TXP40 insecticidal proteins and compositions, encoding polynucleotides and nucleic acid constructs, and related methods

Abstract

The present invention relates to Txp40 protein variants with insecticidal activity, compositions comprising said Txp40 protein variants, polynucleotides and nucleic acid constructs for their expression, plants comprising said polypeptides, polynucleotides and / or nucleic acid constructs and related methods.

Description

[0001] TXP40 INSECTICIDAL PROTEINS AND COMPOSITIONS, ENCODING POLYNUCLEOTIDES AND NUCLEIC ACID CONSTRUCTS, AND RELATED METHODS

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention generally relates to biopesticides. Particularly, the invention is related to biological insecticides, more particularly insecticidal proteins, and methods related to their use.

[0004] BACKGROUND

[0005] Within the agricultural field, there has been a growing concern in recent years about the continuing use of traditional chemistry-based pesticides, particularly related to their environmental impact. Correspondingly, there has been a trend lately for developing biological pesticides, that is, pesticides derived from natural sources instead of synthetic ones, since they are usually more environment-friendly.

[0006] It is known that chemistry-based pesticides present the following disadvantages:

[0007] • Their prolonged use can trigger resistance;

[0008] • They can have undesired environmental effects;

[0009] • They are generally non-specific, harming non-target animals and plants;

[0010] • They have residual effects in the plants that can persist over time.

[0011] In this sense, the use of biological insecticides, based on the metabolic products of microorganisms have the following advantages when compared to chemistry-based pesticides:

[0012] • They can be targeted towards a particular species or genus, presenting no harm to other organisms;

[0013] • They are less aggressive towards the environment, usually being non-toxic to higher organisms and plants.

[0014] Biological insecticides are known in the art, with commercial products already being available in the market. These commercial products are typically based on Bacillus thuringiensis, which exerts an insecticidal effect due to the production of a family of protein toxins referred to as Cry proteins, and they are widely used around the world. However, some insect pests have shown to be resistant to Cry proteins, which is why there is a constant need for further developing biological insecticides to control a wider range of insect pests.

[0015] The proteins belonging to the so-called Txp40 family (Txp40 proteins) are insecticidal proteins produced by certain microorganisms of Photorhabdus spp. and Xenorhabdus spp. In particular, the use of Txp40 proteins and variants thereof has been described in the prior art.

[0016] The following are some examples of these Txp40 proteins and their use as insecticides.

[0017] Mathur C. et al. studies the level of toxicity of a Txp40 protein on Galleria mellonella, wherein the Txp40 is obtained from Photorhabdus luminescens

[0018] Brown S.E. et al. study a gene encoding a Txp40 protein and its level of preservation among Xenorhabdus and Photorhabdus. Furthermore, they analyze the insecticidal activity of the Txp40 protein primarily by injection into the hemocoel of insects, particularly in Spododoptera sp.

[0019] Shankhu P.Y. et al. study a Txp40 protein and its insecticidal applicability against larvae from Helicoperva armigera, Spodoptera litura and Spodoptera exigua. In particular, they use a Txp40 protein obtained from Photorhabdus akhurstii

[0020] Kinkar O.U. et al. characterize a Txp40 protein obtained from Xenorhapdus nematophila, and study the toxic effect of said protein on larvae from Galleria mellonella.

[0021] Park J.M. et al. disclose a Txp40 protein obtained from Xenorhabdus nematophila and its insecticidal activity against larvae of Plutella xylostella.

[0022] Txp40 proteins and variants thereof have also been disclosed in several patent applications, primarily for their use as a pest control, as described in the following documents:

[0023] Patent application US 2004 / 0055036 A1 discloses genes from X. nematophila and P. luminescens that encode proteins with toxic effect, including, among others, a Txp40 protein. Additionally, it describes a method for transforming microorganisms and plants with genes encoding these proteins. Patent application US 2022 / 0324920 A1 discloses the use of a Txp40 protein and variants thereof for the control of Spodoptera pests. In particular, said Txp40 protein is obtained from Photorhabdus luminescens.

[0024] However, the continuous adaptation of pests to their environment pushes a constant need to find new variants of proteins that can remain effective, and functional under certain conditions.

[0025] SUMMARY OF THE INVENTION

[0026] In this sense, the inventors of the present invention have developed variants of a wild type Txp40 protein having the amino acid sequence as set forth in SEQ ID NO: 1 , that exhibit an improved solubility and thermal stability in comparison to the wild type Txp40 protein having the amino acid sequence as set forth in SEQ ID NO: 1 .

[0027] Thus, it is a first aspect of the invention to provide a Txp40 protein variant with insecticidal activity comprising the residues E295, R311 , H331 , Y332.

[0028] In an embodiment, the Txp40 protein variant of further comprises an amino acid sequence as set forth in any one of SEQ ID NO: 2-23.

[0029] In another embodiment, the Txp40 protein variant further comprises a polypeptide as set forth in SEQ ID NO: 24 bound to the N-terminus.

[0030] It is a second aspect of the invention to provide an insecticide composition comprising the Txp40 protein variant of the first aspect, and at least one other agronomically acceptable compound.

[0031] In an embodiment, the insecticide composition comprises

[0032] • an insecticidal agent comprising a Txp40 protein variant of the first aspect of the present invention;

[0033] • a pH buffer,

[0034] • a chelating agent,

[0035] • an emulsifier,

[0036] • an additive,

[0037] • a preservative, and a solvent.

[0038] Preferably, the insecticide composition comprises

[0039] • between 0.1-200 mg / L of an insecticidal agent comprising a Txp40 protein variant of the first aspect of the present invention;

[0040] • between 1 -5 mg / ml of a pH buffer,

[0041] • between 0.1 -0.5 mg / ml of a chelating agent,

[0042] • between 0.01 -10 mg / ml of an emulsifier,

[0043] • between 1 -100 mg / ml of an additive,

[0044] • between 0.01 -1 mg / ml of a preservative, and

[0045] • q.s.f. of a solvent.

[0046] It is a third aspect of the present invention to provide a method for controlling an insect pest comprising the steps of a) providing a Txp40 protein variant of the first aspect or a composition of the second aspect, and b) applying said Txp40 protein variant or composition to a plant or plant part in need thereof.

[0047] In an embodiment, the step b) of the method of the third aspect comprises a plant or plant part selected from the genera Solanum spp., Zea spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., and Allium spp.

[0048] In an embodiment, the method of the third aspect comprises an insect pest selected from Spodoptera spp., Tuta spp., Trialeurodes spp., Anticarsia spp., Helicoperva spp., Spododoptera spp., Tetranychus spp., Bemisia spp., Aphis spp., Myzus spp., Rachiplusia spp., Alphitobus spp. Anthonomus spp., Thirps spp., Franliniella spp., and Dalbulus spp.

[0049] It is a fourth aspect of the present invention to provide a polynucleotide that encodes a Txp40 protein variant of the first aspect, wherein the polynucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 49-70. In an embodiment, the polynucleotide further comprises a nucleotide sequence as set forth in SEQ ID NO: 71 bound to the 5’-end of the polynucleotide.

[0050] It is a fifth aspect of the present invention to provide a nucleic acid construct comprising a polynucleotide of the fourth aspect of the present invention.

[0051] In an embodiment, the nucleic acid construct further comprises a plasmid, expression vector o an expression cassette which contains the polynucleotide of the fourth aspect of the present invention.

[0052] It is a sixth aspect of the present invention to provide a transgenic plant transformed with a nucleic acid construct of the fifth aspect of the present invention, wherein the nucleic acid construct comprises a polynucleotide as set forth in any one of SEQ ID NO: 49-70.

[0053] In an embodiment, the transgenic plant is selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., and Allium spp.

[0054] It is a seventh aspect of the invention to provide a method for controlling an insect pest comprising the steps of a) providing a transgenic plant of the sixth aspect of the present invention, and b) growing the transgenic plant of point a) in conditions that results in the expression of the Txp40 protein variant of the first aspect orthe polynucleotide of the fourth aspect.

[0055] BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows an SDS-PAGE of the soluble (s: soluble) and the insoluble fractions (p: pellet) of some of the variants’ compositions used (Txp40-Var1 , Txp40-Var2, Txp40-Var3, Txp40-Var4, Txp40). The arrows show soluble proteins.

[0057] Figure 2 shows Spodoptera frugiperda control percentage. The insecticidal activity of the different Txp40 mutants was evaluated in in vitro assays with L1 larvae of Spodoptera frugiperda. The increase in control percentage was the result of a combination of reduction in larva size, decreased mobility, arrest at the stage of development, and increased mortality.

[0058] Figure 3 shows an SDS-PAGE analysis of Txp40 and Txp40-Var4 (VAR4)proteins stored for two weeks at 40 and 54 °C. TO: initial time point; 1w: one week of storage; 2w: two weeks of storage; MM: molecular marker. All samples were prepared with the same formulation and normalized based on their optical density. The arrows indicate the proteins remaining after two weeks of storage at 54 °C.

[0059] Figure 4 shows the results of Control efficacy (%) against Dalbulus maidis at 24, 48, and 72 hours for the formulation variants Txp40-Var2, Txp40-Var4, and Txp40-Var7, each evaluated at two doses.

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] Examples given herein shall be interpreted only as exemplary embodiments of the different aspects, objects, and / or methods of the present invention and are not intended to limit the scope of the present invention in any way.

[0062] Any technical terminology used herein shall be understood by the common definition utilized in the art and / or by those skilled in the art, unless otherwise explicitly stated or inferred by context.

[0063] Each and every embodiment of any aspect, object, and / or method of the present invention resulting from the combination of particular embodiments of said aspect, object and / or method described herein are to be considered as falling within the scope of the present invention.

[0064] The present invention is related to variants of a wild type Txp40 protein obtained from Photorhabdus luminescens. The wild type Txp40 protein which the variants of the invention are based on has the amino acid sequence set forth in SEQ ID NO: 1.

[0065] The term “variant” of a protein as used herein refers to a polypeptide that is the result of a variation in the amino acid sequence of the corresponding protein. Said variation may include at least one substitution, insertion, deletion, fusion, or combinations thereof anywhere throughout the sequence of the protein, preferably up to 50 substitutions, insertions, deletions, fusions, or combinations thereof. Alternatively, the term may also comprise a variation in the amino acid sequence such that it has an amino acid sequence with at least 80% sequence identity to the Txp40 protein. The variants of the present invention are variants of the wild type Txp40 protein with the amino acid sequence set forth in SEQ ID NO: 1 , and are also referred to throughout this description as “Txp40 protein variants” or “Txp40 variants”.

[0066] The term “protein” as used herein refers to a polypeptide or an amino acid sequence comprising at least 2 or more amino acids. It may or may not refer to a particular polypeptide or amino acid with a biological activity or function, for example, an enzymatic or structural function.

[0067] The term “insertion” as used herein refers to a peptide or polypeptide that is inserted, i.e., introduced anywhere in a reference amino acid sequence, except in the N-terminal or C-terminal ends.

[0068] The term “deletion” as used herein refers to a peptide or polypeptide that is deleted, i.e., removed from anywhere in the amino acid sequence.

[0069] As used herein, the term “fusion” refers to a peptide or polypeptide that is bound to a protein, polypeptide, or amino acid sequence by either the N-terminal end or the C- terminal end of said protein, polypeptide, or amino acid sequence. Accordingly, a “fusion peptide” is a polypeptide that is fused or bound to a Txp40 protein variant, preferably by either the N-terminal end or the C-terminal end.

[0070] As used herein, the term “stability” and derivatives thereof when referring to a protein, refers to the ability of said protein to keep its conformation, that is, to have a reduced number or lack of alterations in at least one of its primary, secondary, tertiary, or quaternary structures. Said alterations may be result of physical factors, e.g., temperature, chemical, e.g., pH or salinity, or biological, e.g., presence of enzymes. Thus, in particular, the term “thermal stability” refers to the alterations induced by temperature. Meaning that a variant presents an “improved thermal stability” refers to a protein or variant of a protein that presents fewer alterations in its primary, secondary, tertiary, or quaternary structures induced by temperature.

[0071] As used herein, the term “solubility” and derivatives thereof when referring to a protein, refers to the ability of said protein to dissolve in a solvent to form a homogeneous solution at specific conditions of, for example, temperature, salinity, osmolarity and pressure. The solubility of the protein may depend on the chemical properties of the solvent itself, such as its polarity, or on the presence of other components as co-solvent, and may depend on the structure of the Txp40 protein variant structure too. The solubility of a protein can be measured by the concentration of the protein that can be dissolved in a particular solvent or solvent system, or by the biomass yield of protein obtained from a cell culture, i.e., the amount of soluble protein that can be retrieved or purified from a cell culture, either in an absolute or relative values.

[0072] The term “insecticide”, “insecticidal” and variants thereof, as used herein when referring to a substance (e.g. a protein) or a combination of substances (e.g. a composition or formulation) refers to the ability of said substance or combination of substances to inhibit the ability of insect pests to survive, grow, feed, and / or reproduce, or to limit insect- related damage or loss in plants or plant parts. Preferably, these effects are the result of a toxic effect. The term “insecticide” may or may not mean killing insects, although it preferably means killing the insects. Said insects may be in any stage of their life cycle, for example neonate, larva, pupa (if corresponding), or adult.

[0073] The terms “from X to Y”, or “between X and Y” or “from / between X-Y” and similar expressions should be interpreted to include X and Y, unless the context indicate otherwise.

[0074] Throughout the present specification, when referring to a substitution in an amino acid sequence, this will be indicated using the nomenclature “aXp”, where: a is the amino acid located at the position X of an amino acid sequence of reference,

[0075] - X is the position that the amino acid a occupies in the amino acid of the sequence of reference, and p is the amino acid located at the corresponding position to a in the amino acid sequence of interest (i.e., the amino acid which replaces a in the sequence obtained after the substitution), when aligning the amino acid sequences of reference and interest.

[0076] Preferably, the “aXp” nomenclature uses the single-letter code for the amino acids. Although the substitution may also be indicated with a three-letter code.

[0077] For example, when a protein B has a substitution “Q14P” when compared to a protein A, it means that, when aligning the amino acid sequences of proteins A and B, there is a substitution in the position 14 of protein A, wherein the amino acid glutamine (Q) of protein A has been substituted with a proline (P) in the protein B.

[0078] Throughout the present specification, when indicating an insertion in a sequence, this will always be related to a reference sequence. The insertion will be indicated as “Y at 6X”, where

[0079] - Y is the peptide or polypeptide inserted in the amino acid sequence of reference and present in the amino acid sequence of interest,

[0080] 6 is the amino acid located at the position X of an amino acid sequence of reference immediately before the insertion site, and

[0081] - X is the position that the amino acid 6 occupies in the amino acid of the sequence of reference, when aligning the amino acid sequences of reference and interest.

[0082] In an embodiment, the peptide or polypeptide Y inserted in the amino acid sequence comprises at least one amino acid, preferably, between 1 and 50 amino acids.

[0083] Throughout the present specification, when indicating a deletion in a sequence, this will always be related to a reference sequence. The deletion will be indicated as “Y at 6X”, where

[0084] - Y is the peptide or polypeptide deleted from the amino acid sequence of reference and therefore is absent in the amino acid sequence of interest,

[0085] 6 is the amino acid located at the position X of the amino acid sequence of reference immediately before the deletion site, and

[0086] - X is the position that the amino acid 6 occupies in the amino acid of the sequence of reference, when aligning the amino acid sequences of reference and interest.

[0087] In an embodiment, the peptide or polypeptide Y deleted from the amino acid sequence comprises at least one amino acid, preferably, between 1 and 50 amino acids. Throughout the specification, amino acids can be referred to by their name, single-letter code, or triple-letter code as shown in the following table.

[0088]

[0089] Table 1 . Amino acid single-letter code and three-letter code as used herein.

[0090] When a particular position in an amino acid sequence contains an amino acid that has multiple potential variations, the amino acid in question will be denoted by parentheses. Within these parentheses, each of the possible variants forthat amino acid will be listed and separated by commas (,). The deletion of an amino acid in the sequence will be indicated by a dash (-). It is possible for a sequence to include one or multiple positions where an amino acid has multiple potential variants.

[0091] As an example, and without intent of limiting the scope of the present invention, when describing an amino acid sequence as the following expression

[0092] P(P, C)P(D, E, -)P, it is to be understood that the amino acid in the second position of the sequence can be Proline (P) or Cysteine (C), while the amino acid in the fourth position of the sequence can be Aspartate (D), Glutamate (E), or a deletion (-). Thus, the amino acid sequence represented by the expression above can be any one of the following: PPPDP; PPPEP; PPPP; PCPDP; PCPEP; PCPP.

[0093] As used herein, the terms "general" or "generic," when referring to an amino acid sequence, indicate a form of that amino acid sequence that is defined to encompass all the amino acid sequences found within a specific list or group. This is done under the previously mentioned rules for describing sequences.

[0094] The inventors designed variants of the Txp40 protein with greater solubility and thermal stability, without losing its insecticidal effect which results in a safer insecticide (see Examples 4 and 5). This represents a clear advantage over the Txp40 protein that is not obvious nor readily available for a person skilled in the art.

[0095] Thus, a first aspect of the present invention is to provide a Txp40 protein variant with insecticidal activity comprising the residues E295, R311 , H331 , and Y332.

[0096] In an embodiment, said Txp40 protein variant with insecticidal activity comprises an amino acid sequence as set forth in any one of SEQ ID NO: 2-23. More preferably, the Txp40 variant with insecticidal activity comprises an amino acid sequence as set forth in any one of SEQ ID NO: 25-47.

[0097] The Txp40 protein variants of the present invention have highly conserved residues from toxins of the Txp40 type, particularly from the genera Photorhabdus and Xenorhabdus. These highly conserved residues are E295, R311 , H331 , and Y332, and have been identified as highly important for the activity of the Txp40 protein variants, belonging to the C-terminus of the protein. The inventors of the present invention point out that there is no description or suggestion in the prior art on the Txp40 protein’s mechanism of action, but through structural similarities between the C-terminal domain of Txp40 and mRNA-degrading toxins with similar folding, they concluded that these amino acid positions are conserved residues of the toxins which present insecticidal activity.

[0098] The lack of description in the prior art makes this a surprising characteristic of the Txp40 protein and variants thereof. Thus, the Txp40 protein variant of the present invention comprises residues E295, R311 , H331 , and Y332.

[0099] In an embodiment, the Txp40 protein variant of the invention comprises at least one substitution when compared to the Txp40 protein as set forth in SEQ ID NO: 1 in one or more of the following positions; 3, 6, 7, 9, 10, 14, 17, 21 , 22, 23, 25, 26, 29, 30, 31 , 34, 37, 40, 41 , 43, 44, 45, 46, 47, 49, 50, 52, 53, 54, 55, 60, 61 , 63, 64, 69, 70, 73, 76, 77, 79, 80, 81 , 87, 89, 91 , 93, 95, 97, 100, 104, 107, 108, 111 , 112, 114, 118, 119, 120, 122, 124, 126, 127, 129, 139, 140, 141 , 142, 143, 145, 147, 150, 152, 153, 158, 160, 167,

[0100] 169, 171 , 174, 177, 179, 186, 188, 189, 190, 191 , 192, 194, 198, 199, 200, 204, 206,

[0101] 209, 210, 211 , 213, 215, 221 , 225, 230, 231 , 233, 235, 236, 240, 241 , 243, 244, 245,

[0102] 247, 248, 250, 251 , 252, 254, 256, 260, 267, 268, 270, 275, 284, 285, 286, 288, 289,

[0103] 291 , 294, 298, 299, 300, 304, 307, 308, 309, 310, 315, 316, 317, 319, 320, 321 , 322, 324, 326, 327, 330, 333. In another embodiment, the Txp40 protein variant of the present invention comprises the residues E295, R311 , H331 , and Y332 of SEQ ID NO:1 , and an N-terminal fusion peptide as set forth in SEQ ID NO: 24. Thus, the full sequence of a Txp40 protein variant that is bound to an N-terminal fusion peptide as set forth in SEQ ID NO: 24 comprises the amino acids E318, R334, H354 and Y355, which corresponds to residues of E295, R311 , H331 , and Y332 of SEQ ID NO:1 , which have their positions modified by the incorporation of the 23 amino acids in the N-terminal.

[0104] In an embodiment, the Txp40 protein variant comprises an N-terminal fusion peptide as set forth in SEQ ID NO: 24, and at least one substitution when compared to the protein as set forth in SEQ ID NO: 25 in one or more of the following positions; 26, 29, 30, 32, 33, 37, 40, 44, 45, 46, 48, 49, 52, 53, 54, 57, 60, 63, 64, 66, 67, 68, 69, 70, 72, 73, 75, 76, 77, 78, 83, 84, 86, 87, 92, 93, 96, 99, 100, 102, 103, 104, 110, 112, 114, 116, 118, 120, 123, 127, 130, 131 , 134, 135, 137, 141 , 142, 143, 145, 147, 149, 150, 152, 162,

[0105] 163, 164, 165, 166, 168, 170, 173, 175, 176, 181 , 183, 190, 192, 194, 197, 200, 202,

[0106] 209, 211 , 212, 213, 214, 215, 217, 221 , 222, 223, 227, 229, 232, 233, 234, 236, 238,

[0107] 244, 248, 253, 254, 256, 258, 259, 263, 264, 266, 267, 268, 270, 271 , 273, 274, 275,

[0108] 277, 279, 283, 290, 291 , 293, 298, 307, 308, 309, 311 , 312, 314, 317, 321 , 322, 323,

[0109] 327, 330, 331 , 332, 333, 338, 339, 340, 342, 343, 344, 345, 347, 349, 350, 353, 356.

[0110] Unless otherwise specified or inferred by context, the term “-Tag” when referring to or associated with a Txp40 protein or variant thereof, refers to a fusion of said protein or variant thereof with a polypeptide as set forth in SEQ ID NO: 24 (MGSSHHHHHHSSGLVPRGSHMAS) bound to the N-terminus.

[0111] For example, and without intent to limit the scope of the present invention, the protein as set forth in SEQ ID NO: 25, also referred as Txp40-Tag, corresponds to a Txp40 protein as set forth in SEQ ID NO: 1 with a polypeptide as set forth in SEQ ID NO: 24 (MGSSHHHHHHSSGLVPRGSHMAS) bound to the N-terminus.

[0112] In an embodiment, the Txp40 protein variant comprises at least one insertion in one or more of the following positions when compared to the Txp40 protein: 24, 25, 33, 34, 59, 60, 258, 259, 260, 261 , 322, 323. Preferably, said insertion comprises the introduction of between 1 and 24 residues.

[0113] In an embodiment the Txp40 protein or Txp40 protein variant further comprises a fusion, wherein said fusion is a polypeptide bound to the N-terminus of an amino acid sequence, preferably, the polypeptide is bound to the N-terminus of an amino acid sequence as set forth in any one of SEQ ID NO: 1-23.

[0114] In an embodiment, the fusion is with a polypeptide that comprises a His-tag (HHHHHH), or a thrombin cleavage site (LVPRGS), or both. Preferably, the fusion at the N-terminus is an amino acid sequence as set forth in SEQ ID NO: 24.

[0115] The fusion peptide is a polypeptide that increases the production and / or purification of the polypeptide that is fused to. For example, in order to optimize the production and purification in E. Coli BL21 (DE3) strain, the cells are transformed with a vector that codes for a Txp40-Tag protein with an amino acid sequence as set forth in SEQ ID NO: 25. In this sense, the amino acid sequence Txp40-Tag with the amino acid sequence as set forth in SEQ ID NO: 25 comprises:

[0116] • an insecticide Txp40 protein with an amino acid sequence as set forth in SEQ ID NO: 1 , and

[0117] • a fusion peptide at the N-terminal end with an amino acid sequence as set forth in SEQ ID NO: 24.

[0118] Likewise, the amino acid sequences as set forth in any one SEQ ID NO: 26-47 disclosed herein correspond to each one of the proteins with amino acid sequences as set forth in SEQ ID NO: 2-23 fused to the amino acid sequence as set forth in SEQ ID NO: 24 as a fusion at the N-terminal end.

[0119] In an embodiment, the Txp40 protein variants of the present invention have an amino acid sequences with at least 80% sequence identity to the Txp40-Tag protein with an amino acid sequence as set forth in SEQ ID NO: 25. Preferably, between 80%-100% of sequence identity to the Txp40-Tag protein with an amino acid sequence as set forth in SEQ ID NO: 25, more preferably, between 90%-100% of sequence identity to the Txp40- Tag protein with an amino acid sequence as set forth in SEQ ID NO: 25. More preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity to the Txp40-Tag protein with an amino acid sequence as set forth in SEQ ID NO: 25.

[0120] Exemplary embodiments of these Txp40-Tag protein variants are summarized in the following table:

[0121]

[0122] In an embodiment, the solubility can be measured by the concentration of the Txp40 protein variants of the present invention. In an embodiment, the Txp40 protein variants have a solubility of 0.5-500 mg / mL. More preferably, a solubility of 1-100 mg / mL.

[0123] In another embodiment, the solubility can be measured by determining the fraction of Txp40 protein variant that is present in the soluble fraction of purified protein. Said soluble fraction may correspond to at least 1%, preferably at least 10%, more preferably at least 70% of the purified Txp40 protein variant.

[0124] A person skilled in the art would be able to produce the insecticidal Txp40 variant of the present invention in any suitable microorganism, e.g., bacteria like Escherichia spp., Staphilococcus spp., or fungi like Saccharomyces spp., Pichia spp. The production can be performed by any suitable technique known in the art.

[0125] For example, and without intent to limit the scope of the present invention, the production of the insecticidal Txp40 variant of the present invention, in any of its embodiments, can be performed by recombinant technology in a non-pathogenic Escherichia coli bacterial strain, for example, strain BL21 (DE3), alternatively it can be produced in another organism such as yeast, Saccharomyces cerevisiae and Pichia pastoris. The Txp40 protein variants of the present invention may be generated by a person skilled in the art by any technique(s) of mutation known in the state of the art. Examples of mutation techniques include, but are not limited to, random mutagenesis, site-directed mutagenesis, combinatorial mutagenesis, insertional mutagenesis, homologous recombination, CRISPR, gene synthesis, among others. Said mutations may result in at least one substitution, deletion, insertion, fusion, or a combination thereof, introduced in a Txp40 protein as set forth in SEQ ID NO: 1 .

[0126] For the effective use and application of the Txp40 protein variants of the first aspect of the invention as an insecticide, the inventors of the present invention have further developed an agronomically acceptable composition for such purpose.

[0127] Thus, a second aspect of the present invention is to provide an insecticide composition comprising the Txp40 protein variant of the first aspect of the present invention in any one of its embodiments and at least one other agronomically acceptable compound.

[0128] As used herein, the term “agronomically acceptable” when referring to a compound or mixture of compounds shall be understood as a compound or mixture of compounds that is suitable for the manufacturing of an agronomical composition, preferably, the manufacturing of an insecticide composition.

[0129] Agronomically acceptable compounds comprise pH buffers, chelating agents, emulsifiers, preservatives, surfactants, anti-foaming agents, antifreeze agents, colorants, protease inhibitors, dispersing agents, among others.

[0130] In an embodiment, the composition of the second aspect of the present invention comprises

[0131] • an insecticidal agent comprising a Txp40 protein variant of the first aspect of the present invention;

[0132] • a pH buffer selected from Tris-HCI, PIPES, Tricine, MOPS, HEPES or Phosphate buffer

[0133] • a chelating agent, for example, EDTA,

[0134] • an emulsifier selected from Triton, Tween 60, Tween 65, Tween 80, polyethylene glycol (PEG), for example, PEG400, or polyvinyl alcohol (PVA), or any one combination thereof, • an additive selected from maltodextrin, mannitol, sorbitol, monosodium glutamate, glycerol, bovine serum albumin (BSA) and sucrose, a preservative selected from benzisothiazolinone, methylisothiazolinone, iodopropynyl,

[0135] • butylcarbamate, sodium azide, bronopol, methyl paraben, potassium sorbate, or sodium benzoate

[0136] • a solvent, like water.

[0137] Preferably, the composition of the second aspect of the present invention comprises

[0138] • between 0.1-200 mg / L of an insecticidal agent comprising a Txp40 protein variant of the first aspect of the present invention,

[0139] • between 1-5 mg / ml of a pH buffer selected from Tris-HCI, PIPES, Tricine, MOPS, HEPES or Phosphate buffer,

[0140] • between 0.1 -0.5 mg / ml of a chelating agent, for example, EDTA,

[0141] • between 0.01 -10 mg / ml of an emulsifier selected from Triton, Tween 60, Tween 65, Tween 80, polyethylene glycol (PEG), for example, PEG400, or polyvinyl alcohol (PVA), or any one combination thereof,

[0142] • between 1-100 mg / ml of an additive selected from maltodextrin, mannitol, sorbitol, bovine serum albumin (BSA), and sucrose,

[0143] • between 0.01-1 mg / ml of a preservative selected from benzisothiazolinone, sodium azide, bronopol, methyl paraben, potassium sorbate, or sodium benzoate, and

[0144] • q.s.f. of solvent, for example, water.

[0145] It falls within the knowledge of a person skilled in the art to modify or optimize the composition of the present invention in order to better suit a particular need, for example, for the application of the composition on a particular plant species or to have an insecticidal activity against a specific insect pest. These modified or optimized embodiments of the composition shall be understood as falling within the scope of the present invention. Said modifications or optimizations may involve modifying the specific concentration of at least one of the components of the composition, or replacing one component with another one fulfilling the same function within the composition.

[0146] A third aspect of the present invention is to provide a method for controlling an insect pest comprising the steps of a) providing a Txp40 protein variant of the first aspect of the present invention or a composition of the second aspect of the present invention, and b) applying said Txp40 protein variant or composition to a plant or plant part in need thereof.

[0147] The term “controlling an insect pest”, as used herein, means inhibiting the ability of an insect pest to survive, grow, feed, and / or reproduce, or limiting pest-related damage or loss in plants or plant parts. The term “controlling an insect pest” may or may not mean killing said insect pest, although it preferably means the action of killing said pest.

[0148] In an embodiment, the plant or plant part of step b) of the method of the third aspect comprises a plant selected from main row crops, fruits and vegetables, preferably selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., Allium spp. In particular, the plant is selected from Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Zea mays, Oryza sativa, Glycine max, Capsicum annuum, Gossypium hirsutum, Sorghum bicolor, Saccharum officinarum, Helianthus annuus, Oryza sativa, Triticum aestivum, Lactuca sativa, Lens culinaris, Cicer arietinum, Allium sativum, Citrus limon, Citrus sinensis, Pyrus communis, Malus domestica, Prunus domestica, Prunus persica, Prunus armeniaca, Fragaria ananassa, Vaccinium corymbosum.

[0149] In an embodiment, the step b) comprises applying the Txp40 protein variant or the composition to the surface of a plant or a plant part selected from seeds, leaves, flowers, stems, tubers, roots, and the like.

[0150] In an embodiment the step b) comprises applying between 1000-5000 cc / hectare of a Txp40 protein variant of the first aspect of the present invention.

[0151] In an embodiment the method of the third aspect comprises controlling an insect pest selected from the genera Spodoptera spp., Tuta spp., Trialeurodes spp., Anticarsia spp., Helicoperva spp., Spododoptera spp., Tetranychus spp., Bemisia spp., Aphis spp., Myzus spp., Rachiplusia spp., Alphitobus spp. Anthonomus spp., Thirps spp., Franliniella spp., or Dalbulus spp. In a particular embodiment the method of the third aspect comprises controlling an insect pest selected from Tuta absoluta, Trialeurodes vaporariorum, Anticarsia gemmatalis, Helicoverpa armigera, Spodoptera frugiperda, Spodoptera exigua, Spodoptera cosmioides, Tetranychus urticae; Bemisia tabaci; Aphis gossypii, Myzus persicae, Rachiplusia nu, Alphitobius diaperinus, Anthonomus grandis, Helicoverpa zea, Helicoverpa gelotopoeon, Thrips simplex, Frankliniella schultzei Trybom, Frankliniella occidentalis, Frankliniella insularis Franklin, Frankliniella gemina.

[0152] Furthermore, the method of the third aspect comprises controlling an insect pest in any stage of the life cycle, preferably, an insect pest that is on an early-stage larvae, more preferably during neonate or L1 stage.

[0153] In an embodiment, the method of the third aspect comprises contacting an insect with a Txp40 protein variant of the first aspect of the present invention or a composition of the second aspect of the present invention. Said contact may be direct, for example, by directly applying the Txp40 protein variant or composition over an insect, or may be indirect, for example, by applying a Txp40 protein variant or composition over a plant or plant part, and the insect contacting the Txp40 protein variant or composition after a certain amount of time.

[0154] Said contact or contacting comprises a physical interaction between an insect and the Txp40 protein variant or composition. For example, contacting an insect or body part of said insect with the Txp40 protein variant or composition, wherein said body part may be external or internal. Thus, contact between the insect and the Txp40 protein variant or composition includes the ingestion of said protein.

[0155] For an efficient production of the Txp40 protein variant of the first aspect of the invention, the inventors have developed a polynucleotide encoding Txp40 protein variants that can be introduced in a suitable microorganism.

[0156] Thus, a fourth aspect of the invention is to provide a polynucleotide that encodes a Txp40 protein variant of the first aspect of the invention, preferably, a polynucleotide that comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 49-70.

[0157] In a preferred embodiment, the polynucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 73-94.

[0158] Unless otherwise specified or inferred by context, the term “-Tag” when referring to or associated with a polynucleotide that encodes a Txp40 protein or variant thereof, refers to a fusion of said polynucleotide with a nucleotide sequence as set forth in SEQ ID NO: 71 bound to the 5’-end of the polynucleotide. In an embodiment, the polynucleotide that encodes a Txp40 protein or variant thereof comprises the coding region of a His-tag, the coding region, or a thrombin cleavage site, or both. Preferably, the polynucleotide that encodes a Txp40 protein or variant thereof is fused in its 5’-end to a nucleotide sequence as set forth in SEQ ID NO: 71 .

[0159] For example, in order to allow the production of a polypeptide as set forth in SEQ ID NO: 25 in E. Coli BL21 (DE3) strain, the cells are transformed with a vector that comprises a polynucleotide with a nucleotide sequence as set forth in SEQ ID NO: 72. In this sense, the polynucleotide with a nucleotide sequence as set forth in SEQ ID NO: 72 comprises:

[0160] • a polynucleotide with a nucleotide sequence as set forth in SEQ ID NO: 48;

[0161] • a nucleotide sequence as set forth in SEQ ID NO: 71 bound to the 5’-end of the nucleotide sequence of SEQ ID NO: 48.

[0162] Likewise, the nucleotide sequences as set forth in any one SEQ ID NO: 73-94 disclosed herein correspond to each one of the nucleotide sequences as set forth in SEQ ID NO: 49-70, bound to a nucleotide sequence as set forth in SEQ ID NO: 71 at the 5’-end.

[0163] In an embodiment, the polynucleotide of the fourth aspect of the present invention has a nucleotide sequence with at least 80% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 72. Preferably, between 80%-100% of sequence identity to the nucleotide sequence set forth in SEQ ID NO: 72, more preferably, between 90%-100% of sequence identity to the nucleotide sequence set forth in SEQ ID NO: 72. More preferably at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity to the nucleotide sequence set forth in SEQ ID NO: 72.

[0164] Exemplary embodiments of the polynucleotide of the fourth aspect of the present invention encoding specific amino acid (polypeptide) sequences are described in the following table:

[0165]

[0166] The polynucleotide sequences of the fourth aspect of the present invention may be generated by a person skilled in the art by any technique(s) of mutation known in the state of the art. Examples of mutation techniques include, but are not limited to, random mutagenesis, site-directed mutagenesis, combinatorial mutagenesis, insertional mutagenesis, homologous recombination, CRISPR, gene synthesis, among others.

[0167] In order to enable the introduction and expression of the polynucleotide of the fourth aspect of the present invention in a cell such that it produces the Txp40 protein variant, the inventors herein have further developed a nucleic acid construct to be able to carry out these steps. Thus, it is a fifth aspect of the present invention to provide a nucleic acid construct that comprises a polynucleotide of the fourth aspect of the present invention.

[0168] As used herein, the term “nucleic acid construct” refers to a polynucleotide sequence, or an arrangement of nucleic acid like DNA or RNA, that is designed to specifically introduce a specific polynucleotide sequence in a cell, for example, a polynucleotide that encodes a protein of interest for its production and later purification. A nucleic acid construct may contain regulatory elements like promoters and terminators, in addition to the coding sequence of interest. Examples of nucleic acid constructs are plasmids, expression vectors, and cassettes.

[0169] In an embodiment, the nucleic acid construct comprises a polynucleotide that encodes a Txp40 protein variant according to the invention in any of its embodiments, preferably, a polynucleotide that comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 49-70.

[0170] In a preferred embodiment, the nucleic acid construct comprises a polynucleotide having a nucleotide sequence as set forth in any one of SEQ ID NO: 73-94.

[0171] In an embodiment, the nucleic acid construct of the present invention is selected from a plasmid, expression vector, or cassette suitable for the expression of the polynucleotide of the fourth aspect of invention in a microorganism, preferably a bacteria like Escherichia spp., Bacillus spp., or fungi like Saccharomyces spp., or Pichia spp.

[0172] In a preferred embodiment, the nucleic acid construct of the fifth aspect of the present invention enables the production of a Txp40 protein variant of the first aspect of the present invention in a microorganism, preferably a bacteria like Escherichia spp., Bacillus spp., or fungi like Saccharomyces spp., or Pichia spp.

[0173] In another embodiment, the nucleic acid construct of the present invention is selected from a plasmid, expression vector or cassette suitable for the expression of the polynucleotide of the fourth aspect of invention in a plant cell, preferably a plant cell selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., Allium spp. In particular, a plant cell selected from Solanum lycopersicum, Solanum tuberosum, Solarium melongena, Zea mays, Oryza sativa, Glycine max, Capsicum annuum, Gossypium hirsutum, Sorghum bicolor, Saccharum officinarum, Helianthus annuus, Oryza sativa, Triticum aestivum, Lactuca sativa, Lens culinaris, Cicer arietinum, Allium sativum, Citrus limon, Citrus sinensis, Pyrus communis, Malus domestica, Prunus domestica, Prunus persica, Prunus armeniaca, Fragaria ananassa, Vaccinium corymbosum.

[0174] In a preferred embodiment, the nucleic acid construct of the fifth aspect of the present invention enables the production of a Txp40 protein variant of the first aspect of the present invention in a plant cell, preferably a plant cell selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., Allium spp. In particular, a plant cell selected from Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Zea mays, Oryza sativa, Glycine max, Capsicum annuum, Gossypium hirsutum, Sorghum bicolor, Saccharum officinarum, Helianthus annuus, Oryza sativa, Triticum aestivum, Lactuca sativa, Lens culinaris, Cicer arietinum, Allium sativum, Citrus limon, Citrus sinensis, Pyrus communis, Malus domestica, Prunus domestica, Prunus persica, Prunus armeniaca, Fragaria ananassa, Vaccinium corymbosum.

[0175] It falls within the knowledge of a person skilled in the art to modify or optimize the nucleic acid construct of the fifth aspect in order to better suit a particular need, for example, to optimize the expression of the polynucleotide in a particular species by introducing optimized codons, or for its optimal expression under specific conditions, for example, in a specific media culture that contains specific micro or macronutrients. These modified or optimized embodiments of the nucleic acid construct shall be understood as falling within the scope of the present invention.

[0176] It is a sixth aspect of the invention is to provide a transgenic plant transformed with a nucleic acid construct of the fifth aspect of the present invention, wherein the nucleic acid construct comprises a polynucleotide with a nucleotide sequence as set forth in any one of SEQ ID NO: 49-70, more preferably, a polynucleotide with a nucleotide sequence as set forth in any one of SEQ ID NO: 73-94.

[0177] In an embodiment, the transgenic plant is selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., Allium spp. In particular, a transgenic plant selected from Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Zea mays, Oryza sativa, Glycine max, Capsicum annuum, Gossypium hirsutum, Sorghum bicolor, Saccharum officinarum, Helianthus annuus, Oryza sativa, Triticum aestivum, Lactuca sativa, Lens culinaris, Cicer arietinum, Allium sativum, Citrus limon, Citrus sinensis, Pyrus communis, Malus domestica, Prunus domestica, Prunus persica, Prunus armeniaca, Fragaria ananassa, Vaccinium corymbosum.

[0178] It shall be understood that a transgenic plant of the sixth aspect comprises at least one transgenic plant cell that has been successfully transformed with a nucleic acid of the fifth aspect of the invention. Said transgenic plant cell may be located in a specific part of the plant, for example the seeds, leaves, flowers, stems, tubers, or roots. Alternatively, the target plant may comprise only transgenic plant cells.

[0179] As used herein the term “transgenic plant cell” refers to a plant cell that has gone through at least one successful transformation event, and thus contains exogenous genetic material stably integrated in its genome. Said transformation event may result in the introduction of genetic material in any portion of the plant cell genome that does not negatively affect the physiology of the plant cell, i.e., does not cause any form of pathophysiology. Accordingly, the term “transgenic plant” refers to a plant organism of interest that comprises at least one transgenic plant cell. For example, a transgenic plant may comprise only transgenic plant cells.

[0180] It falls within the knowledge of a person skilled in the art to perform the transformation of the at least one plant cell by any means readily available in the state of the art. Exemplary plant cell transformation techniques include, but is not limited to, any one of the following: Agrobacterium-mediated transformation, biolistics (gene gun), CRISPR-Cas9 mediated integration, electroporation, homologous recombination, lentiviral transduction, microinjection, TALENs (Transcription Activator-Like Effector Nucleases), viral vector- mediated transduction, Zinc Finger Nucleases (ZFNs).

[0181] In an embodiment, the transgenic plant comprises a polynucleotide of the fourth aspect of the invention and a plasmid, expression vector, or cassette suitable forthe expression of the polynucleotide of the fourth aspect of invention in at least one plant cell of the transgenic plant. In a particularly preferred embodiment, the transgenic plant comprises a nucleic acid construct of the fifth aspect of the present invention that enables the production of a Txp40 protein variant of the first aspect of the present invention in at least one plant cell of the transgenic plant.

[0182] Thanks to the transformation of a target plant or at least one of the plant cells of the target plant, the inventors of the present invention developed an alternative method for controlling an insect pest that does not involve applying a composition rather by inducing the expression of a polynucleotide that encodes a Txp40 protein variant.

[0183] Thus, a seventh aspect of the present invention is to provide a method for controlling an insect pest comprising the steps of a) providing a transgenic plant of the sixth aspect of the invention, and b) growing the transgenic plant of point a) in conditions that results in the expression of the Txp40 protein variant of the first aspect or the polynucleotide of the fourth aspect.

[0184] The step b) may be performed in such a way that the expression of the Txp40 protein variant of the first aspect or the polynucleotide of the fourth aspect takes place in at least one plant cell of the transgenic plant. Preferably, at least one plant cell located in any one of seeds, leaves, flowers, stems, tubers, or roots, more preferably, at least one plant cell located in the leaves of the transgenic plant.

[0185] It shall be understood that the growing of step b) results in the production of an amount of insecticide Txp40 protein variant such that results in an effective insecticide effect that inhibits the ability of insect pests to survive, grow, feed, and / or reproduce, or limits insect- related damage or loss in plants or plant parts.

[0186] The growing conditions comprise conditions such that the plant expresses a Txp40 protein variant of the first aspect or the polynucleotide of the fourth aspect constantly, or conditions that induce the expression by using specific techniques meaning that the transformed plant will only express the Txp40 protein variant only at specific regulated times, for example, by changing the growing conditions.

[0187] Non-limiting examples of techniques to induce the expression of a polynucleotide that encodes a protein are CRISPR activation (CRISPRa), Doxycycline-inducible system, Ecdysone-inducible system, Gal4 / UAS system, Inducible promoters (e.g., heat-shock, light, chemical inducers), Optogenetic gene control, RNA interference (RNAi) with inducible elements, Steroid-inducible system, Tamoxifen-inducible Cre / lox system, Tetracycline-controlled transcriptional activation (Tet-On / Tet-Off system). These systems may comprise performing further transient or permanent transformations in the transgenic plant, depending on the specifics of each technique, or applying an additional inducing compound to the transgenic plant, as would be known by the person skilled in the art.

[0188] In an embodiment the method of the seventh aspect comprises controlling an insect pest selected from the genera Spodoptera spp., Tuta spp., Trialeurodes spp., Anticarsia spp., Helicoperva spp., Spododoptera spp., Tetranychus spp., Bemisia spp., Aphis spp., Myzus spp., Rachiplusia spp., Alphitobus spp. Anthonomus spp., Thirps spp., Franliniella spp., or Dalbulus spp. In a particular embodiment the method of the seventh aspect comprises controlling an insect pest selected from Tuta absoluta, Trialeurodes vaporariorum, Anticarsia gemmatalis, Helicoverpa armigera, Spodoptera frugiperda, Spodoptera exigua, Spodoptera cosmioides, Tetranychus urticae; Bemisia tabaci; Aphis gossypii, Myzus persicae, Rachiplusia nu, Alphitobius diaperinus, Anthonomus grandis, Helicoverpa zea, Helicoverpa gelotopoeon, Thrips simplex, Frankliniella schultzei Trybom, Frankliniella occidentalis, Frankliniella insularis Franklin, Frankliniella gemina.

[0189] Furthermore, the method of the seventh aspect comprises controlling an insect pest in any stage of the life cycle, preferably, an insect pest that is on an early-stage larvae, more preferably during neonate or L1 stage.

[0190] As it can be appreciated in the following Examples, the Txp40 protein variants provided by the inventors exhibit improved solubility. The increased solubility is advantageous not only during the purification step (see Fig. 1) but also implies a greater bioavailability of functional protein. In addition, the Txp40 protein variants present more thermal stability that implies a longer half-life of the product (see Fig. 3). Furthermore, the Txp40 protein variants exhibit improved insecticidal activity compared to Txp40 (see Fig. 2) which represents a clear advantage. EXAMPLES

[0191] EXAMPLE 1 : PROTEIN VARIANTS WITH IMPROVED SOLUBILITY OR STABILITY

[0192] The following Txp40 variants were generated to achieve improved protein stability:

[0193] Txp40-Var1 is a protein with an amino acid sequence set forth in SEQ ID NO: 2. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein with an amino acid sequence set forth in SEQ ID NO: 1. o Substitutions: Q14P, E23D, V43I, G80R, L139S, R250K, A251 S, V270I, V286I, E320D; o Insertions: PPKL at E235; R at N299

[0194] Txp40-Var2 is a protein with an amino acid sequence set forth in SEQ ID NO: 3. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitutions: Q14P, E23D, I25V, Y37H, H41Y, V43I, D63N, G69D, G80R, D107N, L139S, R250K, A251 S, V270I, V286I, E320D; o Insertions: PPKL at E235; R at N299

[0195] Txp40-Var3 is a protein with an amino acid sequence set forth in SEQ ID NO: 4. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitutions: R10Q, Q14P, E23D, I25V, Y37H, H41Y, V43I, D63N, G69D, G80R, D107N, Q122E, L139S, R186K, R250K, A251 S, V270I, V286I, E320D; o Insertions: LTPDDRG at R10; S at G36; KLPPKSTAMAPSTATTSTTAPS at P237; R at N299.

[0196] Txp40-Var4 is a protein with an amino acid sequence as set forth in SEQ ID NO: 5. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitutions: M1 S, Q14P, E23D, K31 R, Y37T, S40A, H41Y, V43I, I50V, D63N, G69D, E76D, E79D, G80R, D107N, Q122E, I124T, N126D, 1127V, L139S, D141 E, A152S, R186K, S199T, E235K, F243L, E245K, K247R, K248D, R250K, A251 S, G260D, V270I, V286I, L294F, P298Q, N299G, N300D, V304I, K309E, D310H, Q316K, E320D, N321 K, T322I, T324E, H326Q; o Insertions: KLPPMASTATTSTTAPS at P237; R at P298.

[0197] Txp40-Var5 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0198] 6. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitutions: D9P, D34P, E44P, K49L, G80W, D104Y, D107N, G108M, D129G, A152F, R167L, G254N, S268W, T324Y.

[0199] Txp40-Var6 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0200] 7. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitutions: D34P, E44P, K49L, G80W, D104Y, G108M, A152F, R167L, S268W, T324Y

[0201] Txp40-Var7 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0202] 8. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: A47C, F150C.

[0203] Txp40-Var8 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0204] 9. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: A55C, L89C.

[0205] Txp40-Var9 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0206] 10. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: A21 C, L52C. Txp40-Var10 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0207] 11 . This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: R252C, S327C.

[0208] Txp40-Var11 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0209] 12. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: E17C, A53C.

[0210] Txp40-Var12 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0211] 13. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: E17C, A47C, A53C, F150C, R252C, S327C.

[0212] Txp40-Var13 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0213] 14. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: Y45G, 181 H, K111 G, K221 L, T315A, T330S

[0214] Txp40-Var14 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0215] 15. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: L93G, N97Y, G118F, F169Y, K210L, L230A

[0216] Txp40-Var15 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0217] 16. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: R26K, N29D, K73H, L114T, L171 Q, G225E, V241 S, K284I Txp40-Var16 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0218] 17. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: S179A, I188E, S190W, P236R, K275T, T315P, N317K

[0219] Txp40-Var17 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0220] 18. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: T77A, F174K, I177L, Y194R, K200G, K221 R, R240Y, S285G, T288R

[0221] Txp40-Var18 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0222] 19. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: G95Q, Q100T, D142S, E158N, I188S, E198N, P233I, Q244E

[0223] Txp40-Var19 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0224] 20. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: I3R, G60L, I61 M, Y120F, L145F, K213L, K284L, S307H, Q308A, T319V

[0225] Txp40-Var20 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0226] 21 . This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: I3N, P7R, R46S, D140V, E198Y, I206Q, K209L, G225I, A289H

[0227] Txp40-Var21 is a protein with an amino acid sequence as set forth in SEQ ID NO:

[0228] 22. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. Substitution: T6N, G22E, V64M, S91 G, L147M, Q153F, F189V, L211 P, F215L, A231 W, A256M, G291 M, K333N

[0229] Txp40-Var22 is a protein with an amino acid sequence as set forth in SEQ ID NO: 23. This amino acid sequence presents the following substitutions and insertions in comparison to the Txp40 protein amino acid sequence set forth in SEQ ID NO: 1. o Substitution: F30A, Y54N, D70Y, S87N, N112Q, E119S, Y120D, K143Y, N160S, K191 R, N192A, E198G, N204G, S267V

[0230] EXAMPLE 2: INSECTICIDAL PROTEIN PRODUCTION PROTOCOL

[0231] For the production of pesticidal proteins, an E. coli strain BL21 (DE3) was used, which allows high levels of recombinant protein production. This strain was transformed with a plasmid pET28 containing a polynucleotide sequence as set forth any one of SEQ ID NO: 48-70, in order to express any one of the protein sequences as set forth in any one of SEQ ID NO: 1-23.

[0232] The clone to be expressed is inoculated in liquid LB medium supplemented with kanamycin (final concentration 50 pg / mL) and left to grow overnight at 37°C with constant agitation at 200 rpm. The next day, a 1 / 100 dilution of the saturated culture is prepared, also in LB kanamycin medium, and allowed to grow for approximately 2 hours until an optical density (OD) at 600 nm of 1 is reached. At this time, expression is induced with the addition of IPTG (final concentration 0.1 mM), and the same growth conditions are maintained for 4 additional hours. Afterwards, the OD is determined at 600 nm.

[0233] After expression, cell disruption is followed by the application of high pressures. A homogenizer is used for this purpose, at pressures of about 1000 bar.

[0234] EXAMPLE 3: IMPROVED SOLUBILITY AND BIOMASS YIELD OF THE Txp40 PROTEINS VARIANTS OF THE PRESENT INVENTION

[0235] The expression of the mutant proteins showed an increase in the biomass yield obtained with respect to the production of Txp40 under the same conditions. For example, in the case of Txp40-Var7, Txp40-Var8 and Txp40-Var9, the biomass yield obtained was around 2.5 times greater than that of Txp40.

[0236] In addition, some mutants present an increase in the soluble fractions obtained from expression and purification. Figure 1 shows SDS-PAGE of some of the mutant’s fractions (Txp40-Var1 , Txp40-Var2, Txp40-Var3, Txp40-Var4, Txp40). The increase in the soluble fraction implies, among other things, a greater bioavailability of functional protein. Moreover, resulting in greater stability and performance of these proteins.

[0237] EXAMPLE 4: ENHANCED STORAGE STABILITY OF Txp40 PROTEIN VARIANTS OF THE PRESENT INVENTION

[0238] Accelerated Storage Stability Testing

[0239] The disclosed Txp40 variants demonstrate enhanced storage stability compared to the native Txp40 protein, exhibiting significantly reduced heat-induced degradation, i.e., improved thermal stability.

[0240] As shown in Figure 3, when Txp40 samples are stored at elevated temperatures for extended periods, for example, 2 weeks at 54 °C, they undergo almost complete degradation (approximately 96%). In contrast, TxP40-Var4 shows only a 70% decrease, indicating a substantial improvement in stability. Similar results were observed with other variants.

[0241] EXAMPLE 5: INSECTICIDAL ACTIVITY OF THE Txp40 PROTEIN VARIANTS OF THE PRESENT INVENTION

[0242] EXAMPLE 5.1 : INSECTICIDAL ACTIVITY AGAINST Spodoptera frugiperda

[0243] As it has been shown in field trials, the Txp40 protein variants of the present invention not only present an improved solubility and thermal stability, but also retains the insecticidal activity of the Txp40 protein.

[0244] The insecticidal ability of these mutants was tested in insecticidal assays against the lepidopteran Spodoptera frugiperda, one of the most important pests in Argentina, which affects many crops, mainly corn, soybeans, cotton, sorghum, alfalfa, tomatoes, peanuts, and potatoes, among others. The experimental trials used the overlay methodology, where the artificial diet is fully covered with each experimental protein composition. This assay involves early stage larvae (L1 stage). First, in each micro plate well, the diet is inoculated with the appropriate dose of the treatment, it is absorbed, and finally, an insect larva is placed in each treated well. At least 48 Spodoptera larvae were used for each treatment. After a week, the size, stage, and mobility of the insects are evaluated.

[0245] In general, most protein variant compositions presented high efficacy for the control of Spodoptera, but mainly, some of the mutant compositions revealed greater insecticidal activity with respect to Txp40-Tag protein.

[0246] In Figure 2, it can be seen every tested variant effectively controls S. frugiperda. Particularly, it can be seen that variants Txp40-Var2, Txp40-Var3, Txp40-Var4, Txp40- Var5, Txp40-Var6, Txp40-Var7, Txp40-Var8 and Txp40-Var9 exhibit a higher insecticidal activity than the reference Txp40 protein, around 10-12% more than Txp40 in some cases, achieving a high percentage of control of the pest.

[0247] The increase in control percentage was the result of a combination of the reduction in larva size, the decreased mobility, the arrest at the stage of development, and increased mortality. All these factors negatively affect the larvae survival after the addition of an insecticide.

[0248] EXAMPLE 5.2: INSECTICIDAL ACTIVITY AGAINST Dalbulus maidis

[0249] In addition, the insecticidal activity of the different proteins was evaluated using an in vitro assay against Dalbulus maidis. To assess the effect of each treatment, 20 pots were prepared, each containing two sweet corn plants. Each pot received the corresponding treatment, after which three young adult D. maidis were introduced. The pots were maintained at 26 °C, 70% relative humidity, and a 16-hour photoperiod. Evaluations were conducted every 24 hours over three consecutive days, and at each time point, the numbers of live and dead insects were recorded. The chemical standards used were lambda-cyhalothrin and sulfoxaflor, while the biological standard consisted of a mixture of Pseudomonas fluorescens and Pseudomonas chlororaphis.

[0250] The results summarized in Fig. 4 show a consistent increase in mortality percentage over the 24, 48, and 72-hour evaluations. As expected, the chemical standard demonstrated the highest efficacy at all-time points. Some formulation variants achieved control levels comparable to the biological standard and, in several cases, exhibited notably higher control percentages. This suggests that certain variants may offer enhanced insecticidal activity against Dalbulus maidis.

[0251] Based on the descriptions and Examples presented herein, the person skilled in the art would appreciate that the protein variants of the first aspect of the present invention present a higher thermal, chemical and biological stability when compared to a Txp40 protein as set forth in SEQ ID NO: 1 , as described in the prior art. Moreover, this stability is achieved without causing any reduction or hampering in the insecticidal activity of the proteins, being not just a mere alternative but an improvement over what has been described in the prior art.

[0252] LIST OF SEQUENCES

Claims

CLAIMS1) A Txp40 protein variant with insecticidal activity comprising the amino acid residues E295, R311 , H331 , and Y332.2) The Txp40 protein variant of claim 1 , further comprising an amino acid sequence as set forth in any one of SEQ ID NO: 2-23.3) The Txp40 protein variant of claim 1 or 2, further comprising a polypeptide as set forth in SEQ ID NO: 24 bound to the N-terminus of said Txp40 protein variant.4) An insecticide composition comprising a Txp40 protein variant of any one of claims 1-3, and at least one other agronomically acceptable compound.5) The insecticide composition of claim 4, comprising between 0.1-200 mg / L of a Txp40 protein variant of any one of claims 1-3; between 1-5 mg / ml of a pH buffer; between 0.1-0.5 mg / ml of a chelating agent; between 0.01-10 mg / ml of an emulsifier; between 1-100 mg / ml of an additive; between 0.01-1 mg / ml of a preservative; and q.s.f. of a solvent.6) A method for controlling an insect pest comprising the steps of a) providing a Txp40 protein variant of any one of claims 1 -3 or a composition of any one of claims 4-5, and b) applying said Txp40 protein variant or composition to a plant or plant part in need thereof.7) The method according to claim 6, wherein the plant or plant part is selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., Allium spp.8) The method according to any one of claims 6-7, wherein the insect pest is selected from Spodoptera spp., Tuta spp., Trialeurodes spp., Anticarsia spp., Helicoperva spp., Spododoptera spp., Tetranychus spp., Bemisia spp., Aphis spp., Myzus spp., Rachiplusia spp., Alphitobus spp. Anthonomus spp., Thirps spp., Franliniella spp , or Dalbulus spp.9) A polynucleotide that encodes a Txp40 protein variant of claim 1-3, comprising a sequence as set forth in any one of SEQ ID NO: 49-70.10) The polynucleotide of claim 9, further comprising a nucleotide sequence as set forth in SEQ ID NO: 71 bound to the 5’-end of said polynucleotide.11) A nucleic acid construct comprising the polynucleotide of any one of claims 9-10, and a plasmid, expression vector, or expression cassette.12) A transgenic plant transformed with a nucleic acid construct of claim 11 , wherein the nucleic acid construct comprises the polynucleotide as set forth in any one of SEQ ID NO: 49-70.13) The transgenic plant of claim 12, wherein the transgenic plant is selected from the genera Solanum spp., Zea Spp., Oryza spp., Glycine spp., Sorghum spp., Capsicum spp., Triticum spp., Lactuca spp., lens spp., Cicer spp., Gossypium spp., Saccharum spp., Helianthus spp., Citrus spp., Prunus spp., Pyrus spp., Malus spp., Fragaria spp., Vaccinium spp., Allium spp.14) A method for controlling an insect pest comprising the steps of a) providing a transgenic plant of any one of claims 12-13, b) growing the transgenic plant of point a) in conditions that results in the expression of the Txp40 protein variant of any one of claims 1-3, or the polynucleotide of any one of claims 9-10.

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