphorylation to avoid self-ligation / recircularization); the digested vector is then purified by gel filtration. Next, the DNA fragment of interest, e.g., the polynucleotide encoding CRIP or peptide-IA, is amplified by PCR, and any excess enzymes, primers, unincorporated dNTPs, short-lived PCR products, and / or salts are removed from the PCR reaction using techniques known to those skilled in the art (e.g., by using a PCR cleanup kit). The DNA fragment of interest is ligated to a vector by generating a mixture comprising: approximately 20 ng of vector; approximately 100 ng to 1,000 ng of the DNA fragment of interest; 2 μL of 10× buffer (i.e., 30 mM Tris-HCl, 4 mM MgCl2, 26 μM NAD, 1 mM DTT, 50 μg / ml BSA, pH 8, stored at 25°C); 1 μL of T4 DNA ligase; and a total volume of 20 μL is achieved by adding H2O. The ligation reaction mixture can then be incubated at room temperature for 2 hours or overnight at 16°C. The ligation reaction mixture (i.e., approximately 1 μL) can then be transformed into competent cells, for example by electroporation or chemical methods, and colony PCR can then be performed to identify vectors containing the DNA fragment of interest.

[0395] In some implementations, the polynucleotide encoding CRIP or peptide-IA, along with other DNA fragments that together constitute the CRIP or peptide-IA expression ORF, can be programmed for secretion from a host yeast cell. An exemplary method for designing a CRIP or peptide-IA expression ORF is as follows: the ORF can begin with a signal peptide sequence, followed by a DNA sequence encoding a Kex2 cleavage site (lysine-arginine), and then a CRIP or peptide-IA polynucleotide transgene with a glycine-serine codon added at the 5' end and a stop codon added at the 3' end. All these elements are then expressed as a fusion peptide as a single open reading frame (ORF) in the yeast cell. The α-mating factor (αMF) signaling sequence is most commonly used to promote the metabolic processing of recombinant insecticidal peptides through the endogenous secretion pathway of recombinant yeast. That is, the expressed fusion peptide usually enters the endoplasmic reticulum, where the α-mating factor signaling sequence is removed by signal peptidase activity, and then the resulting pro-insectic peptide is transported to the Golgi apparatus, where the aforementioned lysine-arginine dipeptide is completely removed by the Kex2 endopeptide, and then the mature polypeptide (i.e., CRIP or peptide-IA) is secreted from the cell.

[0396] In some implementations, peptide expression levels in recombinant yeast cells can be enhanced by codon optimization based on a specific host yeast species. The natural frequency of codons observed in the endogenous open reading frames of a given host organism does not necessarily need to be optimized for efficient expression. Furthermore, different yeast species (e.g., Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, etc.) have different optimal codons for efficient expression. Therefore, codon optimization for CRIP or peptide-IA expression ORFs should be considered, including sequence elements encoding the signal sequence, the Kex2 cleavage site, and CRIP or peptide-IA, as they are initially translated into a fusion peptide in recombinant yeast cells.

[0397] In some implementations, codon-optimized CRIPs or peptide-IA expression ORFs can be ligated into yeast-specific expression vectors for yeast expression. Many expression vectors are available for yeast expression, including appendage vectors and integrative vectors, and they are typically designed for specific yeast strains. A suitable expression vector should be carefully selected based on the specific yeast expression system to be used for peptide production. In some implementations, integrative vectors can be used, which integrate into the chromosome of the transformed yeast cells and remain stable during cycles of cell division and proliferation. The integrative DNA sequence is homologous to the target genomic DNA locus in the transformed yeast species, and such integrative sequences include pLAC4, 25S rDNA, pAOX1, and TRP2. The transgene of the insecticidal peptide can be located adjacent to the integrative DNA sequence (insertion vector) or within the integrative DNA sequence (replacement vector).

[0398] In some embodiments, the expression vector may contain E. coli elements for preparing DNA in *E. coli*, such as *E. coli* origin of replication, antibiotic selection markers, etc. In some embodiments, the vector may contain an array of sequence elements required for expressing the transgene of interest, such as transcription promoters, terminators, yeast selection markers, integrated DNA sequences homologous to host yeast DNA, etc. Many suitable yeast promoters are available, including natural and engineered promoters, such as yeast promoters like pLAC4, pAOX1, pUPP, pADH1, pTEF, pGal1, etc., and other promoters that may be used in some embodiments.

[0399] In some embodiments, selection methods such as acetamide protrophic selection; bleomycin resistance selection; genimycin resistance selection; norsinosin resistance selection; uracil deficiency selection; and / or other selection methods may be used. For example, in some embodiments, the *Aspergillus nidulans* amdS gene may be used as a selection marker. Exemplary methods using selection markers are found in U.S. Patent Nos. 6,548,285 (filed April 3, 1997); 6,165,715 (filed June 22, 1998); and 6,110,707 (filed January 17, 1997), the disclosures of which are incorporated herein by reference in their entirety.

[0400] In some implementations, a polynucleotide encoding CRIP or peptide-IA can be inserted into the pKLAC1 plasmid. pKLAC1 can be obtained from New England... Commercially available from Inc. (NEB#E1000). pKLAC1 is designed for high-level expression of recombinant proteins (e.g., CRIP or peptide-IA) in *Kluyveromyces lactis*. The pKLAC1 plasmid can be ordered alone or as part of a *Kluyveromyces lactis* protein expression kit. The pKLAC1 plasmid can be linearized using SacII or BstXI restriction enzymes and has an MCS downstream of the αMF secretion signal. The αMF secretion signal directs the recombinant protein into the secretion pathway, where it is then cleaved via Kex2 to produce, for example, CRIP or peptide-IA. Kex2 is a calcium-dependent serine protease involved in activating the secretion pathway and is commercially available. Project No. 450-45).

[0401] In some embodiments, a polynucleotide encoding CRIP or peptide-IA can be inserted into the pKlac1 plasmid, or subcloned into the pKlac1 plasmid after yeast colonies transformed with a pKLAC1 plasmid linked to a polynucleotide encoding CRIP or peptide-IA are selected. Yeast colonies transformed with a pKLAC1 plasmid linked to a polynucleotide encoding CRIP or peptide-IA, such as *Kluyveromyces lactis*, can be selected based on acetamipase (amdS), which allows the transformed yeast cells to grow in YCB medium containing acetamide as their sole nitrogen source. Once positive yeast colonies transformed with a pKLAC1 plasmid linked to a polynucleotide encoding CRIP or peptide-IA are identified...

[0402] In some embodiments, the polynucleotide encoding CRIP or peptide-IA can be inserted into other commercially available plasmids and / or vectors that are readily available to those skilled in the art, for example, plasmids can be obtained from Addgene (a non-profit plasmid library). and Promega TM get.

[0403] In some implementations, the polynucleotide encoding TVP can be inserted into other commercially available plasmids and / or vectors that are readily available to those skilled in the art, for example, plasmids can be obtained from Addgene (a non-profit plasmid library). and Promega TM get.

[0404] In some embodiments, yeast cells transformed with one or more CRIP expression cassettes can produce CRIP in yeast cultures at yields of at least 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, and 200 mg / L per liter of culture medium. / L, at least 500mg / L, at least 750mg / L, at least 1,000mg / L, at least 1,250mg / L, at least 1,500mg / L, at least 1,750mg / L, at least 2,000mg / L, at least 2,500mg / L, at least 3,000mg / L, at least 3,500mg / L, at least 4,000mg / L, at least 4,500mg / L, at least 5,000mg / L, at least 5,500mg / L, at least 6,000mg / L, at least 6,500mg / L 0 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 1 CRIP at concentrations of 7,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L.

[0405] In some embodiments, one or more expression cassettes containing a polynucleotide operable to express CRIP can be inserted into the vector, resulting in a CRIP yield per liter of culture medium (yeast fermentation broth supernatant) in the following ranges: about 100 mg / L CRIP to about 100,000 mg / L CRIP; about 110 mg / L to about 100,000 mg / L; about 120 mg / L to about 100,000 mg / L; about 130 mg / L to about 100,000 mg / L; about 140 mg / L to about 100,000 mg / L; about 150 mg / L to about 100,000 mg / L; about 160 mg / L to about 100,000 mg / L; about 170 mg / L About 100,000 mg / L; about 180 mg / L to about 100,000 mg / L; about 190 mg / L to about 100,000 mg / L; about 200 mg / L to about 100,000 mg / L; about 250 mg / L to about 100,000 mg / L; about 500 mg / L to about 100,000 mg / L; about 750 mg / L to about 100,000 mg / L; about 1000 mg / L to about 100,000 mg / L; about 1000 mg / L to about 100,000 mg / L; about 1500 mg / L to about 100,000 mg / L; about 2000 mg / L to about 100,000 mg / L; about 2500 mg / L to about 1 00,000 mg / L; about 3,000 mg / L to about 100,000 mg / L; about 3,500 mg / L to about 100,000 mg / L; about 4,000 mg / L to about 100,000 mg / L; about 4,500 mg / L to about 100,000 mg / L; about 5,000 mg / L to about 100,000 mg / L; about 5,500 mg / L to about 100,000 mg / L; about 6,000 mg / L to about 100,000 mg / L; about 6,500 mg / L to about 100,000 mg / L; about 7,000 mg / L to about 100,000 mg / L; about 7,500 mg / L to about 100,000 mg / L; about 8,000 mg / L From about 100,000 mg / L; from about 8,500 mg / L to about 100,000 mg / L; from about 9,000 mg / L to about 100,000 mg / L; from about 9,500 mg / L to about 100,000 mg / L; from about 10,000 mg / L to about 100,000 mg / L; from about 10,500 mg / L to about 100,000 mg / L; from about 11,000 mg / L to about 100,000 mg / L; from about 11,500 mg / L to about 100,000 mg / L; from about 12,000 mg / L to about 100,000 mg / L; from about 12,500 mg / L to about 100,000 mg / L; from about 13,000 mg / L to about 100,000 mg / L;Approximately 13,500 mg / L to approximately 100,000 mg / L; approximately 14,000 mg / L to approximately 100,000 mg / L; approximately 14,500 mg / L to approximately 100,000 mg / L; approximately 15,000 mg / L to approximately 100,000 mg / L; approximately 15,500 mg / L to approximately 100,000 mg / L; approximately 16,000 mg / L to approximately 100,000 mg / L; approximately 16,500 mg / L to approximately 100,000 mg / L; approximately 17,000 mg / L to approximately 100,000 mg / L; approximately 17,500 mg / L to approximately 100,000 mg / L; approximately 18,000 mg / L to approximately 100,000 mg / L; approximately 18,5 ... mg / L to about 100,000 mg / L; about 19,000 mg / L to about 100,000 mg / L; about 19,500 mg / L to about 100,000 mg / L; about 20,000 mg / L to about 100,000 mg / L; about 20,500 mg / L to about 100,000 mg / L; about 21,000 mg / L to about 100,000 mg / L; about 21,500 mg / L to about 100,000 mg / L; about 22,000 mg / L to about 100,000 mg / L; about 22,500 mg / L to about 100,000 mg / L; about 23,000 mg / L to about 100,000 mg / L; about 23,500 mg / L to about 100,000 mg / L. 100,000 mg / L; about 24,000 mg / L to about 100,000 mg / L; about 24,500 mg / L to about 100,000 mg / L; about 25,000 mg / L to about 100,000 mg / L; about 25,500 mg / L to about 100,000 mg / L; about 26,000 mg / L to about 100,000 mg / L; about 26,500 mg / L to about 100,000 mg / L; about 27,000 mg / L to about 100,000 mg / L; about 27,500 mg / L to about 100,000 mg / L; about 28,000 mg / L to about 100,000 mg / L; about 28,500 mg / L to about 100,000 mg / L 0 mg / L; about 29,000 mg / L to about 100,000 mg / L; about 29,500 mg / L to about 100,000 mg / L; about 30,000 mg / L to about 100,000 mg / L; about 30,500 mg / L to about 100,000 mg / L; about 31,000 mg / L to about 100,000 mg / L; about 31,500 mg / L to about 100,000 mg / L; about 32,000 mg / L to about 100,000 mg / L; about 32,500 mg / L to about 100,000 mg / L; about 33,000 mg / L to about 100,000 mg / L; about 33,500 mg / L to about 100,000 mg / L;Approximately 34,000 mg / L to approximately 100,000 mg / L; approximately 34,500 mg / L to approximately 100,000 mg / L; approximately 35,000 mg / L to approximately 100,000 mg / L; approximately 35,500 mg / L to approximately 100,000 mg / L; approximately 36,000 mg / L to approximately 100,000 mg / L; approximately 36,500 mg / L to approximately 100,000 mg / L; approximately 37,000 mg / L to approximately 100,000 mg / L; approximately 37,500 mg / L to approximately 100,000 mg / L; approximately 38,000 mg / L to approximately 100,000 mg / L; approximately 38,500 mg / L to approximately 100,000 mg / L; approximately 39,000 mg / L to approximately 100,000 mg / L; approximately 39,000 mg / L to approximately 100,000 mg / L; approximately 39,000 mg / L to approximately 100,000 mg / L; approximately 34,5 ... mg / L to about 100,000 mg / L; about 39,500 mg / L to about 100,000 mg / L; about 40,000 mg / L to about 100,000 mg / L; about 40,500 mg / L to about 100,000 mg / L; about 41,000 mg / L to about 100,000 mg / L; about 41,500 mg / L to about 100,000 mg / L; about 42,000 mg / L to about 100,000 mg / L; about 42,500 mg / L to about 100,000 mg / L; about 43,000 mg / L to about 100,000 mg / L; about 43,500 mg / L to about 100,000 mg / L; about 44,000 mg / L to about 100,000 mg / L; about 44,500 mg / L to about 100,000 mg / L; about 45,000 mg / L to about 100,000 mg / L; about 45,500 mg / L to about 100,000 mg / L; about 46,000 mg / L to about 100,000 mg / L; about 46,500 mg / L to about 100,000 mg / L; about 47,000 mg / L to about 100,000 mg / L; about 47,500 mg / L to about 100,000 mg / L; about 48,000 mg / L to about 100,000 mg / L; about 48,500 mg / L to about 100,000 mg / L; about 49,000 mg / L to about 100,000 mg / L. 0 mg / L; about 49,500 mg / L to about 100,000 mg / L; about 50,000 mg / L to about 100,000 mg / L; about 50,500 mg / L to about 100,000 mg / L; about 51,000 mg / L to about 100,000 mg / L; about 51,500 mg / L to about 100,000 mg / L; about 52,000 mg / L to about 100,000 mg / L; about 52,500 mg / L to about 100,000 mg / L; about 53,000 mg / L to about 100,000 mg / L; about 53,500 mg / L to about 100,000 mg / L; about 54,000 mg / L to about 100,000 mg / L;Approximately 54,500 mg / L to approximately 100,000 mg / L; approximately 55,000 mg / L to approximately 100,000 mg / L; approximately 55,500 mg / L to approximately 100,000 mg / L; approximately 56,000 mg / L to approximately 100,000 mg / L; approximately 56,500 mg / L to approximately 100,000 mg / L; approximately 57,000 mg / L to approximately 100,000 mg / L; approximately 57,500 mg / L to approximately 100,000 mg / L; approximately 58,000 mg / L to approximately 100,000 mg / L; approximately 58,500 mg / L to approximately 100,000 mg / L; approximately 59,000 mg / L to approximately 100,000 mg / L; approximately 59,5 ... mg / L to about 100,000 mg / L; about 60,000 mg / L to about 100,000 mg / L; about 60,500 mg / L to about 100,000 mg / L; about 61,000 mg / L to about 100,000 mg / L; about 61,500 mg / L to about 100,000 mg / L; about 62,000 mg / L to about 100,000 mg / L; about 62,500 mg / L to about 100,000 mg / L; about 63,000 mg / L to about 100,000 mg / L; about 63,500 mg / L to about 100,000 mg / L; about 64,000 mg / L to about 100,000 mg / L; about 64,500 mg / L to about 100,000 mg / L. 100,000 mg / L; about 65,000 mg / L to about 100,000 mg / L; about 65,500 mg / L to about 100,000 mg / L; about 66,000 mg / L to about 100,000 mg / L; about 66,500 mg / L to about 100,000 mg / L; about 67,000 mg / L to about 100,000 mg / L; about 67,500 mg / L to about 100,000 mg / L; about 68,000 mg / L to about 100,000 mg / L; about 68,500 mg / L to about 100,000 mg / L; about 69,000 mg / L to about 100,000 mg / L; about 69,500 mg / L to about 100,000 mg / L 0 mg / L; about 70,000 mg / L to about 100,000 mg / L; about 70,500 mg / L to about 100,000 mg / L; about 71,000 mg / L to about 100,000 mg / L; about 71,500 mg / L to about 100,000 mg / L; about 72,000 mg / L to about 100,000 mg / L; about 72,500 mg / L to about 100,000 mg / L; about 73,000 mg / L to about 100,000 mg / L; about 73,500 mg / L to about 100,000 mg / L; about 74,000 mg / L to about 100,000 mg / L; about 74,500 mg / L to about 100,000 mg / L;Approximately 75,000 mg / L to approximately 100,000 mg / L; approximately 75,500 mg / L to approximately 100,000 mg / L; approximately 76,000 mg / L to approximately 100,000 mg / L; approximately 76,500 mg / L to approximately 100,000 mg / L; approximately 77,000 mg / L to approximately 100,000 mg / L; approximately 77,500 mg / L to approximately 100,000 mg / L; approximately 78,000 mg / L to approximately 100,000 mg / L; approximately 78,500 mg / L to approximately 100,000 mg / L; approximately 79,000 mg / L to approximately 100,000 mg / L; approximately 79,500 mg / L to approximately 100,000 mg / L; approximately 80,000 mg / L mg / L to about 100,000 mg / L; about 80,500 mg / L to about 100,000 mg / L; about 81,000 mg / L to about 100,000 mg / L; about 81,500 mg / L to about 100,000 mg / L; about 82,000 mg / L to about 100,000 mg / L; about 82,500 mg / L to about 100,000 mg / L; about 83,000 mg / L to about 100,000 mg / L; about 83,500 mg / L to about 100,000 mg / L; about 84,000 mg / L to about 100,000 mg / L; about 84,500 mg / L to about 100,000 mg / L; about 85,000 mg / L to about 100,000 mg / L; about 85,500 mg / L to about 100,000 mg / L; about 86,000 mg / L to about 100,000 mg / L; about 86,500 mg / L to about 100,000 mg / L; about 87,000 mg / L to about 100,000 mg / L; about 87,500 mg / L to about 100,000 mg / L; about 88,000 mg / L to about 100,000 mg / L; about 88,500 mg / L to about 100,000 mg / L; about 89,000 mg / L to about 100,000 mg / L; about 89,500 mg / L to about 100,000 mg / L; about 90,000 mg / L to about 100,000 mg / L 0 mg / L; about 90,500 mg / L to about 100,000 mg / L; about 91,000 mg / L to about 100,000 mg / L; about 91,500 mg / L to about 100,000 mg / L; about 92,000 mg / L to about 100,000 mg / L; about 92,500 mg / L to about 100,000 mg / L; about 93,000 mg / L to about 100,000 mg / L; about 93,500 mg / L to about 100,000 mg / L; about 94,000 mg / L to about 100,000 mg / L; about 94,500 mg / L to about 100,000 mg / L; about 95,000 mg / L to about 100,000 mg / L;About 95,500 mg / L to about 100,000 mg / L; about 96,000 mg / L to about 100,000 mg / L; about 96,500 mg / L to about 100,000 mg / L; about 97,000 mg / L to about 100,000 mg / L; about 97,500 mg / L to about 100,000 mg / L; about 98,000 mg / L to about 100,000 mg / L; about 98,500 mg / L to about 100,000 mg / L; about 99,000 mg / L to about 100,000 mg / L; or about 99,500 mg / L to about 100,000 mg / L.

[0406] In some embodiments, one or more expression cassettes containing a polynucleotide operable to express CRIP can be inserted into the vector, resulting in a yield of CRIP per liter of culture medium (yeast fermentation broth supernatant) in the following ranges: about 100 mg / L to about 100,000 mg / L of CRIP; about 100 mg / L to about 99,500 mg / L; about 100 mg / L to about 99,000 mg / L; about 100 mg / L to about 98,500 mg / L; about 100 mg / L to about 98,000 mg / L; about 100 mg / L to about 97,500 mg / L; about 100 mg / L to about 97,000 mg / L; about 100 mg / L to about 96,500 mg / L; about 100 mg / L to about 96000 mg / L; about 100 mg / L to about 95500 mg / L; about 100 mg / L to about 95000 mg / L; about 100 mg / L to about 94500 mg / L; about 100 mg / L to about 94000 mg / L; about 100 mg / L to about 93500 mg / L; about 100 mg / L to about 93000 mg / L; about 100 mg / L to about 92500 mg / L; about 100 mg / L to about 92000 mg / L; about 100 mg / L to about 91500 mg / L; about 100 mg / L to about 91000 mg / L; about 100 mg / L to about 90500 mg / L; about 100 mg / L L to about 90,000 mg / L; about 100 mg / L to about 89,500 mg / L; about 100 mg / L to about 89,000 mg / L; about 100 mg / L to about 88,500 mg / L; about 100 mg / L to about 88,000 mg / L; about 100 mg / L to about 87,500 mg / L; about 100 mg / L to about 87,000 mg / L; about 100 mg / L to about 86,500 mg / L; about 100 mg / L to about 86,000 mg / L; about 100 mg / L to about 85,500 mg / L; about 100 mg / L to about 85,000 mg / L; about 100 mg / L to about 84,500 mg / L; about 100 mg / L to about 840,000 mg / L. 00 mg / L; about 100 mg / L to about 83500 mg / L; about 100 mg / L to about 83000 mg / L; about 100 mg / L to about 82500 mg / L; about 100 mg / L to about 82000 mg / L; about 100 mg / L to about 81500 mg / L; about 100 mg / L to about 81000 mg / L; about 100 mg / L to about 80500 mg / L; about 100 mg / L to about 80000 mg / L; about 100 mg / L to about 79500 mg / L; about 100 mg / L to about 79000 mg / L; about 100 mg / L to about 78500 mg / L; about 100 mg / L to about 78000 mg / L;About 100 mg / L to about 77500 mg / L; about 100 mg / L to about 77000 mg / L; about 100 mg / L to about 76500 mg / L; about 100 mg / L to about 76000 mg / L; about 100 mg / L to about 75500 mg / L; about 100 mg / L to about 75000 mg / L; about 100 mg / L to about 74500 mg / L; about 100 mg / L to about 74000 mg / L; about 100 mg / L to about 73500 mg / L; about 100 mg / L to about 73000 mg / L; about 100 mg / L to about 72500 mg / L; about 100 mg / L to about 72000 mg / L; about 100 mg / L to about 71500 mg / L; about 100 mg / L to about 71000 mg / L; about 100 mg / L to about 70500 mg / L; about 100 mg / L to about 70000 mg / L; about 100 mg / L to about 69500 ​​mg / L; about 100 mg / L to about 69000 mg / L; about 100 mg / L to about 68500 mg / L; about 100 mg / L to about 68000 mg / L; about 100 mg / L to about 67500 mg / L; about 100 mg / L to about 67000 mg / L; about 100 mg / L to about 66500 mg / L; about 100 mg / L to about 66000 mg / L; about 100 mg / L to about 65500 mg / L; About 100 mg / L to about 65000 mg / L; about 100 mg / L to about 64500 mg / L; about 100 mg / L to about 64000 mg / L; about 100 mg / L to about 63500 mg / L; about 100 mg / L to about 63000 mg / L; about 100 mg / L to about 62500 mg / L; about 100 mg / L to about 62000 mg / L; about 100 mg / L to about 61500 mg / L; about 100 mg / L to about 61000 mg / L; about 100 mg / L to about 60500 mg / L; about 100 mg / L to about 60000 mg / L; about 100 mg / L to about 59500 mg / L; about 100 mg / L to about 59000 mg / L; about 100 mg / L to about 58500 mg / L; about 100 mg / L to about 58000 mg / L; about 100 mg / L to about 57500 mg / L; about 100 mg / L to about 57000 mg / L; about 100 mg / L to about 56500 mg / L; about 100 mg / L to about 56000 mg / L; about 100 mg / L to about 55500 mg / L; about 100 mg / L to about 55000 mg / L; about 100 mg / L to about 54500 mg / L; about 100 mg / L to about 54000 mg / L; about 100 mg / L to about 53500 mg / L; about 100 mg / L to about 53000 mg / L;About 100 mg / L to about 52500 mg / L; about 100 mg / L to about 52000 mg / L; about 100 mg / L to about 51500 mg / L; about 100 mg / L to about 51000 mg / L; about 100 mg / L to about 50500 mg / L; about 100 mg / L to about 50000 mg / L; about 100 mg / L to about 49500 mg / L; about 100 mg / L to about 49000 mg / L; about 100 mg / L to about 48500 mg / L; about 100 mg / L to about 48000 mg / L; about 100 mg / L to about 47500 mg / L; about 100 mg / L to about 47000 mg / L; about 100 mg / L to about 46500 mg / L; about 100 mg / L to about 46000 mg / L; about 100 mg / L to about 45500 mg / L; about 100 mg / L to about 45000 mg / L; about 100 mg / L to about 44500 mg / L; about 100 mg / L to about 44000 mg / L; about 100 mg / L to about 43500 mg / L; about 100 mg / L to about 43000 mg / L; about 100 mg / L to about 42500 mg / L; about 100 mg / L to about 42000 mg / L; about 100 mg / L to about 41500 mg / L; about 100 mg / L to about 41000 mg / L; about 100 mg / L to about 40500 mg / L; About 100 mg / L to about 40000 mg / L; about 100 mg / L to about 39500 mg / L; about 100 mg / L to about 39000 mg / L; about 100 mg / L to about 38500 mg / L; about 100 mg / L to about 38000 mg / L; about 100 mg / L to about 37500 mg / L; about 100 mg / L to about 37000 mg / L; about 100 mg / L to about 36500 mg / L; about 100 mg / L to about 36000 mg / L; about 100 mg / L to about 35500 mg / L; about 100 mg / L to about 35000 mg / L; about 100 mg / L to about 34500 mg / L; about 100 mg / L to about 34000 mg / L; about 100 mg / L to about 33500 mg / L; about 100 mg / L to about 33000 mg / L; about 100 mg / L to about 32500 mg / L; about 100 mg / L to about 32000 mg / L; about 100 mg / L to about 31500 mg / L; about 100 mg / L to about 31000 mg / L; about 100 mg / L to about 30500 mg / L; about 100 mg / L to about 30000 mg / L; about 100 mg / L to about 29500 mg / L; about 100 mg / L to about 29000 mg / L; about 100 mg / L to about 28500 mg / L; about 100 mg / L to about 28000 mg / L;About 100 mg / L to about 27500 mg / L; about 100 mg / L to about 27000 mg / L; about 100 mg / L to about 26500 mg / L; about 100 mg / L to about 26000 mg / L; about 100 mg / L to about 25500 mg / L; about 100 mg / L to about 25000 mg / L; about 100 mg / L to about 24500 mg / L; about 100 mg / L to about 24000 mg / L; about 100 mg / L to about 23500 mg / L; about 100 mg / L to about 23000 mg / L; about 100 mg / L to about 22500 mg / L; about 100 mg / L to about 22000 mg / L; about 100 mg / L to about 21500 mg / L; about 100 mg / L to about 21000 mg / L; about 100 mg / L to about 20500 mg / L; about 100 mg / L to about 20000 mg / L; about 100 mg / L to about 19500 mg / L; about 100 mg / L to about 19000 mg / L; about 100 mg / L to about 18500 mg / L; about 100 mg / L to about 18000 mg / L; about 100 mg / L to about 17500 mg / L; about 100 mg / L to about 17000 mg / L; about 100 mg / L to about 16500 mg / L; about 100 mg / L to about 16000 mg / L; about 100 mg / L to about 155 00 mg / L; about 100 mg / L to about 15000 mg / L; about 100 mg / L to about 14500 mg / L; about 100 mg / L to about 14000 mg / L; about 100 mg / L to about 13500 mg / L; about 100 mg / L to about 13000 mg / L; about 100 mg / L to about 12500 mg / L; about 100 mg / L to about 12000 mg / L; about 100 mg / L to about 11500 mg / L; about 100 mg / L to about 11000 mg / L; about 100 mg / L to about 10500 mg / L; about 100 mg / L to about 10000 mg / L; about 100 mg / L to about 9500 mg / L; About 100 mg / L to about 9000 mg / L; about 100 mg / L to about 8500 mg / L; about 100 mg / L to about 8000 mg / L; about 100 mg / L to about 7500 mg / L; about 100 mg / L to about 7000 mg / L; about 100 mg / L to about 6500 mg / L; about 100 mg / L to about 6000 mg / L; about 100 mg / L to about 5500 mg / L; about 100 mg / L to about 5000 mg / L; about 100 mg / L to about 4500 mg / L; about 100 mg / L to about 4000 mg / L; about 100 mg / L to about 3500 mg / L; about 100 mg / L to about 3000 mg / L;About 100 mg / L to about 2500 mg / L; about 100 mg / L to about 2000 mg / L; about 100 mg / L to about 1500 mg / L; about 100 mg / L to about 1000 mg / L; about 100 mg / L to about 1000 mg / L; about 100 mg / L to about 750 mg / L; about 100 mg / L to about 500 mg / L; about 100 mg / L to about 250 mg / L; about 100 mg / L to about 100 mg / L; or about 100 mg / L to about 110 mg / L.

[0407] In addition to the DNA polynucleotide sequence encoding CRIP or peptide-IA, additional DNA fragments, called regulatory elements, can be cloned into vectors that allow for enhanced expression of exogenous DNA or transgenes; examples of such additional DNA fragments include (1) promoter, terminator, and / or enhancer elements; (2) suitable mRNA stabilizing polyadenylation signals; (3) internal ribosome entry sites (IRES); (4) introns; and (5) posttranscriptional regulatory elements. The combination of the DNA fragment of interest with any of the aforementioned cis-acting elements is called an “expression cassette.”

[0408] A single expression cassette may contain one or more of the aforementioned regulatory elements and a polynucleotide operable to express CRIP or peptide-IA. For example, in some embodiments, the CRIP or peptide-IA expression cassette may contain a polynucleotide operable to express CRIP or peptide-IA, and an α-MF signal; a Kex2 site; a LAC4 terminator; an ADN1 promoter; and an acetamase (amdS) selection marker—sidelined at the 5' and 3' ends of the LAC4 promoter.

[0409] In some embodiments, a plurality of expression cassettes cloned into the vector may be present. For example, in some embodiments, a first expression cassette may be present, which contains a polynucleotide operable to express CRIP or peptide-IA. In alternative embodiments, two cassettes operable to encode CRIP or peptide-IA are present (i.e., dual expression cassettes). In other embodiments, three expression cassettes operable to encode CRIP or peptide-IA are present (i.e., triple expression cassettes).

[0410] In some implementations, dual expression cassettes can be generated by subcloning a second CRIP or peptide-IA expression cassette into a vector containing a first CRIP or peptide-IA expression cassette.

[0411] In some implementations, a three-expression cassette can be generated by subcloning a third CRIP or peptide-IA expression cassette into a vector containing the first and second CRIPs or peptide-IA expression cassettes.

[0412] In some embodiments, yeast cells transformed with one or more CRIP or peptide-IA expression cassettes can produce CRIP or peptide-IA in yeast cultures at yields of at least 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, or 190 mg / L per liter of yeast culture medium. / L, 200mg / L, at least 500mg / L, at least 750mg / L, at least 1,000mg / L, at least 1,250mg / L, at least 1,500mg / L, at least 1,750mg / L, at least 2,000mg / L, at least 2,500mg / L, at least 3,000mg / L, at least 3,500mg / L, at least 4,000mg / L, at least 4,500mg / L, at least 5,000mg / L, at least 5,500mg / L, at least 6,000mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 1 CRIP or peptide-IA at concentrations of 7,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L.

[0413] In some embodiments, one or more expression cassettes containing a polynucleotide operable to express CRIP or peptide-IA may be inserted into a vector (e.g., pKlac1 plasmid), resulting in a yield of approximately 100 mg / L CRIP or peptide-IA (yeast fermentation broth supernatant). For example, in some embodiments, two expression cassettes containing a polynucleotide operable to express CRIP or peptide-IA may be inserted into a vector (e.g., pKS482 plasmid), resulting in a yield of approximately 2 g / L CRIP or peptide-IA (yeast fermentation broth supernatant). Alternatively, in some embodiments, three expression cassettes containing a polynucleotide operable to express CRIP or peptide-IA may be inserted into a vector (e.g., pKlac1T plasmid).

[0414] In some implementations, multiple CRIP or peptide-IA expression cassettes can be transfected into yeast to enable the integration of one or more copies of optimized CRIP or peptide-IA transgenes into the *Kluyveromyces lactis* genome. An exemplary method for introducing multiple CRIP or peptide-IA expression cassettes into the *Kluyveromyces lactis* genome is as follows: A CRIP or peptide-IA expression cassette DNA sequence is synthesized, containing a complete LAC4 promoter element, a codon-optimized CRIP or peptide-IA expression ORF element, and a pLAC4 terminator element; the complete expression cassette is ligated to the pKlac1 vector between the SalI and KpnI restriction sites downstream of the pLAC4 terminator of pKS477, thereby generating a double transgene CRIP or peptide-IA expression vector pKS482; the double transgene vector pKS482 is then linearized using the SacII restriction endonuclease and transformed into *Kluyveromyces lactis* strain YCT306 by electroporation. The resulting yeast colonies were then grown on YCB agar plates supplemented with 5 mM acetamide. Only cells expressing acetamonase could effectively utilize acetamide as a nitrogen metabolic source. To evaluate the yeast colonies, approximately 100 to 400 colonies were picked from pKS482 yeast plates. Inoculum from each colony was cultured individually in 2.2 mL of a defined *Kluyveromyces lactis* medium supplemented with 2% sugar alcohol as a carbon source. The cultures were incubated at 23.5 °C with shaking at 280 rpm for six days, at which point the cell density in the culture reached its maximum level, as indicated by the absorbance at 600 nm (OD600). Cells were then removed from the cultures by centrifugation at 4,000 rpm for 10 min, and the resulting supernatant (conditioned medium) was filtered through a 0.2 μM membrane for HPLC yield analysis.

[0415] Chemical synthesis of peptides

[0416] Peptide synthesis or chemical synthesis, or peptides and / or polypeptides, can be used to produce CRIPs or peptide-IAs: these methods can be performed by those skilled in the art and / or by using commercial suppliers (e.g., The process can be carried out in Piscataway, New Jersey. For example, in some embodiments, chemical peptide synthesis can be achieved using liquid-phase peptide synthesis (LPPS) or solid-phase peptide synthesis (SPPS).

[0417] In some implementations, peptide synthesis is typically achieved by using a strategy in which the carboxyl group of a subsequent amino acid is coupled to the N-terminus of the preceding amino acid to produce a nascent polypeptide chain—a process that is the opposite of the type of peptide synthesis that occurs in nature.

[0418] Peptide deprotection is an important first step in peptide chemical synthesis. Peptide deprotection is the process of blocking the reactive groups of amino acids using chemicals to prevent the functional groups of the amino acids from participating in unwanted or non-specific reactions or side reactions; in other words, the amino acid is "protected" from participating in these undesirable reactions.

[0419] Before synthesizing peptide chains, amino acids must be "deprotected" to allow chain formation (i.e., amino acid binding). Chemicals used to protect the N-terminus include 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc), each of which can be removed by using a weak base (e.g., piperidine) and a moderately strong acid (e.g., trifluoroacetic acid (TFA)).

[0420] The required C-terminal protecting agent depends on the type of chemical peptide synthesis strategy used: for example, LPPS requires protection of the C-terminal amino acid, while SPPS does not, because the solid support acts as the protecting group. Side-chain amino acids require several different protecting groups, which vary based on the individual peptide sequence and N-terminal protection strategy; however, protecting groups used for side-chain amino acids are typically based on tert-butyl (tBu) or benzyl (Bzl) protecting groups.

[0421] Amino acid coupling is the next step in the peptide synthesis process. To achieve amino acid coupling, the C-terminal carboxylic acid of the introduced amino acid must be activated: this can be accomplished using carbodiimides such as diisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC), which react with the carboxyl group of the introduced amino acid to form an O-acylisourea intermediate. The O-acylisourea intermediate is then replaced by nucleophilic attack of the primary amino group at the N-terminus of the growing peptide chain. The reactive intermediate generated by the carbodiimide can lead to racemization of the amino acid. To avoid racemization of the amino acid, a reagent such as 1-hydroxybenzotriazole (HOBt) is added to react with the O-acylisourea intermediate. Other coupling agents that can be used include 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) and benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), as well as additional activating bases. Finally, after the amino acids are deprotected and coupled,

[0422] At the end of the synthesis process, protecting groups must be removed from the peptide—usually through acid hydrolysis. The reagents required for peptide cleavage depend on the protection scheme used and the overall synthetic method. For example, in some embodiments, hydrogen bromide (HBr), hydrogen fluoride (HF), or trifluoromethanesulfonic acid (TFMSA) can be used to cleave the Bzl and Boc groups. Alternatively, in other embodiments, weaker acids such as TFA can achieve the hydrolysis of the tBut and Fmoc groups. Finally, the peptide can be purified based on its physiological and chemical properties (e.g., charge, size, hydrophobicity, etc.). Techniques that can be used to purify peptides include purification techniques such as reversed-phase chromatography (RPC); size exclusion chromatography; partition chromatography; high-performance liquid chromatography (HPLC); and ion-exchange chromatography (IEC).

[0423] Exemplary methods for peptide synthesis can be found in: Anderson GW and McGregor AC, 1957, “T-butyloxycarbonylamino acids and their use in peptide synthesis,” Journal of the American Chemical Society, Vol. 79, pp. 6180-6183; Carpino LA, 1957, “Oxidative reactions of hydrazines. Iv. Elimination of nitrogen from 1,1-disubstituted-2-arenesulfonhydrazides 1-4,” Journal of the American Chemical Society, Vol. 79, pp. 4427-4431; McKay FC and Albertson NF, 1957, “New amine-masking groups for peptide synthesis,” Journal of the American Chemical Society, Vol. 79, pp. 4686-4690; Merrifield RB, 1963, “Solid phase peptide synthesis. I. The synthesis of a tetrapeptide,” Journal of the American Chemical Society. Chemical Society, Vol. 85: pp. 2149-2154; Carpino LA and Han GY, 1972, “9-fluorenylmethoxycarbonyl amino-protecting group”, The Journal of Organic Chemistry, Vol. 37: pp. 3404-3409; and A Lloyd-Williams P.The disclosures of these documents and patents, including, “Chemical approaches to the synthesis of peptides and proteins,” Boca Raton: CRC Press, 1997, p. 278; U.S. Patent Nos. 3,714,140 (filed March 16, 1971); 4,411,994 (filed June 8, 1978); 7,785,832 (filed January 20, 2006); 8,314,208 (filed February 10, 2006); 10,442,834 (filed October 2, 2015); and U.S. Patent Application 2005 / 0165215 (filed December 23, 2004), are incorporated herein by reference in their entirety.

[0424] Other exemplary methods for generating polynucleotides, peptides, and CRIPs can be found in U.S. Patent Application Publication No. 20150148288A1, the entire disclosure of which is incorporated herein by reference.

[0425] Any of the methods described herein can be used to produce any of the CRIP, CRIP-insecticide protein, or peptide-IA described herein.

[0426] Cell culture and transformation techniques

[0427] Both the terms “transformation” and “transfection” describe the process of introducing exogenous and / or heterologous DNA or RNA into a host organism. Generally, those skilled in the art sometimes retain the term “transformation” to describe the process of introducing exogenous and / or heterologous DNA or RNA into bacterial cells, and the term “transfection” to describe the process of introducing exogenous and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the terms “transformation” and “transfection” are used synonymously, regardless of whether the method describes the introduction of exogenous and / or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plant, or animal).

[0428] In some implementations, host cells may be transformed using the following methods: electroporation; cell squeezing; microscopic injection; puncture; use of hydrostatic pressure; acoustic perforation; optical transfection; continuous infusion; lipid transfection; use of viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus; chemical phosphate method; endocytosis via DEAE-glucan or polyethyleneimine (PEI); protoplast fusion; hydrodynamic delivery; magnetic transfection; nucleolar transfection; and / or others. Exemplary methods for transfection and / or transformation technologies can be found in: Makrides, “Gene Transfer and Expression in Mammalian Cells”, Elvesier, 2003; Wong, TK and Neumann, E., “Electric field mediated gene transfer”, Biochem. Biophys. Res. Commun., Vol. 107: pp. 584-587, 1982; Potter and Heller, “Transfection by Electroporation”, Curr Protoc Mol Biol., May 2003, Chapter: Unit 9.3; Kim and Eberwine, “Mammalian cell transfection: the present and the future”, Anal Bioanal Chem., August 2010, Vol. 397, No. 8: pp. 3173-3178. The full text of each of these publications is incorporated herein by reference.

[0429] Electroporation is a technique in which an electric current is applied to cells to make the cell membrane permeable; this, in turn, allows foreign DNA to be introduced into the cells. Electroporation is well known to those skilled in the art, and the tools and apparatus required to perform electroporation are commercially available (e.g., Gene Pulser Xcell). TM Electroporation system, For electroporation Transfection systems, Thermo-Fisher Scientific; and other tools and / or devices. Exemplary methods of electroporation are shown in: Potter and Heller, “Transfection by Electroporation,” Curr Protoc MolBiol., May 2003, Chapter: Unit 9.3; Saito, “Electroporation Methods in Neuroscience,” Springer Press, 2015; Pakhomov et al., “Advanced Electroporation Techniques in Biology and Medicine,” Taylor & Francis, 2017; the full text of these publications is incorporated herein by reference.

[0430] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding CRIP or peptide-IA into yeast, for example, cloning CRIP or peptide-IA into the pKlac1 plasmid and transforming it into *Kluyveromyces lactis* cells via electroporation. This can be achieved by inoculating the yeast culture with approximately 10 mL to 200 mL of yeast extract peptone dextran (YEPD) with a suitable yeast species such as *Kluyveromyces lactis*, *Kluyveromyces marxianus*, *Saccharomyces cerevisiae*, or *Pichia pastoris*, and incubating at 30°C on a shaker until the early exponential phase of the yeast culture (e.g., approximately 0.6 to 2 × 10⁻⁶). 8 The goal is to achieve a cell / mL ratio; harvest yeast in sterile centrifuge tubes and centrifuge at 3000 rpm for 5 minutes at 4°C (Note: keep cells frozen during this procedure), wash cells with 40 mL of ice-cold sterile deionized water, and granulate cells at 23,000 rpm for 5 minutes; repeat the washing steps and resuspend the cells in 20 mL of 1M fermentable sugar, such as galactose, maltose, latotriose, sucrose, fructose, or glucose. Resuspend the cells to 3 × 10⁻⁶ rpm for 5 minutes with an appropriate volume of chilled 1M fermentable sugar, such as galactose, maltose, latrotriose, sucrose, fructose, or glucose and / or sugar alcohol, such as erythritol, hydrogenated starch hydrolysate, isomaltitol, lactitol, maltitol, mannitol, and xylitol. 9Achieving a final cell density of cells / mL; in a pre-chilled 0.2 cm electroporation cuvette, mix 40 μl of yeast suspension with approximately 1 μl to 4 μl of carrier containing approximately 1 μg of a linear polynucleotide encoding CRIP or peptide-IA (Note: ensure the sample is in contact with both sides of the aluminum cuvette); apply a single pulse of 2000 V with an optimal time constant of 5 ms for the RC circuit, then recover the cells in 0.5 mL of LYED and 0.5 mL of 1 M fermentable sugars, such as galactose, maltose, latrotriose, sucrose, fructose, or glucose and / or sugar alcohols, such as erythritol, hydrogenated starch hydrolysate, isomaltose, lactitol, maltitol, mannitol, and a mixture of xylitol, and then plate onto a selective plate.

[0431] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding CRIP or peptide-IA into plant protoplasts via the following steps: incubating sterile plant material in a protoplast solution (e.g., approximately 8 mL of 10 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 5.5; 0.01% (w / v) pectylase; 1% (w / v) dissociation enzyme; 40 mM CaCl2; and 0.4 M mannitol), and adding the mixture to a rotary shaker at 30°C for approximately 3 to 6 hours to produce protoplasts; removing debris by filtration through an 80 μm nylon sieve; and using approximately 4 mL of plant electroporation buffer (e.g., 5 mM... The sieve was rinsed with CaCl2, 0.4M mannitol, and PBS; protoplasts were combined in a sterile 15mL conical centrifuge tube and centrifuged at approximately 300×g for about 5 minutes; after centrifugation, the supernatant was discarded and the protoplasts were washed with 5mL of plant electroporation buffer; the protoplasts were then precipitated at approximately 1.5×10⁻⁶ ppm per mL of liquid. 6 Up to 2×10 6 One protoplast is resuspended in plant electroporation buffer; approximately 0.5 mL of protoplast suspension is transferred to one or more electroporation cuvettes placed on ice, and the vector is added (Note: For stable transformation, the vector should be linearized using any of the above restrictive methods, and approximately 1 μg to 10 μg of vector can be used; for transient expression, the vector can be kept in its supercoiled state, and approximately 10 μg to 40 μg of vector can be used); the vector is mixed with the protoplast suspension; the cuvette is placed in the electroporation apparatus and electroporated once or multiple times at approximately 1 kV to 2 kV (initially a capacitor of 3 μF to 25 μF can be used to optimize the reaction); the cuvette is returned to ice; the transformed cells are diluted 20-fold in complete culture medium; and the protoplasts are harvested after approximately 48 hours.

[0432] Host cells

[0433] The methods, compositions, CRIPs, and peptide-IAs of the present invention can be implemented in any cell type (e.g., eukaryotic or prokaryotic cells).

[0434] In some embodiments, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA is a prokaryote. For example, in some embodiments, the host cell may be an archaea or eubacterium, such as a Gram-negative or Gram-positive organism. Examples of useful bacteria include Escherichia (e.g., Escherichia coli), Bacilli (e.g., Bacillus subtilis), Enterobacteriaceae, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus.

[0435] In some embodiments, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be a single-celled cell. For example, in some embodiments, the host cell can be a bacterial cell, such as Gram-positive bacteria.

[0436] In some embodiments, the host cell may be bacteria selected from the following genera: Candidatus Chloracidobacterium, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Mycobacterium, Propionibacterium, Streptomyces, Aquifex, Bacteroides, Porphyromonas, and Bacteroides. Ooides, Porphyromonas, Flavobacterium, Chlamydia, Prosthecobacter, Verrucomicrobium, Chloroflexus, Chroococcus, Merismopedia, Synechococcus, Anabaena, Nostoc, Spirulina, Trichodesmium, Pleurotus rocapsa), Prochlorococcus, Prochloron, Bacillus, Listeria, Staphylococcus, Clostridium, Dehalobacter, Epulopiscium, Ruminococcus, Enterococcus, Lactobacillus, Streptococcus, Erysipelothrix, Mycoplasma ycoplasma, Leptospirillum, Nitrospira, Thermodesulfobacterium, Gemmata, Pirellula, Planctomyces, Calobacter, Agrobacterium, Bradyrhizobium, Brucella, Methylobacterium, ProsthecomicrobiumRhizobium, Rhodopseudomonas, Sinorhizobium, Rhodobacter, Roseobacter, Acetobacter, Rhodospirillum, Rickettsia, Rickettsia conorii, mitochondria, Wolbachia, Erythrobacter, Erythromicrobium, Sphingomonas, Alcaligenes, Burkholderia, Leptothrix, Sphaerotilus, Thiobacillus, Neisseria, Nitrosomonas, Gallionella, Spirillum, Azoarcus, Aeromonas, Succinomonas, Succinivibrio, Ruminobacter, Nitrosococcus, Thiocapsa, Enterobacter, Escherichia coli The following bacteria are listed: *Richia*, *Klebsiella*, *Salmonella*, *Shigella*, *Wigglesworthia*, *Yersinia*, *Coxiella*, *Legionella*, *Halomonas*, *Pasteurella*, *Acinetobacter*, *Azotobacter*, *Pseudomonas*, *Psychrobacter*, *Beggiatoa*, *Thiomargarita*, *Vibrio*, *Xanthomonas*, *Bdellovibrio*, *Campylobacter*, *Helicobacter*, *Myxococcus*, *Desulfosarcina*, and *Geobacter*.Desulfuromonas, Borrelia, Leptospira, Treponema, Petrotoga, Thermotoga, Deinococcus, or Thermus.

[0437] In some embodiments, the host cells used for producing CRIP, CRIP-insecticide protein, or peptide-IA may be selected from one of the following bacterial species: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptomyces lividans, and Streptomyces gravidans. Streptomyces murinus, Streptomyces coelicolor, Streptomyces albicans, Streptomyces griseus, Streptomyces plicatosporus, Escherichia albertii, Escherichia blattae, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia senegalensis, Escherichia vulneris, Pseudomonas abietaniphila, Pseudomonas agarici, Pseudomonas agarolyticus, Pseudomonas basophila *Pseudomonas alcaliphila*, *Pseudomonas salginovora*, *Pseudomonas andersonii*, *Pseudomonas antarctica*, *Pseudomonas*Pseudomonas asplenii, Pseudomonas azelaica, Pseudomonas batumici, Pseudomonas borealis, Pseudomonas brassicacearum, Pseudomonas chloritidismutans, Pseudomonas cremoricolorata, Pseudomonas diterpeniphila, Pseudomonas filiscindens, Pseudomonas frederiksbergensis, Pseudomonas gingeri, Pseudomonas graminis, Pseudomonas grimontii, Pseudomonas halodenitrificans, Pseudomonas halophila, Pseudomonas *Pseudomonas hibiscicola*, *Pseudomonas hydrogenovora*, *Pseudomonas indica*, *Pseudomonas japonica*, *Pseudomonas jessenii*, *Pseudomonas kilonensis*, *Pseudomonas koreensis*, *Pseudomonas lini*, *Pseudomonas lurida*, *Pseudomonas lutea*, *Pseudomonas marginata*, *Pseudomonas meridiana*, *Pseudomonas mesoacidophila*, *Pseudomonas pachastrellae*, *Pseudomonas palleroniana*, *Pseudomonas parafulva*, *Pseudomonas* pavonanceae, Pseudomonas proteolyica, Pseudomonas psychrophila, Pseudomonas psychrotolerans, Pseudomonas pudica, Pseudomonas*Pseudomonas rathonis*, *Pseudomonas reactans*, *Pseudomonas rhizosphaerae*, *Pseudomonas salmononii*, *Pseudomonas thermaerum*, *Pseudomonas thermocarboxydovorans*, *Pseudomonas thermotolerans*, *Pseudomonas thivervalensis*, *Pseudomonas umsongensis*, *Pseudomonas vancouverensis*, *Pseudomonas wisconsinensis*, *Pseudomonas xanthomarina*, *Pseudomonas xiamenensis*, *Pseudomonas aeruginosa*, *Pseudomonas alcaligenes*, *Pseudomonas spp.* Anguilliseptica, Pseudomonas citronellolis, Pseudomonas flavescens, Pseudomonas jinjuensis, Pseudomonas mendocina, Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans, Pseudomonas straminae, Pseudomonas aurantiaca, Pseudomonas chlororaphis, Pseudomonas fragi, Pseudomonas lundii *Pseudomonas lundensis*, *Pseudomonas taetrolens*, *Pseudomonas azotoformans*, *Pseudomonas brenneri*, *Pseudomonas**Pseudomonas congelans*, *Pseudomonas corrugata*, *Pseudomonas costantinii*, *Pseudomonas extremorientalis*, *Pseudomonas fluorescens*, *Pseudomonas fulgida*, *Pseudomonas gessardii*, *Pseudomonas libanensis*, *Pseudomonas mandelii*, *Pseudomonas marginalis*, *Pseudomonas mediterranea*, *Pseudomonas migulae*, *Pseudomonas mucidolens*, *Pseudomonas orientalis* *Pseudomonas orientalis*, *Pseudomonas poae*, *Pseudomonas rhodesiae*, *Pseudomonas synxantha*, *Pseudomonas tolaasii*, *Pseudomonas trivialis*, *Pseudomonas veronii*, *Pseudomonas denitrificans*, *Pseudomonas pertucinogena*, *Pseudomonas fulva*, *Pseudomonas monteilii*, *Pseudomonas mosselii*, *Pseudomonas oryzihabitans*, *Pseudomonas proteomorpha*. *Pseudomonas plecoglossicida*, *Pseudomonas putida*, *Pseudomonas balearica*, *Pseudomonas luteola*, or *Pseudomonas stutzeri*. *Pseudomonas avellanae*, *Pseudomonas cannabinoids*.Pseudomonas cannabina, Pseudomonas caricapapyae, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas fuscovaginae, Pseudomonas tremae, or Pseudomonas viridiflava.

[0438] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be a eukaryote.

[0439] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be a cell belonging to a different branch: posterior flagellates; green plants (e.g., algae and plants); Amebozoa; phylum Leptoglossi; phylum Cystica; phylum Marine flagellates; phylum Anisochondria; superphylum Platycerium; or kingdom Archaea.

[0440] In some implementations, the procedures and methods described herein may be performed using a host cell, such as a metazoan, a choanoflagellata, or a fungus.

[0441] In some embodiments, the procedures and methods described herein may be performed using a host cell, such as a fungus. For example, in some embodiments, the host cell may be a cell belonging to a eukaryotic phylum: Ascomycota, Basidiomycota, Chytridiomycota, Microsporidiales, or Zygomycota.

[0442] In some implementations, the procedures and methods described herein may be performed using a host cell that is a fungus belonging to one of the following genera: Aspergillus, Cladosporium, Magnaphorthe, Morchella, Neurospora, Penicillium, Saccharomyces, Cryptococcus, or Ustilago.

[0443] In some implementations, the procedures and methods described herein may be performed using a host cell, which is a fungus belonging to one of the following species: *Saccharomyces cerevisiae*, *Saccharomyces boulardi*, *Saccharomyces uvarum*; *Aspergillus flavus*, *Aspergillus terreus*, *Aspergillus awamori*; *Cladosporium elatum*, *Cl. Herbarum*, *Cl. Sphaerospermum*, and *Cl. Cladosporioides*; *Magnaporthe grise*, *Magnaporthe oryzae*, *Magnaporthe rhizophila*; *Morchella deliciosa*, *Morchella esculenta*, *Morchella cuspidatum*. (conica); Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma; Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[0444] In some implementations, the procedures and methods described herein may be performed using a host cell, such as Kluyveromyces lactis, Kluyveromyces martensii, Saccharomyces cerevisiae, or Pichia pastoris.

[0445] In some implementations, the host cells used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be fungi belonging to one of the following genera: Aspergillus, Cladosporium, Rice blast fungus, Morel, Neurospora, Penicillium, Yeast, Cryptococcus, or Ustilago.

[0446] In some embodiments, the host cell used for producing CRIP, CRIP-insecticide protein, or peptide-IA can be a member of the family Saccharomycetaceae. For example, in some embodiments, the host cell can be one of the following genera within the family Saccharomycetaceae: *Brettanomyces*, *Candida*, *Citeromyces*, *Cyniclomyces*, *Debaryomyces*, *Issatchenkia*, *Kazachstania*, *Kluyveromyces*, *Komagataella*, and *Kuraishi*. a) Genus Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, or Zygotorulaspora.

[0447] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be one of the following: Aspergillus flavus, Aspergillus terreus, Aspergillus avocado, Cladosporium sphaerospermum, Cladosporium cladosporioides, rice blast fungus, rice blast fungus, Magnaphorthe rhizophila, morel, morel, morel, morel, morel, Neurospora tetrasperma, penicillin, Penicillium chrysogenum, Penicillium loudi, or Penicillium scabra.

[0448] In some implementations, the host cells used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be species of the genus Candida. For example, the host cell can be one of the following: *Candida albicans*, *Candida ascalaphidarum*, *Candida amphixiae*, *Candida antarctica*, *Candida argentea*, *Candida atlantica*, *Candida atmosphaerica*, *Candida auris*, *Candida blankii*, *Candida blattae*, *Candida bracarensis*, *Candida bromeliacearum*, *Candida carpophila*, *Candida carvajalis*, *Candida cerambycidarum*, *Candida chauliodes*, *Candida corydalis*, *Candida docephalis*. Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, or Candidakefyr.

[0449] In some implementations, the host cell used for producing CRIP, CRIP-insecticide protein, or peptide-IA can be a species of the genus Kluyveromyces. For example, the host cell can be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marx, Kluyveromyces nonfermentans, or Kluyveromyces wickerhamii.

[0450] In some implementations, the host cell used for producing CRIP, CRIP-insecticide protein, or peptide-IA can be a species of the genus Pichia. For example, the host cell can be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmehmeri, Pichia pastoris, Pichia methanolica, or Pichia subpelliculosa.

[0451] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be a species of yeast. For example, the host cell can be one of the following: *Saccharomyces arboricolus*, *Saccharomyces bayanus*, *Saccharomyces bulderi*, *Saccharomyces cariocanus*, *Saccharomyces cariocus*, *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae varboulardii*, *Saccharomyces chevalieri*, *Saccharomyces dairenensis*, *Saccharomyces ellipsoideus*, *Saccharomyces eubayanus*, *Saccharomyces exiguous*, *Saccharomyces florentinus*, *Saccharomyces fragilis*, *Saccharomyces kudriazweiensis*. Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces pencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, or Saccharomyces zonatus.

[0452] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be one of the following: Saccharomyces cerevisiae, Pichia pastoris, Pichia methanata, Schizosaccharizoi saccharizoi, or Hansenula anomala.

[0453] Using yeast cells as a host organism to produce recombinant CRIP or peptide-IA is a well-known method among those skilled in the art. In some embodiments, the methods and compositions described herein can be performed using any yeast species, including but not limited to any species of the genera *Saccharomyces*, *Pichia pastoris*, *Kluyveromyces*, *Hansenula*, *Yersinia*, or *Schizosaccharomyces*, and the genus *Saccharomyces* includes any species of the genus *Saccharomyces*, such as *Saccharomyces cerevisiae* species selected from the following strains: INVSC1, YNN27, S150-2B, W303-1B, CG25, W3124, JRY188, BJ5464, AH22, GRF18, W303-1A, and BJ3505. In some implementations, members of the genus *Pichia* include any species of the genus *Pichia*, such as *Pichia* species and *Pichia pastoris*, for example, *Pichia pastoris* selected from the following strains: Bg08, Y-11430, X-33, GS115, GS190, JC220, JC254, GS200, JC227, JC300, JC301, JC302, JC303, JC 304, JC305, JC306, JC307, JC308, YJN165, KM71, MC100-3, SMD1163, SMD1165, SMD1168, GS241, MS105, any PEP4 knockout strain and any PRB1 knockout strain, and Pichia pastoris selected from the following strains: Bg08, X-33, SMD1168 and KM71. In some implementations, any species of the genus *Kluyveromyces* can be used to carry out the methods described herein, including any species of the genus *Kluyveromyces*, such as *Kluyveromyces lactis*, and we teach that strains of *Kluyveromyces lactis* may, but are not required to, be selected from the following strains: GG799, YCT306, YCT284, YCT389, YCT390, YCT569, YCT598, NRRL Y-1140, MW98-8C, MS1, CBS293.91, Y721, MD2 / 1, PM6-7A, WM37, K6, K7, 22AR1, 22A295-1, SD11, MG1 / 2, MSK110, JA6, CMK5, HP101, HP108, and PM6-3C, in addition to *Kluyveromyces lactis* species, also selected from GG799, YCT306, and NRRL Y-1140.

[0454] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be Aspergillus oryzae.

[0455] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be Aspergillus japonicas.

[0456] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be Aspergillus niger.

[0457] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be Bacillus licheniformis.

[0458] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be Bacillus subtilis.

[0459] In some implementations, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA can be Trichoderma reesei.

[0460] In some embodiments, the procedures and methods described herein may be performed using a host cell, which is yeast, including but not limited to any species of the genus *Hansenula*, and preferably *Hansenula polymorpha*. In some embodiments, the procedures and methods described herein may be performed using any species of yeast, including but not limited to any species of the genus *Yarrowia*, such as *Yarrowia lipolytica*. In some embodiments, the procedures and methods described herein may be performed using any species of yeast, including but not limited to any species of the genus *Schizostomyces*, and preferably *Schizostomyces macranthum*.

[0461] Yeast cell culture

[0462] In some implementations, yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, and Pichia pastoris can be used as host organisms. Yeast cell culture techniques are well known to those skilled in the art. Exemplary methods for yeast cell culture can be found in Evans, Yeast Protocols, Springer, 1996; Bill, Recombinant Protein Production in Yeast, Springer, 2012; Hagan et al., Fission Yeast: A Laboratory Manual, CSH Press, 2016; Konishi et al., “Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture”, Biosci Biotechnol Biochem., 2014, Vol. 78, No. 6: pp. 1090-1093; Dymond, “Saccharomyces cerevisiae growth media”, Methods Enzymol., 2013, Vol. 533: pp. 191-204; Looke et al., “Extraction of genomic DNA from yeasts for The full text of these references is incorporated herein by reference in “PCR-based applications”, Biotechniques, May 2011, Vol. 50, No. 5: pp. 325-328; and Romanos et al., “Culture of yeast for the production of heterologous proteins”, Curr ProtocCell Biol., September 2, 2014, Vol. 64, No. 20.9: pp. 1-16.

[0463] The formulations of yeast cell fermentation medium and primary strain are as follows: (1) MSM medium formulation: 2 g / L sodium citrate dihydrate; 1 g / L calcium sulfate dihydrate (0.79 g / L anhydrous calcium sulfate); 42.9 g / L potassium dihydrogen phosphate; 5.17 g / L ammonium sulfate; 14.33 g / L potassium sulfate; 11.7 g / L magnesium sulfate heptahydrate; 2 mL / L PTM1 microsalt solution; 0.4 ppm biotin (from 500×, 200 ppm stock solution); 1% to 2% pure glycerol or other carbon source. (2) PTM1 trace salt solution: Copper sulfate-5H2O 6.0g; Sodium iodide 0.08g; Manganese sulfate-H2O 3.0g; Sodium molybdate-2H2O 0.2g; Boric acid 0.02g; Cobalt chloride 0.5g; Zinc chloride 20.0g; Ferrous sulfate-7H2O 65.0g; Biotin 0.2g; Sulfuric acid 5.0ml; Add water to a final volume of 1 liter. An exemplary composition of Kluyveromyces lactis Determined Components Medium (DMSor) is as follows: 11.83 g / L KH₂PO₄, 2.299 g / L K₂HPO₄, 20 g / L fermentable sugars (e.g., galactose, maltose, latrotriose, sucrose, fructose, or glucose and / or sugar alcohols, such as erythritol, hydrogenated starch hydrolysate, isomaltitol, lactitol, maltitol, mannitol, and xylitol), 1 g / L MgSO₄·7H₂O, 10 g / L (NH₄)SO₄, 0.33 g / L CaCl₂·2H₂O, 1 g / L NaCl, 1 g / L KCl, 5 mg / L CuSO₄·5H₂O, 30 mg / L MnSO₄·H₂O, 10 mg / L ZnCl₂, 1 mg / L KI, 2 mg / L CoCl2·6H2O, 8 mg / L Na2MoO4·2H2O, 0.4 mg / L H3BO3, 15 mg / L FeCl3·6H2O, 0.8 mg / L Biotin, 20 mg / L Calcium Pantothenate, 15 mg / L Thiamine, 16 mg / L Inositol, 10 mg / L Nicotinic Acid, and 4 mg / L Pyridoxine.

[0464] Yeast cells can be cultured in 48-well deep-well plates and sealed with sterile, breathable caps after inoculation. Yeast colonies, such as *Kluyveromyces lactis* colonies, can be picked from the plates and inoculated into the deep-well plates with 2.2 mL of DMSO medium per well. The inoculated deep-well plates can be grown for 6 days at 23.5°C in a refrigerated incubator shaker with shaking at 280 rpm. On day 6 post-inoculation, the conditioned medium should be harvested by centrifugation at 4000 rpm for 10 minutes followed by filtration through a filter plate with a 0.22 μM membrane, and the filtered medium should be analyzed by HPLC.

[0465] Yeast transformation, peptide purification and analysis

[0466] An exemplary method for yeast transformation is as follows: An expression vector carrying a CRIP ORF, a CRIP-insecticide ORF, or a peptide-IA ORF is transformed into yeast cells. First, the expression vector is typically linearized by specific restriction enzyme cleavage to facilitate chromosomal integration via homologous recombination. The linear expression vector is then transformed into yeast cells via chemical or electroporation transformation, and integrated into the target site of the yeast genome via homologous recombination. Integration can occur multiple times at the same chromosomal locus; therefore, the genome of the transformed yeast cells may contain multiple copies of the CRIP or peptide-IA expression cassette. Successfully transformed yeast cells can be identified using growth conditions favorable to selection markers engineered into the expression vector and co-integrated with the CRIP, CRIP-insecticide ORF, or peptide-IA ORF into the yeast chromosome; examples of such markers include, but are not limited to, acetamide protrophic, bleomycin resistance, genimycin resistance, norsinolytic resistance, and uracil protrophic.

[0467] Due to the influence of unpredictable and variable factors—such as epigenetic modifications of genes and gene networks, and variations in the number of integration events occurring in individual cells within a population undergoing a transformation procedure—individual yeast colonies undergoing a given transformation process will differ in their ability to produce CRIP ORF, CRIP-insecticidal protein ORF, or peptide-IA ORF. Therefore, high-yielding strains should be screened from transgenic yeast colonies carrying CRIP or peptide-IA transgenes. Two effective methods for this screening—each relying on the growth of small-scale cultures of transgenic yeast to provide conditioned medium samples for subsequent analysis—use reverse-phase HPLC or a housefly injection procedure to analyze conditioned medium samples from positive transgenic yeast colonies.

[0468] Transgenic yeast culture can be performed using 14 mL round-bottom polypropylene tubes, with 5 mL to 10 mL of definitive-component medium added to each tube, or in 48-well deep-well plates, with 2.2 mL of definitive-component medium added to each well. Definitive-component medium free of crude protein extracts or byproducts such as yeast extract or peptone is used for culturing to reduce the protein background in the conditioned medium harvested for subsequent screening steps. Cultures are performed at optimal temperatures, for example, 23.5 °C for Kluyveromyces lactis, for approximately 5 to 6 days until maximum cell density is reached. CRIP, or peptide-IA, will now be produced by the transformed yeast cells and secreted from the cells into the growth medium. To prepare samples for screening, cells are removed from the culture by centrifugation, and the supernatant is collected as conditioned medium, then washed by filtration through a 0.22 μm filter membrane, and then prepared for strain screening.

[0469] In some implementations, positive yeast colonies transformed with CRIP or peptide-IA can be screened by reversed-phase HPLC (rpHPLC) screening of putative yeast colonies. In this screening method, an HPLC column with a C18 bonded phase can be used. Acetonitrile and water are used as the mobile phase solvent, and the peptide is detected using a UV absorbance detector set at 220 nm. An appropriate amount of conditioned medium sample is loaded into the rpHPLC system and eluted with a linear gradient of mobile phase solvents. The corresponding peak area of ​​the insecticidal peptide in the HPLC chromatography is used to quantify the concentration of CRIP or peptide-IA in the conditioned medium. Known amounts of pure CRIP or peptide-IA are run through the same rpHPLC column using the same HPLC protocol to confirm the peptide retention time and generate a standard peptide HPLC profile for quantification.

[0470] An exemplary reverse-phase HPLC screening method for positive Kluyveromyces lactis cells is as follows: CRIP ORF, CRIP-insecticide ORF, or peptide-IA ORF can be inserted into the expression vector pKLAC1 and transformed into Kluyveromyces lactis strain YCT306 from New England Biolabs (Ipswich, MA, USA). The pKLAC1 vector is an integrative expression vector. Once the CRIP or peptide-IA transgene is cloned into pKLAC1 and transformed into YCT306, their expression is controlled by the LAC4 promoter. The resulting transformed colonies produce a precursor peptide containing an α-mating factor signal peptide, a Kex2 cleavage site, and mature CRIP or peptide-IA. The α-mating factor signal peptide guides the precursor peptide into the endogenous secretion pathway, and the mature CRIP or peptide-IA is released into the growth medium.

[0471] In some implementations, codon optimization for CRIP or peptide-IA expression can be performed in two rounds. For example, in the first round, based on some common features of highly expressed DNA sequences, multiple variants of CRIP or peptide-IA expression ORFs expressing α-mating factor signal peptides, Kex2 cleavage sites, and CRIP or peptide-IA are designed, and their expression levels are evaluated in Kluyveromyces lactis strain YCT306, thereby generating an initial Kluyveromyces lactis expression algorithm. In the second round of optimization, additional variant CRIP or peptide-IA expression ORFs can be designed based on the initial Kluyveromyces lactis expression algorithm to further fine-tune the Kluyveromyces lactis expression algorithm and identify the optimal ORF for CRIP or peptide-IA expression in Kluyveromyces lactis. In some implementations, the DNA sequence obtained from the above optimization may have an open reading frame encoding α-MF signal peptide, Kex2 cleavage site, and CRIP, CRIP insecticidal protein, or peptide-IA, and can be cloned into the pKLAC1 vector using Hind III instead of I restriction sites, thereby generating a CRIP or peptide-IA expression vector.

[0472] In some implementations, the yeast *Pichia pastoris* can be transformed using CRIP, CRIP-insecticidal protein, or peptide-IA expression cassettes. An exemplary method for transforming *Pichia pastoris* is as follows: Vectors pJUGαKR and pJUZαKR can be used to transform CRIP or peptide-IA into *Pichia pastoris*. The pJUGαKR and pJUZαKR vectors are available from Biogrammatics (Carlsbad, California, USA). Both vectors are integrative vectors and use the uracil ribosyltransferase promoter (pUPP) to enhance heterologous transgene expression. The only difference between the vectors is that pJUGαKR provides G418 resistance to the host yeast, while pJUZαKR provides Zeocin resistance. Complementary oligonucleotide pairs encoding CRIP or peptide-IA are designed and synthesized for subcloning into both yeast expression vectors. Hybridization was performed by mixing the corresponding complementary oligonucleotides in 30 mM NaCl, 10 mM Tris-Cl (all final concentrations), pH 8 to a final concentration of 20 μM, then incubating at 95 °C for 20 min, followed by incubation at 92 °C for 9 h, and ending at 17 °C, with a temperature drop of 3 °C every 20 min. The hybridization reaction produced DNA fragments encoding CRIP or peptide-IA. Both Pichia pastoris vectors were digested with BsaI-HF restriction enzyme, and the resulting double-stranded DNA product was subcloned into linearized Pichia pastoris vectors using standard procedures. After confirming the subcloned sequence, plasmids were transfected aliquots into Pichia pastoris strain Bg08 via electroporation. The resulting transformants were cultured and screened as described herein, selected based on resistance to Zeocin or G418 conferred by elements engineered into vectors pJUZαKR and pJUGαKR, respectively.

[0473] The following provides a detailed description of the ORF and its components.

[0474] Screening and evaluation of yeast peptide production

[0475] Peptide yield can be determined by any method known to those skilled in the art (e.g., capillary gel electrophoresis (CGE), Western blot analysis, etc.). Activity assays as described herein and known in the art can also provide information about peptide yield. In some embodiments, these or any other methods known in the art can be used to evaluate peptide yield.

[0476] Quantitative assays

[0477] In some implementations, and without limitation, CRIP peptide yield can be measured using the following methods: HPLC; mass spectrometry (MS) and related techniques; LC / MS / MS; reversed-phase protein array (RPPA); immunohistochemistry; ELISA; suspension bead array, mass spectrometry; dot blot; SDS-PAGE; capillary gel electrophoresis (CGE); protein blot analysis; Bradford assay; measurement of UV absorbance at 260 nm; Lowry assay; Smith copper / dioctylcholine assay; secretion assay; Pierce protein assay; biuret reaction, etc. Exemplary methods for protein quantification are provided in Stoscheck, C., 1990, “Quantification of Protein,” Methods in Enzymology, Vol. 182, pp. 50–68; Lowry, O., Rosebrough, A., Farr, A., and Randall, R., 1951, J. Biol. Chem., Vol. 193, p. 265; Smith, P. et al., 1985, Anal. Biochem., Vol. 150, pp. 76–85; Bradford, M., 1976, “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,” Anal. Biochem., Vol. 72, pp. 248–254; Cabib, E., and Polacheck, I., 1984, “Protein assay for dilute.” "Solutions", Methods in Enzymology, Vol. 104: pp. 318-328; Turcanu, Victor, Williams, Neil A., 2001, "Cell identification and isolation on the basis of cytokine secretion: A novel tool for investigating immune responses", Nature Medicine, Vol. 7, No. 3: pp. 373-376; US Patent No. 6,391,649; The full text of these documents and patents is incorporated herein by reference.

[0478] In other embodiments, CRIP peptide yield can be quantified and / or assessed using methods including, but not limited to, the following: the amount of recombinant protein per volume of culture (e.g., grams or milligrams of protein per liter of culture); the percentage or fraction of insoluble precipitate of recombinant protein obtained after cell lysis (e.g., the amount / quantity of protein in the insoluble component of the recombinant protein extraction supernatant); the percentage or fraction of active protein (e.g., the amount / analysis of active protein for protein quantity); the percentage or fraction of total cellular protein (TCP); and / or the percentage or ratio of protein / cell quantity to dry biomass.

[0479] In some implementations, yield is expressed in culture volume, taking into account cell density, particularly when comparing yields between different cultures.

[0480] In some embodiments, the present invention provides a method for producing a heterologous polypeptide that is at least about 5%, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or more of total cellular protein (TCP). "Total cellular protein percentage" is the percentage of the amount of heterologous polypeptide in the host cell relative to the aggregated cellular protein. The determination of the total cellular protein percentage is well known in the art.

[0481] "Total cellular protein (tcp)" or "total cellular protein percentage (%tcp)" is the percentage of protein or polypeptide in the host cell relative to the total cellular protein. Methods for determining the total cellular protein percentage are well known in the art.

[0482] In some implementations, HPLC can be used to quantify peptide yield. For example, in some implementations, the yield of CRIP or peptide-IA can be evaluated using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 × 100 mm C18 reversed-phase analytical HPLC column and an autoinjector. An illustrative use of the Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 × 100 mm C18 reversed-phase analytical HPLC column and an autoinjector is as follows: A filtered conditioned medium sample from transformed Kluyveromyces lactis cells is analyzed using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 × 100 mm C18 reversed-phase analytical HPLC column and an autoinjector by analyzing HPLC-grade water and acetonitrile containing 0.1% trifluoroacetic acid (consisting of the two mobile phase solvents for HPLC analysis); the peak areas of both CRIP and peptide-IA are analyzed using HPLC chromatography and then used to calculate the peptide concentration in the conditioned medium, which can be further normalized to the corresponding final cell density (e.g., determined by OD600 measurement) as the normalized peptide yield.

[0483] Activity assays

[0484] In some implementations, a housefly injection assay can be used to screen positive yeast colonies transformed with CRIP or peptide-IA. When the dose is measured by injection through the dorsal thoracic cavity, CRIP or peptide-IA paralyzes / kills the housefly. The potency of CRIP or peptide-IA can be measured by the mean paralyzing / lethal dose (PD) of the peptide. 50 / LD 50 Defined as either a knockout rate or mortality rate of 50% in injected houseflies, CRIP or peptide-IA are commonly used in housefly injection assays to generate a standard dose-response curve from which PD can be determined. 50 / LD 50 Values. PD was analyzed using standard dose-response curves from pure CRIP or peptide-IA. 50 / LD 50 The values, determined by housefly injection using serial dilutions of the corresponding conditioned medium, enable the quantification of CRIP or peptide-IA produced by yeast transformation.

[0485] An exemplary housefly injection bioassay is performed as follows: Conditioned medium is serially diluted to produce a full dose-response curve from the housefly injection bioassay. Adult houseflies (common houseflies) are fixed with CO2 prior to injection, and 12-18 mg of houseflies are selected for injection. Using a micro-application device fitted with a 1 cc syringe and a 30-gauge needle, 0.5 μL of the serially diluted conditioned medium sample is injected into each housefly through the body wall of the dorsal thorax. The injected houseflies are placed in a sealed container with moistened filter paper and a breathing hole, and their condition is assessed by knockdown ratio or by mortality score at 24 hours post-injection. Standardized yields are calculated. Peptide yield refers to the concentration of peptides in the conditioned medium, expressed in mg / L. However, peptide yield is not always sufficient to accurately compare strain productivity. Individual strains may have different growth rates, and therefore, cell densities may differ between cultures when harvested. Cultures with high cell densities may produce higher concentrations of peptides in the medium, even if the peptide productivity of that strain is lower than that of another strain with a higher yield. Therefore, the term "normalized yield" is generated by dividing peptide yield by the cell density in the corresponding culture, and this allows for a better comparison of peptide productivity between strains. Cell density is expressed as absorbance at 600 nm, in "A" (absorbance units).

[0486] Screening yeast colonies that have undergone CRIP or peptide-IA conversion allows for the identification of high-yielding yeast strains from hundreds of potential colonies. When using the optimized fermentation medium and conditions described herein, these strains can be fermented in a bioreactor to achieve CRIP or peptide-IA yields of at least 4 g / L, at least 3 g / L, or at least 2 g / L. Higher productivity (expressed in mg / L) can be from about 100 mg / L to about 100,000 mg / L; or from about 100 mg / L to about 90,000 mg / L; or from about 100 mg / L to about 80,000 mg / L; or from about 100 mg / L to about 70,000 mg / L; or from about 100 mg / L to about 60,000 mg / L; or from about 100 mg / L to about 50,000 mg / L; or from about 100 mg / L to about 40,000 mg / L; or from about 100 mg / L to about 30,000 mg / L; or from about 100 mg / L to about 20,000 mg / L; or from about 100 mg / L to about 17,500 mg / L; or about 100 mg / L to about 15,000 mg / L; or about 100 mg / L to about 12,500 mg / L; or about 100 mg / L to about 10,000 mg / L; or about 100 mg / L to about 9,000 mg / L; or about 100 mg / L to about 8,000 mg / L; or about 100 mg / L to about 7,000 mg / L; or about 100 mg / L to about 6,000 mg / L; or about 100 mg / L to about 5,000 mg / L; or about 100 mg / L to about 3,000 mg / L; or about 100 mg / L to 2,000 mg / L; or about 100 mg / L to 1,500 mg / L; or about 100 mg / L to 1,000 mg / L; or about 100 mg / L to 750 mg / L; or about 100 mg / L to 500 mg / L; or about 150 mg / L to 100,000 mg / L; or about 200 mg / L to 100,000 mg / L; or about 300 mg / L to 100,000 mg / L; or about 400 mg / L to 100,000 mg / L; or about 500 mg / L to 100,000 mg / L; or about 750 mg / L to 100,000 mg / L / L; or about 1,000 mg / L to 100,000 mg / L; or about 1,250 mg / L to 100,000 mg / L; or about 1,500 mg / L to 100,000 mg / L; or about 2,000 mg / L to 100,000 mg / L; or about 2,500 mg / L to 100,000 mg / L; or about 3,000 mg / L to 100,000 mg / L; or about 3,500 mg / L to 100,000 mg / L; or about 4,000 mg / L to 100,000 mg / L; or about 4,500 mg / L to 100,000 mg / L;Or approximately 5,000 mg / L to 100,000 mg / L; or approximately 6,000 mg / L to 100,000 mg / L; or approximately 7,000 mg / L to 100,000 mg / L; or approximately 8,000 mg / L to 100,000 mg / L; or approximately 9,000 mg / L to 100,000 mg / L; or approximately 10,000 mg / L to 100,000 mg / L; or approximately 12,500 mg / L to 100,000 mg / L; or approximately 15,000 mg / L to 100,000 mg / L; or approximately 17,500 mg / L to 100,000 mg / L; or approximately 20,000 mg / L to 100,000 mg / L. 000 mg / L; or about 30,000 mg / L to 100,000 mg / L; or about 40,000 mg / L to 100,000 mg / L; or about 50,000 mg / L to 100,000 mg / L; or about 60,000 mg / L to 100,000 mg / L; or about 70,000 mg / L to 100,000 mg / L; or about 80,000 mg / L to 100,000 mg / L; or about 90,000 mg / L to 100,000 mg / L; or any range of values ​​provided by the same or similar production method used to produce the peptide before conversion, or even higher yields achievable with the peptide before conversion.

[0487] Any of the foregoing methods can be used and / or customized to produce CRIP and / or peptide-IA (e.g., insecticides that facilitate such methods, such as polymers of amino acids, peptides, and / or proteins). For example, any of the foregoing methods can be used to produce, generate, manufacture, express, transcribe, translate, synthesize, or otherwise generate any of the CRIP or peptide-IA described herein, including but not limited to ACTX peptides (e.g., U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, rκ-ACTX-Hv1c, ω-ACTX-Hv1a, and / or ω-ACTX-Hv1a+2); Γ-CNTX-Pn1a; U1-funnel web spider toxin-Ta1b; TVP; Av2; Av3; AVP; and / or Bt toxins (e.g., Cry toxin, Cyt toxin, or Vip).

[0488] Culture and fermentation conditions

[0489] Cell culture techniques are well known in the art. In some embodiments, the culture methods and / or materials will necessarily need to be adapted based on the selected host cells; and such adaptations (e.g., changes in pH, temperature, culture medium concentration, etc.) are well known to those skilled in the art. In some embodiments, any known culture technique can be used to produce the CRIP, CRIP-insecticide protein, or peptide-IA of the present invention.

[0490] Exemplary cultivation methods are provided in U.S. Patent Nos. 3,933,590; 3,946,780; 4,988,623; 5,153,131; 5,153,133; 5,155,034; 5,316,905; 5,330,908; 6,159,724; 7,419,801; 9,320,816; 9,714,408; and 10,563,169; the disclosures of these patents are incorporated herein by reference in their entirety.

[0491] Yeast cultures

[0492] Yeast cell culture techniques are well known to those skilled in the art. Exemplary methods for yeast cell culture can be found in Evans, Yeast Protocols, Springer, 1996; Bill, Recombinant Protein Production in Yeast, Springer, 2012; Hagan et al., Fission Yeast: A Laboratory Manual, CSHPress, 2016; Konishi et al., “Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture”, Biosci Biotechnol Biochem., 2014, Vol. 78, No. 6: pp. 1090-1093; Dymond, “Saccharomyces cerevisiae growth media”, Methods Enzymol., 2013, Vol. 533: pp. 191-204; Looke et al., “Extraction of genomic DNA from yeasts for PCR-based "Applications", Biotechniques, May 2011, Vol. 50, No. 5: pp. 325-328; and Romanos et al., "Culture of yeast for the production of heterologous proteins", Curr Protoc Cell Biol., September 2, 2014, Vol. 64, No. 20.9: pp. 1-16. The full text of these documents is incorporated herein by reference.

[0493] Yeast can be cultured in a variety of media. For example, in some embodiments, yeast can be cultured in the following media: basal medium; YPD medium; yeast auxotrophic medium; yeast nitrogen-based (YNB, with or without amino acids); YEPD medium; ADE D medium; ADE DS" medium; LEU D medium; HISD medium; or mineral salt medium.

[0494] In some embodiments, the yeast can be cultured in a basal medium. In some embodiments, the basal medium may contain: 2% sugar; phosphate buffer, pH 6.0; magnesium sulfate; calcium chloride; ammonium sulfate; sodium chloride; potassium chloride; copper sulfate; manganese sulfate; zinc chloride; potassium iodide; cobalt chloride; sodium molybdate; boric acid; ferric chloride; biotin; calcium pantothenate; thiamine; inositol; nicotinic acid; and pyridoxine.

[0495] In some implementations, the yeast can be cultured in YPD medium. YPD medium contains bacterial peptone, yeast extract, and glucose.

[0496] In some embodiments, yeast can be cultured in a yeast auxotrophic medium that can be used to distinguish auxotrophic mutant strains that cannot grow without specific medium components transformed with a plasmid that allows the transformants to grow on a medium lacking the required components.

[0497] In some implementations, yeast can be cultured using yeast nitrogen-based (YNB, with or without amino acids), which contains nitrogen, vitamins, trace elements, and salts.

[0498] In some embodiments, the culture medium may be YEPD medium, such as a medium containing 2% D-glucose, 2% BACTO peptone (Difco Laboratories, Detroit, MI), 1% BACTO yeast extract (Difco), 0.004% adenine and 0.006% L-leucine or a variant thereof, wherein the carbon source is a sugar alcohol, such as glycerol or sorbitol.

[0499] In some embodiments, the culture medium may be an ADE D medium, for example, a medium containing 0.056% Ade-Trp-Thr powder, 0.67% amino acid-free yeast nitrogen, 2% D-glucose and 0.5% 200× tryptophan, threonine solution or a variant thereof, wherein the carbon source is a sugar alcohol, such as glycerol or sorbitol.

[0500] In some embodiments, the culture medium may be an ADE DS medium, for example, a medium containing 0.056% Ade-Trp-Thr powder, 0.67% amino acid-free yeast nitrogen, 2% D-glucose, 0.5% 200× tryptophan, threonine solution and 18.22% D-sorbitol or a variant thereof, wherein the carbon source is entirely a sugar alcohol, such as glycerol or sorbitol.

[0501] In some embodiments, the culture medium may be LEU D medium, for example, a medium containing 0.052% Leu-Trp-Thr powder, 0.67% amino acid-free yeast nitrogen, 2% D-glucose and 0.5% 200× tryptophan, threonine solution or a variant thereof, wherein the carbon source is a sugar alcohol, such as glycerol or sorbitol.

[0502] In some embodiments, the culture medium may be HISD medium, for example, a medium containing 0.052% His-Trp-Thr powder, 0.67% amino acid-free yeast nitrogen, 2% D-glucose and 0.5% 200× tryptophan, threonine solution or a variant thereof, wherein the carbon source is a sugar alcohol, such as glycerol or sorbitol.

[0503] In some implementations, mineral salt media may be used. Mineral salt media consist of mineral salts and a carbon source, such as glucose, sucrose, or glycerol. Examples of mineral salt media include, for example, M9 medium, Pseudomonas aeruginosa medium (ATCC179), and Davis-Mingioli medium. See Davis and Mingioli, 1950, J. Bact., Vol. 60: pp. 17-28. The mineral salts used to prepare mineral salt media include those selected from, for example, potassium phosphate, ammonium sulfate or ammonium chloride, magnesium sulfate or magnesium chloride, and trace minerals such as calcium chloride, borates, and sulfates of iron, copper, manganese, and zinc. Typically, organic nitrogen sources, such as peptone, tryptone, amino acids, or yeast extracts, are not included in mineral salt media. Instead, inorganic nitrogen sources are used, and these may be selected from, for example, ammonium salts, ammonia water, and gaseous ammonia. Mineral salt media will typically contain glucose or glycerol as a carbon source.

[0504] Compared to mineral salt media, basal media may also contain inorganic salts and carbon sources, but may be supplemented with, for example, low levels of amino acids, vitamins, peptones, or other components, although these are added at very low levels. Media can be prepared using methods described in the art, for example, U.S. Patent Application Publication No. 2006 / 0040352, the entire disclosure of which is incorporated herein by reference. A detailed description of the culture procedures and mineral salt media useful in the methods of the present invention is found in Riesenberg, D et al., 1991, “High celldensity cultivation of Escherichia coli at controlled specific growth rate,” J. Biotechnol., Vol. 20, No. 1: pp. 17-27.

[0505] In some implementations, *Kluyveromyces lactis* is grown in a basal medium supplemented with 2% glucose, galactose, sorbitol, or glycerol as the sole carbon source. Cultures are incubated at 30°C until mid-log (24 to 48 hours) for β-galactosidase measurement, or at 23.5°C for 6 days for heterologous protein expression.

[0506] In some implementations, yeast cells can be cultured in 48-well deep-well plates and sealed with sterile, breathable caps after inoculation. Yeast colonies, such as *Kluyveromyces lactis* colonies, can be picked from the plates and inoculated into the deep-well plates with 2.2 mL of DMSO medium per well. The inoculated deep-well plates can be grown for 6 days at 23.5°C in a refrigerated incubator shaker with shaking at 280 rpm. On day 6 post-inoculation, the conditioned medium should be harvested by centrifugation at 4000 rpm for 10 minutes followed by filtration through a filter plate with a 0.22 μM membrane, wherein the filtered medium is then analyzed by HPLC.

[0507] In some implementations, yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, etc., can be used as host organisms, and / or yeast modified using the methods described herein.

[0508] Temperature and pH conditions will vary depending on the stage of culture and the chosen host cell species. Variables in cell culture, such as temperature and pH, are readily known to those skilled in the art.

[0509] pH level is important in yeast culture. Those skilled in the art will understand that the culture process includes not only the initiation of yeast culture but also its maintenance. Yeast culture can be started at any pH level; however, because the culture medium tends to become more acidic (i.e., decrease pH) over time, pH levels must be carefully monitored during the culture process.

[0510] In some embodiments of the invention, the yeast is grown in a medium with a pH level determined based on the species of yeast used, the stage of culture, and / or the temperature. Thus, in some embodiments, the pH level may fall in the range of about 2 to about 10. Those skilled in the art will recognize that the optimal pH for most microorganisms is close to neutral (pH 7.0). However, in some embodiments, some fungal species prefer an acidic environment: therefore, in some embodiments, the pH may be in the range of 2 to 6.5. In some embodiments, the pH may be in the range of about 4 to about 4.5. Some fungal species (e.g., molds) can grow at a pH of about 2 to about 8.5, but acidic pH is preferred. See Mountney and Gould, Practical food microbiology and technology, 1988, 3rd edition; and Pena et al., “Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae”, FEMS Yeast Res., March 2015, Vol. 15, No. 2: fou005.

[0511] In other embodiments, the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8.

[0512] In some embodiments, the pH of the culture medium may be at least 5.5. In other aspects, the pH level of the culture medium may be about 5.5. In other aspects, the pH level of the culture medium may be between 4 and 8. In some cases, the pH level of the culture is maintained between 5.5 and 8. In other aspects, the pH level of the culture medium is between 6 and 8. In some cases, the pH level of the culture medium is maintained between 6 and 8. In some embodiments, yeast is grown and / or maintained at a pH level between 6.1 and 8.1. In some embodiments, yeast is grown and / or maintained at a pH level between 6.2 and 8.2. In some embodiments, yeast is grown and / or maintained at a pH level between 6.3 and 8.3. In some embodiments, yeast is grown and / or maintained at a pH level between 6.4 and 8.4. In some embodiments, yeast is grown and / or maintained at a pH level between 5.5 and 8.5. In some embodiments, yeast is grown and / or maintained at a pH level between 6.5 and 8.5. In some embodiments, the yeast is grown at a pH level of approximately 5.6, 5.7, 5.8, or 5.9. In some embodiments, the yeast is grown at a pH level of approximately 6. In some embodiments, the yeast is grown at a pH level of approximately 6.5. In some embodiments, the yeast is grown at a pH level of approximately 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the yeast is grown at a pH level of approximately 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the yeast is grown at a pH level higher than 8.

[0513] In some embodiments, the pH of the culture medium can be in the pH range of 2 to 8.5. In some embodiments, the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[0514] Exemplary methods for yeast cultivation can be found in: U.S. Patent No. 5,436,136, entitled “Repressible yeast promoters” (filed December 20, 1991, assigned to Ciba-Geigy Corporation); U.S. Patent No. 6,645,739, entitled “Yeast expression systems, methods of producing polypeptides in yeast, and compositions relating to the same” (filed July 26, 2001, assigned to Phoenix Pharmacologies, Inc., Lexington, KY); and U.S. Patent No. 10,023,836, entitled “Medium for yeasts” (filed August 23, 2013, assigned to Yamaguchi University); the disclosures of these patents are incorporated herein by reference in their entirety.

[0515] Fermentation

[0516] This invention considers culturing the host organism in any fermentation manner. For example, batch, fed-batch, semi-continuous, and continuous fermentation modes may be employed.

[0517] Fermentation can be carried out on any scale. The methods and techniques considered according to the present invention can be used for recombinant protein expression at any scale. Thus, in some embodiments, for example, fermentation volumes of microliters, milliliters, centiliters, and deciliters can be used, and fermentation volumes of 1 liter and larger can be used.

[0518] In some embodiments, the fermentation volume is about 1 liter or more. For example, in some embodiments, the fermentation volume is about 1 liter to about 100 liters. In some embodiments, the fermentation volume is about 1 liter, about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, or about 10 liters. In some embodiments, the fermentation volume is about 1 liter to about 5 liters, about 1 liter to about 10 liters, about 1 liter to about 25 liters, about 1 liter to about 50 liters, about 1 liter to about 75 liters, about 10 liters to about 25 liters, about 25 liters to about 50 liters, or about 50 liters to about 100 liters. In other embodiments, the fermentation volume is 5 liters or more, 10 liters, 15 liters, 20 liters, 25 liters, 50 liters, 75 liters, 100 liters, 200 liters, 500 liters, 1,000 liters, 2,000 liters, 5,000 liters, 10,000 liters, or 50,000 liters.

[0519] In some embodiments, the fermentation medium may be a nutrient solution for growing and / or maintaining cells. Without limitation, the solution typically provides at least one component from one or more of the following categories: (1) an energy source, typically in the form of a carbon source, such as glucose; (2) all essential amino acids, and typically a basic set of twenty amino acids; (3) vitamins and / or other organic compounds required at low concentrations; (4) free fatty acids or lipids, such as linoleic acid; and (5) trace elements, wherein trace elements are defined as inorganic compounds or naturally occurring elements typically required at very low concentrations (typically in the micromolar range).

[0520] In some embodiments, the fermentation medium may be the same as a cell culture medium or any other culture medium described herein. In some embodiments, the fermentation medium may be different from a cell culture medium. In some embodiments, the fermentation medium may be modified to suit large-scale protein production.

[0521] In some embodiments, the fermentation medium may be selectively supplemented with one or more components from any of the following categories: (1) hormones and other growth factors, such as serum, insulin, transferrin, etc.; (2) salts, such as magnesium, calcium and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases, such as adenosine, thymidine, etc.; (5) protein and tissue hydrolysates, such as peptones or peptone mixtures that may be obtained from purified gelatin, plant material or animal by-products; (6) antibiotics, such as gentamicin; and (7) cell protectants, such as Pranic polyol.

[0522] In some embodiments, the pH of the fermentation medium can be maintained using pH buffers and methods known to those skilled in the art. Ammonia can also be used to control the pH during fermentation. In some embodiments, the pH of the fermentation medium will be selected based on the preferred pH of the organism used. Thus, in some embodiments, and depending on the host cell and temperature, the pH can be in the range of about 1 to about 10.

[0523] In some embodiments, the pH range of the fermentation medium can be from 2 to 8.5. In some embodiments, the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[0524] In other embodiments, the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8.

[0525] In some implementations, for example when using E. coli, the optimal pH range is between 6.5 and 7.5, depending on the temperature.

[0526] In other implementations, for example, when using a yeast strain, the pH may be in the range of about 4.0 to 8.0.

[0527] In some implementations, a neutral pH, i.e., a pH of about 7.0, can be used.

[0528] Those skilled in the art will recognize that during fermentation, pH levels can drift due to the conversion and production of substrates and metabolic compounds.

[0529] In some embodiments, the fermentation medium may be supplemented with buffer solutions or other chemicals to prevent pH changes. For example, in some embodiments, Ca(OH)₂, CaCO₃, NaOH, or NH₄OH may be added to the fermentation medium to neutralize the formation of acidic compounds, such as those present in some yeast species, during industrial processes.

[0530] Temperature is another important consideration in the fermentation process; and, as with pH, ​​temperature will depend on the type of host cell chosen.

[0531] In some embodiments, the fermentation temperature is maintained between about 4°C and about 42°C. In some embodiments, the fermentation temperature is about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C.

[0532] In other embodiments, the fermentation temperature is maintained at approximately 25°C to approximately 27°C, approximately 25°C to approximately 28°C, approximately 25°C to approximately 29°C, approximately 25°C to approximately 30°C, approximately 25°C to approximately 31°C, approximately 25°C to approximately 32°C, approximately 25°C to approximately 33°C, approximately 26°C to approximately 28°C, approximately 26°C to approximately 29°C, approximately 26°C to approximately 30°C, approximately 26°C to approximately 31°C, approximately 26°C to approximately 32°C, approximately 27°C to approximately 29°C, and approximately 27°C to approximately 30°C. 0°C, about 27°C to about 31°C, about 27°C to about 32°C, about 26°C to about 33°C, about 28°C to about 30°C, about 28°C to about 31°C, about 28°C to about 32°C, about 29°C to about 31°C, about 29°C to about 32°C, about 29°C to about 33°C, about 30°C to about 32°C, about 30°C to about 33°C, about 31°C to about 33°C, about 31°C to about 32°C, about 30°C to about 33°C, or about 32°C to about 33°C.

[0533] In other implementations, the temperature changes during fermentation, for example, depending on the stage of fermentation.

[0534] Fermentation can be achieved using a variety of microorganisms known to those skilled in the art. Microorganisms suitable for large-scale production of CRIP, CRIP-insecticide protein, or peptide-IA include any of the microorganisms listed herein. In some embodiments, non-limiting examples of microorganisms include strains from the following genera: yeast species (including but not limited to brewer's yeast (baker's yeast), saccharifying yeast (S. distaticus), grape juice yeast), Kluyveromyces (including but not limited to Kluyveromyces marxi and Kluyveromyces fragilis), Candida (including but not limited to Candida pseudotropicalis and Candida brassicae), Pichia stipitis (a close relative of Candida shehatae), Clavispora (including but not limited to Candida lusitaniae and Candida opuntiae), P. tannophilus (including but not limited to P. tannophilus), and Bacillus (including but not limited to B. clausenii). Other suitable microorganisms include, for example, *Zymomonasmobilis*, *Clostridium* species (including but not limited to *C. thermocellum*, *C. saccharobutylacetonicum*, *C. saccharobutylicum*, *C. Puniceum*, *C. beijernckii*, and *C. acetobutylicum*), *Moniliella pollinis*, *Moniliella megachiliensis*, *Lactobacillus* species, *Yersinia lipolytica*, *Aureobasidium* species, *Trichosporonoides* species, *Trigonopsis variabilis*, *Trichosporon* species, *Moniliella acetoabutans* species, *Typhula variabilis*, and *Candida*. Species of the class *Magnolias*, *Ustilaginomycetes*, *Pseudozyma tsukubaensis*, yeast species of the genera *Zygosaccharomyces*, *Debaliyces*, *Hansenula* and *Pichia*, and fungi of the genus *Torula*.See, for example, Philippidis, GP, 1996, “Cellulose bioconversion technology”, Handbook on Bioethanol: Production and Utilization, edited by Wyman, CE, Taylor & Francis, Washington, DC, pp. 179-212.

[0535] Fermentation media can be selected according to the needs of the host cell and / or the end user. Any necessary supplements other than, for example, carbon can be introduced alone at appropriate concentrations or as a mixture with another supplement or culture medium such as a complex nitrogen source.

[0536] Yeast fermentation

[0537] Fermentation methods using yeast are well known to those skilled in the art. In some embodiments, batch fermentation may be used according to the methods provided herein; in other embodiments, a continuous fermentation program may be used.

[0538] In some embodiments, batch fermentation methods can be used to produce the CRIP, CRIP-insecticide protein, or peptide-IA of the present invention. In short, batch fermentation refers to a type of fermentation carried out in a closed system, wherein the composition of the culture medium is determined at the start of fermentation and is not subject to human intervention during fermentation (i.e., the culture medium is inoculated with one or more yeast cells at the start of fermentation, and fermentation is allowed to continue without user interruption). Typically, in batch fermentation systems, the metabolite and biomass composition of the system continuously changes until fermentation stops. In batch culture, yeast cells progress through a lag phase to a high logarithmic growth phase and finally to a stationary phase, during which the growth rate decreases or stops. If left untreated, the yeast cells in the stationary phase will eventually die. In batch methods, yeast cells in the logarithmic phase are typically responsible for the bulk synthesis of the final product.

[0539] In some embodiments, fed-batch fermentation can be used to produce the CRIP, CRIP-insecticide protein, or peptide-IA of the present invention. In short, fed-batch fermentation is similar to a typical batch process (as described above); however, in a fed-batch process, the substrate is added incrementally as fermentation proceeds. Fed-batch fermentation is useful when catabolite inhibitors can suppress yeast cell metabolism, and when a limited amount of substrate in the culture medium is desired. Typically, substrate concentration measurements in fed-batch systems are estimated based on changes in measurable factors reflecting metabolism, such as pH, dissolved oxygen, exhaust gas (e.g., CO2) partial pressure, etc.

[0540] In some embodiments, a fed-batch fermentation process can be used to produce CRIP, CRIP-insecticide protein, or peptide-IA as follows: In a 10L bioreactor purged with a N2 / CO2 mixture, a production organism (e.g., modified yeast cells) is cultured using a 5L liquid medium containing 5 g / L potassium phosphate, 2.5 g / L ammonium chloride, 0.5 g / L magnesium sulfate, and 30 g / L corn steep liquor, with initial and first carbon source concentrations of 20 g / L. As the modified yeast cells grow and utilize the carbon source, an additional 70% of the carbon source mixture is then fed into the bioreactor at a rate approximately equal to the carbon source consumption. The bioreactor temperature is typically maintained at 30°C. Growth continues for approximately 24 hours or longer, and the heteropeptide reaches the desired concentration, e.g., a cell density between approximately 5 g / L and 10 g / L. After the culture period, the fermenter contents can be passed through a cell separation unit such as a centrifuge to remove cells and cell debris, and the fermentation broth can be transferred to a product separation unit. The separation of the heteropeptide can be performed using standard separation procedures well known in the art.

[0541] In some embodiments, continuous fermentation can be used to produce the CRIP, CRIP-insecticide protein, or peptide-IA of the present invention. In short, continuous fermentation refers to fermentation using an open system in which fermentation medium is continuously added to a bioreactor while approximately an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation typically maintains the culture at a high density, where yeast cells are primarily in the logarithmic growth phase. Generally, continuous fermentation methods are employed to maintain steady-state growth conditions, and the loss of yeast cells due to medium withdrawal should be balanced with the cell growth rate during fermentation.

[0542] In some embodiments, a continuous fermentation method can be used to produce CRIP, CRIP-insecticide protein, or peptide-IA as follows: A modified yeast strain can be cultured using bioreactor equipment and a culture medium composition, although the initial first and second carbon sources are approximately, for example, 30 g / L to 50 g / L. When the carbon sources are depleted, a feed medium of the same composition is continuously supplied at a rate of approximately 0.5 L / h to 1 L / h, and the liquid is withdrawn at the same rate. The concentration of the heterologous peptide in the bioreactor is typically kept constant along with the cell density. The temperature is typically maintained at 30°C, and the pH is typically maintained at approximately 4.5 using concentrated NaOH and HCl, as needed.

[0543] In some implementations, when producing CRIP, CRIP-insecticide protein, or peptide-IA, the bioreactor can be operated continuously, for example, for about a month, with daily or as-needed sampling to ensure consistency of the target compound concentration. In continuous mode, the fermenter contents are continuously removed as fresh feed medium is supplied. The effluent stream containing cells, culture medium, and heterologous peptides can then undergo a continuous product separation procedure, with or without cell and cell debris removal, and can be carried out using continuous separation methods well known in the art to separate the organic product from the peptide of interest.

[0544] In some embodiments, yeast cells operable to express CRIP, CRIP-insecticidal protein, or peptide-IA can be grown, for example, using a fed-batch process in an aerobic bioreactor. Briefly, the reactor is filled to approximately 20% to approximately 70% of its capacity with a culture medium containing a carbon source and other reagents. Temperature and pH are maintained using one or more chemicals described herein. Oxygen levels are maintained by intermittently purging air in conjunction with stirring.

[0545] For example, in some embodiments, the present invention provides a method for using a fed-batch process in an aerobic bioreactor, wherein the reactor is filled to approximately 20%; 21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%; 32%; 33%; 34%; 35%; 36%; 37%; 38%; 39%; 40%; 41%; 42%; 43%; 44%; 45%; 46%; 47%; 48%; 49%; 50%; 51%; 52%; 53%; 54%; 55%; 56%; 57%; 58%; 59%; 60%; 61%; 62%; 63%; 64%; 65%; 66%; 67%; 68%; 69%; or 70% of its capacity.

[0546] In some embodiments, the present invention provides a fed-batch fermentation method using an aerobic bioreactor to produce CRIP, CRIP-insecticide protein, or peptide-IA, wherein the culture medium is a rich medium. For example, in some embodiments, the carbon source may be glucose, sorbitol, or lactose.

[0547] In some implementations, the amount of glucose may be approximately 2 g / L; 3 g / L; 4 g / L; 5 g / L; 6 g / L; 7 g / L; 8 g / L; 9 g / L; 10 g / L; 11 g / L; 12 g / L; 13 g / L; 14 g / L; 15 g / L; 16 g / L; 17 g / L; 18 g / L; 19 g / L; 20 g / L; 21 g / L; 22 g / L; 23 g / L; 24 g / L; 25 g / L; 26 g / L; 27 g / L; 28 g / L; 29 g / L; or 30 g / L of culture medium.

[0548] In some implementations, the amount of sorbitol may be about 2 g / L; 3 g / L; 4 g / L; 5 ...

Claims

1. A combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA).

2. The combination according to claim 1, wherein the IA is a virus, bacterial toxin; fungal toxin; lectin; neem (Azadirachta indica) compound; boron compound; or a combination thereof.

3. The combination according to claim 2, wherein the virus is a particulate virus, the bacterial toxin is Bacillus thuringiensis (Bt) toxin protein or Photorhabdus toxin; and the fungal toxin is Beauveria toxin; Choose one of them: (1) The granuloviruses selected were: cotton brown-banded leafroller (Adoxophyes orana) granulovirus; yellow cutworm (Agrotis segetum) granulovirus; cabbage white butterfly (Artogeia rapae) granulovirus; European white butterfly (Pieris brassicae) granulovirus; spruce leafroller (Choristoneura fumiferana) granulovirus; western spruce leafroller (Choristoneura occidentalis) granulovirus; poplar leafroller (Clostera anachoreta) granulovirus; Clostera anastomosis granulovirus A; Clostera anastomosis granulovirus (Henan); Clostera anastomosis granulovirus B; rice leaf roller (Cnaphalocrocis) Granuloviruses of the following species: * *Cryptophlebialeucotreta* (Apple leafroller); *Cydia pomonella* (Codling moth); *Cydia pomonella* (Mexican isolate); *Diatraea saccharalis* (Sugarcane borer); *Epinotia aporema* (Night roller); *Erinnyis ello* (Cassava hawk moth); *Harrisina brillians* (Grape leaf spot moth); *Helicoverpa armigera* (Cotton bollworm); *Lacanobia oleracea* (Grass armyworm); *Mocis latipes* (South American hairy-legged armyworm); *Mythimna unipuncta* (Gross armyworm) A; *Pseudalatia unipuncta* (Gross armyworm); *Mythimna* (Gross armyworm) Granulovirus B (unipuncta); Granulovirus of Mythimna unipuncta; Granulovirus of Phthorimaea operculella; Granulovirus of Plodia interpunctella; Granulovirus of Plutella xylostella; Granulovirus of Spodopterafrugiperda; Granulovirus of Spodoptera litura; Granulovirus of Trichoplusia ni;Granulovirus LBIV-12 of *Trichoplusia ni* (Powdered Armyworm); Granulovirus of *Xestia c-nigrum* (Fighting Tiger); Unclassified β-baculovirus (Betabaculovirus); Granulovirus of *Achaea janata* (Flying Armyworm); Granulovirus of *Adoxophyes honmai* (Tea Leaf Roller); Granulovirus of *Agrotis exclamationis* (Police Armyworm); Granulovirus of *Amelia pallorana* (Jumping Mantis); Granulovirus of *Andraca bipunctata* (Tea Silkworm); Granulovirus of *Autographa gamma* (Striped Armyworm); Granulovirus of *Caloptilia theivora* (Tea Fine Moth); Granulovirus of *Choristoneura murinana* (European Spruce Leaf Roller); β-baculovirus of *Choristoneura viridis* (Oak Green Leaf Roller); Granulovirus of *Clostera anstomosis* (Mountain Leaf Roller); Granulovirus of *Cnephasia* (Omnivorous Cloud Roller). Granuloviruses from the genus *Longana*; *Estigmene acrea*; *Euxoa ochrogaster*; *Heliothis armigera*; *Hoplodrina ambigua*; *Hyphantria cunea*; *Natada nararia*; *Nephelodes emmedonia*; *Pandemis limitata*; *Peridorma morpontora*; *Pieris rapae*; *Plathypena scabra*; *Pseudaletia* β-baculovirus; *Scotogramma trifolii*; *Spodoptera* Androgea granulovirus; cotton leafworm (Spodoptera littoralis) granulovirus; Andean potato tuber moth (Tecia solanivora) granulovirus; or Mocis sp. granulovirus; (2) Bt toxin protein, wherein the Bt toxin protein is isolated from one or more of the following fermentation solids, spores or toxins: Bacillus thuringiensis Goldsteine ​​(Btk); Bacillus thuringiensis Tenebrionibacterium (Btt); Bacillus thuringiensis Israel (Bti); Bacillus thuringiensis Ayuzawa (Btk); Bacillus thuringiensis Ayuzawa / Pacific (Btk); Bacillus thuringiensis Allais (Btk); Bacillus thuringiensis Amagin (Btk); Bacillus thuringiensis Andalou (Btk); Bacillus thuringiensis Argentinus (Btk); Bacillus thuringiensis Asturiensis (Btk). Bacillus thuringiensis azorensis; Bacillus thuringiensis balearica; Bacillus thuringiensis berliner; Bacillus thuringiensis bolivia; Bacillus thuringiensis brasilensis; Bacillus thuringiensis Cameroon variant; Bacillus thuringiensis Canada variant; Bacillus thuringiensis chanpaisis variant; Bacillus thuringiensis Sinica variant; Bacillus thuringiensis Colmer variant; Bacillus thuringiensis coreanensis variant; Bacillus thuringiensis Darcota variant; Bacillus thuringiensis Darmstadt variant; Bacillus thuringiensis Songshu variant; Bacillus thuringiensis insecticidal variant; Bacillus thuringiensis insecticidal / subtoxic variant; Bacillus thuringiensis tussockworm variant; Bacillus thuringiensis fukuokaensis variant; Bacillus thuringiensis galechiae variant; Bacillus thuringiensis wax moth variant; Bacillus thuringiensis graciosensis variant; Bacillus thuringiensis Bacillus thuringiensis Guiyang variant; Bacillus thuringiensis higo variant; Bacillus thuringiensis Central China variant; Bacillus thuringiensis iberica variant; Bacillus thuringiensis Indian variant; Bacillus thuringiensis Israel / Limu variant; Bacillus thuringiensis Japan variant; Bacillus thuringiensis jegathesan variant; Bacillus thuringiensis Jinghong variant; Bacillus thuringiensis Kenya variant; Bacillus thuringiensis kim variant; Bacillus thuringiensis kumamtoensis variant; Bacillus thuringiensis kunthalanags3 variant; Bacillus thuringiensis kunthalaRX24 variant; Bacillus thuringiensis kunthalaRX27 variant; Bacillus thuringiensis kun Bacillus thalaRX28 variant; Bacillus thalamus Kyushu variant; Bacillus thalamus Leei variant; Bacillus thalamus Londrina variant; Bacillus thalamus Malayensis variant; Bacillus thalamus Medellin variant; Bacillus thalamus Mexico variant; Bacillus thalamus Mogi variant; Bacillus thalamus Monterey variant; Bacillus thalamus Morrison variant; Bacillus thalamus Muju variant; Bacillus thalamus Navarensis variant; Bacillus thalamus Neoleonensis variant; Bacillus thalamus Nigeriensis variant; Bacillus thalamus Novosibirsk variant; Bacillus thalamus Bacillus thuringiensis ostriniae; Bacillus thuringiensis oswaldocruzi; Bacillus thuringiensis pahangi; Bacillus thuringiensis Pakistani; Bacillus thuringiensis palmanyolensis; Bacillus thuringiensis pingluonsis; Bacillus thuringiensis pirenaica; Bacillus thuringiensis polonniensis; Bacillus thuringiensis pondicheriensis; Bacillus thuringiensis pulsiensis; Bacillus thuringiensis rongseni; Bacillus thuringiensis roskildiensis; Bacillus thuringiensis Santiago.Bacillus thuringiensis Seoul variant; Bacillus thuringiensis Shandong variant; Bacillus thuringiensis Silu variant; Bacillus thuringiensis sinensis variant; Bacillus thuringiensis sooncheon variant; Bacillus thuringiensis sudden-down variant; Bacillus thuringiensis sudden-down / Songshu variant; Bacillus thuringiensis subviral variant; Bacillus thuringiensis sumiyoshiensis variant; Bacillus thuringiensis sylvestriensis variant; Bacillus thuringiensis Thailandlandensis variant; Bacillus thuringiensis Thompson variant; Bacillus thuringiensis Bacillus thuringiensis variant; Bacillus thuringiensis (Bt) Limu variant; Bt. thuringiensis toguchini variant; Bt. thuringiensis Northeast variant; Bt. thuringiensis Duowo variant; Bt. thuringiensis Tomano variant; Bt. thuringiensis Vazensis variant; Bt. thuringiensis Wratislaviensis variant; Bt. thuringiensis Wuhan variant; Bt. thuringiensis Xiaguangiensis variant; Bt. thuringiensis Yosoo variant; Bt. thuringiensis Yunnan variant; Bt. thuringiensis Zhaodong variant; and Bt. thuringiensis Konkukian variant toxin; (3) Beauveria bassiana toxin, wherein the Beauveria bassiana toxin is selected from: Beauveria alba toxin; Beauveria amorpha toxin; Beauveria arenaria toxin; Beauveria asiatica toxin; Beauveria australis toxin; Beauveria bassiana toxin; Cordyceps bassiana toxin; Beauveria brongniartii toxin; Beauveria brumptii toxin; Beauveria caledonica toxin; Beauveria chiromensis toxin; Beauveria coccorum toxin; Beauveria cretacea toxin; Beauveria cylindrospora toxin; Beauveria delacroixii toxin; Beauveria densa toxin; Beauveria dependens toxin; Beauveria doryphorae toxin; Beauveria effusa toxin; Beauveria epigaea toxin; Beauveria felina toxin; Beauveria geodes toxin; Beauveria globulifera toxin; Beauveria heimii toxin; Beauveria hoplocheli toxin; Beauveria kipukae toxin; Beauveria laxa toxin; Beauveria malawiensis toxin; Beauveria medogensis toxin; Beauveria melolonthae toxin; Beauveria nubicola toxin; Beauveria oryzae toxin; Beauveria paradoxa toxin; Beauveria paranensis toxin; Beauveria parasitica toxin; Beauveria petelotii toxin; Beauveria pseudobassiana toxin; Beauveria rileyi toxin; Beauveria rubra toxin; Beauveria shiotae toxin; Beauveria sobolifera toxin; Beauveria spicata toxin; Beauveria stephanoderis toxin; Beauveria sulfurescens toxin; Beauveria sungii toxin; Beauveria tenella toxin; Beauveria tundrensis toxin;Beauveria velata toxin; Beauveria varroae toxin; Beauveria vermiconia toxin; Beauveria vexans toxin; Beauveria viannai toxin; or Beauveria virella toxin; (4) Lectins, wherein the lectins are selected from: snowdrop (Galanthus nivalis) lectin (GNA); elderberry (Sambucus nigra) lectin (SNA); Korean locust (Maackia amurensis)-II (MAL-II); Erythrina cristagalli lectin (ECL); castor bean (Ricinus communis) lectin-I (RCA); peanut lectin (PNA); wheat germ lectin (WGA); Griffonia simplicifolia-II (GSL-II); ConA; Lentil (Lens culinaris) lectin (LCA); Mannose-binding lectin (MBL); BanLec; Galactoglobulin; Common bean (Phaseolus vulgaris) leukocyte lectin (PHA-L); Common bean erythrocyte lectin (PHA-E); and Datura stramonium (Datura stramonium) lectin (DSL); (5) Azadirachtin compounds, wherein the azadirachtin compounds are selected from: azadirachtin dione; Azadiradionolide; Deacetylated gadurin; azadirachtin B; Desfuranoazadiradione; epoxide neemfruit dione; gadurin; Mahmooodin; Neemfruitin A; Neemfruitin B; azadirachtin lactone; azadirachtin; Nimolicinol; Ohchinin Acetate; Salannol; alpha-Nimolactone; beta-Nimolactone; 2',3'-dihydroazadirachtin; 3-deacetylatedazadirachtin; 6-deacetylated azadirachtin; 7-Acetyl-16,17-dehydro-16-hydroxyneotrichilenone; 7-Benzoylnimbocinol; 7 -Deacetyl-7-benzoylepinimolicinol; 7-Deacetyl-7-benzoylgadurin; 7-Deacetyl-17-epinimolicinol; 15-hydroxyepinimolicinol; 17-epinimolicinol; 17-epinimolicinol; 20,21,22,23-tetrahydro-23-oxoepinimolicinol; 22,23-Dihydronimocinol; or 28-deoxoepinimolicinol; (6) A boron compound, wherein the boron compound is selected from: borax; boric acid; disodium octaborate; sodium borate; sodium metaborate; sodium tetraborate decahydrate; boron oxide; boron carbide; boron nitride; boron tribromide; boron trichloride; or boron trifluoride; or (7) Photorhabdus toxins, wherein the photorhabdus toxins are selected from: Photorhabdus akhurstii toxin; Photorhabdus asymbiotica toxin; Photorhabdus asymbiotica subsp. asymbiotica toxin; Photorhabdus asymbiotica ATCC43949 toxin; Photorhabdus australis toxin; Photorhabdus australis DSM 17609 toxin; Photorhabdus bodei toxin; Photorhabdus caribbeanensis toxin; Photorhabdus cinerea toxin; Photorhabdus hainanensis toxin; Photorhabdus heterorhabditis toxin; Photorhabdus kayaii toxin; Photorhabdus khanii toxin; Photorhabdus khanii NC19 toxin; Photorhabdus khanii subsp. guanajuatensis toxin; Photorhabdus kleinii toxin; Photorhabdus laumondii toxin; Photorhabdus laumondii subsp. clarkei toxin; Photorhabdus laumondii subsp. laumondii toxin; Photorhabdus laumondii subsp. Laumondii TTO1 toxin; Photorhabdus luminescens BA1 toxin; Photorhabdus NBAII H75HRPL105 toxin; Photorhabdus NBAII HiPL101 toxin; Photorhabdus luminescens subsp. luminescens toxin; Photorhabdus luminescens subsp. ATCC29999 toxin; Photorhabdus luminescens subsp. Mexican subsp.(mexicana) toxin; Photorhabdus sonorensis subsp. toxin; Photorhabdus namnaonensis toxin; Photorhabdus noenieputensis toxin; Photorhabdus stackebrandtii toxin; Photorhabdus tasmaniensis toxin; Photorhabdus temperata toxin; Photorhabdus J3 toxin; Photorhabdus phorame subsp. toxin; Photorhabdus temperata subsp. temperata toxin; Photorhabdus M1021 toxin; Photorhabdus Meg1 toxin; Photorhabdus thracensis toxin; unclassified Photorhabdus toxins; Species of the genus Photorhabdus sp.) toxins; *Gymnospermum* species 3014 toxin; *Gymnospermum* species 3240 toxin; *Gymnospermum* species Az29 toxin; *Gymnospermum* species BS21 toxin; *Gymnospermum* species CbKj163 toxin; *Gymnospermum* species CRCIA-P01 toxin; *Gymnospermum* species ENY toxin; *Gymnospermum* species FL2122 toxin; *Gymnospermum* species FL480 toxin; *Gymnospermum* species FsIw96 toxin; *Gymnospermum* species GDd233 toxin. Species H3086 toxin; Species H3107 toxin; Species H3240 toxin; Species HB301 toxin; Species HB78 toxin; Species HB89 toxin; Species HIT toxin; Species HO1 toxin; Species HUG-39 toxin; Species IT toxin; Species JUN toxin; Species KcTs129 toxin; Species KJ13.1 TH toxins; KJ14.3 TH toxin for *Bacillus* species; KJ24.5 TH toxin for *Bacillus* species; KJ29.1 TH toxin for *Bacillus* species; KJ37.1 TH toxin for *Bacillus* species; KJ7.1 TH toxin for *Bacillus* species; KJ8.2 TH toxin for *Bacillus* species. KJ9.1 TH toxin from *Bacillus* species; KJ9.2 TH toxin from *Bacillus* species; KK1.3 TH toxin of *Gymnospermum* species; KK1.4 TH toxin of *Gymnospermum* species; KMD74 toxin from *Gymnospermum* species; KOH toxin from *Gymnospermum* species; MID10 toxin from *Gymnospermum* species; MOL toxin from *Gymnospermum* species; MSW_058 toxin of *Gymnospermum* species; MSW_079 toxin of *Gymnospermum* species; NK2.1 TH toxin of *Bacillus* species; NK2.5 TH toxin of *Bacillus* species; NnMt2h toxin from *Gymnospermum* species; NP1 toxin from *Gymnospermum* species; OH10 toxin from *Gymnospermum* species; OnIr40 toxin from *Gymnospermum* species; OnKn2 toxin from *Gymnospermum* species; PB10.1 TH toxin from *Gymnospermum* species; PB16.3TH toxin of *Bacillus* species; PB17.1 TH toxin of *Bacillus* species; PB17.3 TH toxin of *Bacillus* species; PB2.5 TH toxin of *Bacillus* species; PB22.4 TH toxin of *Bacillus* species; PB22.5 TH toxin of *Bacillus* species; PB32.1 TH toxin of *Bacillus* species; PB33.1 TH toxin of *Bacillus* species; PB33.4 TH toxin of *Bacillus* species; PB37.4 TH toxin of *Gymnospermum* species; PB39.2 TH toxin of *Bacillus* species; PB4.5TH toxin from species of the genus *Gymnospermum*; PB41.4 TH toxin of *Bacillus* species; PB45.5 TH toxin of *Bacillus* species; PB47.1 TH toxin of *Bacillus* species; PB47.3 TH toxin of *Bacillus* species; PB5.1 TH toxin of *Gymnospermum* species; PB5.4 TH toxin of *Bacillus* species; PB50.4 TH toxin of *Bacillus* species; PB51.4 TH toxin of *Bacillus* species; PB52.2 TH toxin of *Bacillus* species; PB54.4 TH toxin of *Bacillus* species; PB58.2 TH toxin of *Bacillus* species; PB58.4 TH toxin of *Bacillus* species; PB58.5 TH toxin of *Bacillus* species; PB59.2 TH toxin of *Bacillus* species; PB6.5TH toxin from species of the genus *Gymnospermum*; PB67.2 TH toxin of *Bacillus* species; PB67.4 TH toxin of *Bacillus* species; PB68.1 TH toxin of *Bacillus* species; PB7.5 TH toxin of *Bacillus* species; PB76.1 TH toxin of *Bacillus* species; PB76.4 TH toxin of *Bacillus* species; PB76.5 TH toxin of *Bacillus* species; PB78.2 TH toxin of *Bacillus* species; PB80.3 TH toxin of *Bacillus* species; PB80.4 TH toxin of *Gymnospermum* species; Pjun toxin of *Gymnospermum* species; RW14-46 toxin of *Gymnospermum* species; S10-54 toxin of *Gymnospermum* species; S12-55 toxin of *Gymnospermum* species; S14-60 toxin of *Gymnospermum* species; S15-56 toxin of *Gymnospermum* species; S5P8-50 toxin of *Gymnospermum* species; S7-51 toxin of *Gymnospermum* species; S8-52 toxin of *Gymnospermum* species; S9-53 toxin of *Gymnospermum* species; SJ2 toxin of *Gymnospermum* species; SN259 toxin of *Gymnospermum* species; SP1.5 TH toxin of *Gymnospermum* species; *Phyllostachys* species SP16.4 TH toxin; *Phyllostachys* species SP21.5 TH toxin; *Phyllostachys* species SP3.4 TH toxin; *Phyllostachys* species SP4.5 TH toxin; *Phyllostachys* species SP7.3 TH toxin; *Phyllostachys* species TyKb140 toxin; *Phyllostachys* species UK76 toxin; *Phyllostachys* species VMG toxin; *Phyllostachys* species WA21C toxin; *Phyllostachys* species WkSs43 toxin; *Phyllostachys* species Wx13 toxin; *Phyllostachys* species X4 toxin; *Phyllostachys* species YNb90 toxin; *Phyllostachys* species ZM toxin; or one or more proteins derived from the luminescent *Phyllostachys* toxin complex (Tca).

4. The combination according to claim 3, wherein the IA is: (a) Codling moth granulovirus (CpGV), optionally wherein the codling moth granulovirus (CpGV) is the codling moth granulovirus isolate V22 virus; (b) Bt toxin protein comprising one or more fermentation solids, spores or toxins isolated from Bacillus thuringiensis Goldsdee (Btk), Bacillus thuringiensis Tenebrionibacterium (Btt) or Bacillus thuringiensis Israel (Bti). (c) Luminescent rod-shaped bacterial toxin complex (Tca); Beauveria bassiana toxin; (d) Snowdrop lectin (GNA); (e) Neem compounds; or (f) Boron compounds; Choose one of them: (i) The Bt toxin protein is isolated from one or more of the following fermentation solids, spores or toxins: Bacillus thuringiensis Goldsde subsp. (Btk) strain EVB-113-19; Bacillus thuringiensis Beta thuringiensis subsp. (Btt) strain NB-176; Bacillus thuringiensis Israel subsp. (Bti) strain BMP144, or has a Bt toxin protein as shown in any of SEQ ID NO:412-587; (ii) The luminescent rod-shaped bacterial toxin complex (Tca) comprises TcaA protein (SEQ ID NO:616), TcaB protein (SEQ ID NO:617), TcaC protein (SEQ ID NO:618), and TcaZ protein (SEQ ID NO:619); (iii) The beauveria bassiana toxin described herein has the chemical formula C. 45 H 57 Beauveria bassiana toxin of N3O9, chemical formula C 46 H 59 Beauveria bassiana A toxin of N3O9; chemical formula C 47 H 61 Beauveria bassiana B toxin of N3O9, or beauveria bassiana toxin isolated from spores of Beauveria bassiana strain ANT-03. (iv) The snowdrop lectin (GNA) has the amino acid sequence shown in SEQ ID NO:35; (v) The neem compound is azadirachtin; or (vi) The boron compound mentioned is boric acid.

5. The combination according to any one of claims 1-4, wherein the CRIP is U1-funnel-web spider venom-Ta1b peptide; U1-funnel-web spider venom-Ta1b variant polypeptide (TVP); anemone venom; Av3 variant polypeptide (AVP); Brazilian wandering spider (Phoneutria) venom; or Atracotoxin (ACTX).

6. The combination according to claim 5, wherein the CRIP is: (i) Wild-type U1-funnel web spider toxin-Ta1b peptide having the amino acid sequence shown in SEQ ID NO:1; (ii) A variant polypeptide of U1-funnel-web spider toxin-Ta1b, which, relative to the wild-type sequence of U1-funnel-web spider toxin-Ta1b as shown in SEQ ID NO:1, contains an R9Q amino acid substitution. (iii) U1-funnel web spider toxin-Ta1b variant polypeptide having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NO:2-15, 49-53, 621-622, 624-628, 631-640, 642-651 or 653-654; (iv) Wild-type Av3 toxin having the amino acid sequence shown in SEQ ID NO:44; (v) Av3 variant polypeptide (AVP), which contains R1K substitution and C-terminal deletion relative to the wild-type sequence of Av3 as shown in SEQ ID NO:44; (vi)Γ-CNTX-Pn1a, which has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:65; (vii)ACTX, which has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NO:60-64 or 594.

7. The combination according to any one of claims 1-6, wherein the combination is formulated into separate compositions or single compositions, wherein each of the separate compositions or single compositions further comprises at least one excipient; The separate compositions or the single compositions are formulated into powders, granules, pellets, sprays, emulsions, colloids, solutions, or combinations thereof; and The separate compositions are formulated using the same excipient or different excipients.

8. The combination according to claim 1, wherein the combination comprises: (1) an insecticide (IA) protein or virus fully expressed in a plant; and a cysteine-rich insecticidal peptide (CRIP) formulated into a composition comprising at least one excipient; (2) a cysteine-rich insecticidal peptide (CRIP) fully expressed in a plant; and an insecticide (IA) formulated into a composition comprising at least one excipient; or (3) an insecticide (IA) protein or virus and a cysteine-rich insecticidal peptide (CRIP) both fully expressed in the same plant.

9. A method for combating, preventing, or suppressing pests, the method comprising providing: an insecticidally effective amount of a composition comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA); wherein the IA is selected from any one of claims 2-4; and the CRIP is selected from any one of claims 5-6; and then applying the combination to: a pest, a location of the pest, a food source of the pest, a habitat of the pest, or a breeding ground of the pest; a pest-vulnerable plant, seed, part of a plant, a location of a plant, or the environment of a plant; a pest-vulnerable animal, a location of an animal, or the environment of an animal; or a combination thereof.

10. The method according to claim 9, wherein: (a) wherein the composition is formulated into separate compositions or a single composition; wherein each of the separate compositions or the single composition further comprises at least one excipient; Optionally, the separate composition or the single composition is formulated as a powder, granule, pellet, granule, spray, emulsion, colloid, solution or combination thereof; Optionally, the separate compositions are formulated using the same excipient or different excipients; Optionally, the separate compositions are applied separately, sequentially, simultaneously, in parallel, or at staggered times; or (b) The composition thereof comprises: (1) an insecticide (IA) protein or virus fully expressed in a plant; and a cysteine-rich insecticide peptide (CRIP) formulated into a composition comprising at least one excipient; (2) a cysteine-rich insecticide peptide (CRIP) fully expressed in a plant; and an insecticide (IA) formulated into a composition comprising at least one excipient; or (3) an insecticide (IA) protein or virus and a cysteine-rich insecticide peptide (CRIP) both fully expressed in the same plant.

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