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WO2024194831A1 - Expression de protéines myofibrillaires animales dans des plantes - Google Patents

Expression de protéines myofibrillaires animales dans des plantes Download PDF

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Publication number
WO2024194831A1
WO2024194831A1 PCT/IB2024/052721 IB2024052721W WO2024194831A1 WO 2024194831 A1 WO2024194831 A1 WO 2024194831A1 IB 2024052721 W IB2024052721 W IB 2024052721W WO 2024194831 A1 WO2024194831 A1 WO 2024194831A1
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Prior art keywords
promoter
terminator
plant
nucleic acid
recombinant
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Inventor
Gaston PALADINI
Henk HOOGENKAMP
Martin Salinas
Amit DHINGRA
Bruce Williamson BENAVIDES
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Moolec Science Ltd
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Moolec Science Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the present disclosure is related to the heterologous expression of myofibrillar proteins in non-animal hosts.
  • the present disclosure provides customized expression cassettes for the production of myofibrillar proteins in transgenic organisms. These expression cassettes comprise selected regulatory elements and codon-optimized protein coding sequences.
  • Meat is an important source of protein and other nutrients in human diets.
  • Genetic engineering is a tangible and expedient approach that can be used to produce myofibrillar animal proteins in different host organisms that can subsequently improve the organoleptic and nutritional features of meat alternatives.
  • the subject disclosure described herein provides a solution to produce myofibrillar proteins in transgenic organisms.
  • the recombinant myofibrillar proteins expressed herein are useful for application in the traditional meat industry in order to reduce the amount of meat and in non-animal foods.
  • the international patent application WO2021138674 discloses the expression of a muscle myosin coiled-coil domain and muscle actin coiled- coil domain in Escherichia coli and Pichia pastoris.
  • the international patent application WO2021226690 provides a method to obtain fibrillar animal proteins using a genetically modified Saccharomyces cerevisiae. Another study discloses the production of functional skeletal muscle tropomyosin in S. cerevisiae (Alegria et al., (2003) Protein Expr. Purif.
  • the invention disclosed herein provides a novel approach to produce recombinant myofibrillar proteins, with higher expression yields and by using superior hosts than previously reported in the art.
  • a method to produce a recombinant myofibrillar protein in a plant comprising:
  • the recombinant myofibrillar protein is selected from the group consisting of actin (e.g., beta-actin, gamma-actin), myosin (e.g., non-muscle and muscle myosin II), actinin (e.g., alpha-actinin), nebulin, Cap Z, m-protein, c-protein, M- protein, tropomodulin, troponin (e.g., troponin T, troponin I, and troponin C), tropomyosin, keratin and the fragments thereof.
  • actin e.g., beta-actin, gamma-actin
  • myosin e.g., non-muscle and muscle myosin II
  • actinin e.g., alpha-actinin
  • nebulin ebulin
  • Cap Z m-protein, c-protein, M- protein
  • tropomodulin troponin (e.g.
  • the nucleic acid encoding for the recombinant myofibrillar protein is operatively linked to at least one regulatory element selected from the group of: promoter, terminator, enhancers, a storage signal to chloroplasts, a storage signal to vacuoles, and the combination thereof.
  • the plant comprises or is selected from the group consisting of: Arabidopsis, Nicotiana, Glycine, Pisum, Oryza, Hordeum, Triticum, Zea, Secal, Avena, Beta, Pastinaca, Vigna, Arachis, Lens, Phaseolus, Cicer, Medicago, Eruca, Sinapis, Brassica, Lactuca, Solanum, Ipomoea, Manihot, and triticale.
  • the plant comprises or is selected from the group consisting of: Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum L., Glycine max, Pisum sativum, Oryza sativa, Hordeum vulgare, Triticum aestivum, Zea mays, Secale cereale, Avena sativa, Beta vulgaris, Pastinaca sativa, Vigna angularis, Vigna radiata, Arachis hypogaea, Lens culinaris, Phaseolus vulgaris, Cicer arietinum, Medicago sativa, Eruca vesicaria, Sinapis alba, Brassica juncea, Brassica nigra, Lactuca sativa, Solanum tuberosum, Ipomoea batatas, Manihot esculenta, triticale, Triticum spelta, and legume plants.
  • the plant comprises or is selected from the group consisting of: Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum L., Oryza sativa, Hordeum vulgare, Triticum aestivum, Zea mays, Secale cereale, Avena sativa, Beta vulgaris, Pastinaca sativa, Eruca vesicaria, Sinapis alba, Brassica juncea, Brassica nigra, Lactuca sativa, Solanum tuberosum, Ipomoea batatas, Manihot esculenta, triticale, Triticum spelta, and legume plants.
  • the legume plant is selected from the genus consisting of: Phaseolus, Pisum, Vigna, Cajanus, Lens, Cicer, Vicia, Arachis, Glycine, Macrotyloma, Mucuna, Lupinus, Ceratonia, Canavalia, Cyamopsis, Lablab, Psophocarpus, Clitoria, Lathryus, Trifolium, Medicago, Melilotus, and Tamarindus.
  • the plant is a legume plant selected from the group consisting of: Phaseolus vulgaris, Phaseolus lunatus, Phaseolus coccineus, Phaseolus acutifolius, Pisum sativum, Vigna radiata, Vigna mungo, Vigna uniguiculata, Vigna aconitifoilia, Vigna angularis, Cajanus cajan, Lens culinaris, Cicer arietinum, Vicia faba, Vicia ervilia, Vicia sativa, Arachis hypogaea, Glycine max, Macrotyloma uniflorum, Mucuna pruriens, Lupinus albus, Lupinus mutabilis, Ceratonia siliqua, Canavalia gladiata, Canavalia ensiformis, Cyamopsis tetragonoloba, Lablab purpureus, Psosphocarpus tetranoglobulus, Clitoria ternatea,
  • the nucleic acid encoding for the myofibrillar protein comprises a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences selected from the group consisting of SEQ ID NOs: 32 to 73, 92-93 and/or 98.
  • the promoter is operatively linked to the 5’ end of the nucleic acid encoding for the recombinant myofibrillar protein, and the terminator is located at the 3’ end of the nucleic acid encoding for the recombinant myofibrillar protein followed by a region attachment sequence.
  • the promoter and the storage signal to vacuoles is operatively linked to the 5’ end of the nucleic acid encoding for the recombinant myofibrillar protein, and a KDEL fused to the terminator is located at the 3’ end of the nucleic acid encoding for the recombinant myofibrillar protein followed by a region attachment sequence.
  • the promoter and the storage signal to chloroplasts is operatively linked to the 5’ end of the nucleic acid encoding for the recombinant myofibrillar protein, and the terminator is located at the 3’ end of the nucleic acid encoding for the recombinant myofibrillar protein followed by a region attachment sequence.
  • the nucleic acid encoding for the myofibrillar protein is cloned into a vector, wherein said vector comprises a sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NOs: 83 to 91, 95-97, and/or 99-104.
  • a transgenic plant comprising a nucleic acid encoding for a recombinant myofibrillar protein, wherein said nucleic acid is operatively linked to at least one regulatory element selected of the group consisting of: promoter, terminator, enhancers, a storage signal to chloroplasts, a storage signal to vacuoles, and combinations thereof.
  • said recombinant myofibrillar protein comprises or is selected from the group consisting of: actin (beta-actin, gamma-actin), myosin (nonmuscle and muscle myosin II), actinin (alpha-actinin), nebulin, Cap Z, m-protein, c- protein, M-protein, tropomodulin, troponin (T, I, and C), tropomyosin, keratin and the fragments thereof.
  • the transgenic plant, plant cell, plant part, or plant tissue is selected from the group consisting of: Arabidopsis, Nicotiana, Glycine, Pisum, Oryza, Hordeum, Triticum, Zea, Secal, Avena, Beta, Pastinaca, Vigna, Arachis, Lens, Phaseolus, Cicer, Medicago, Eruca, Sinapis, Brassica, Lactuca, Solanum, Ipomoea, Manihot, and triticale.
  • the transgenic plant, plant cell, plant part, or plant tissue is selected from the group consisting of: Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum L., Glycine max, Pisum sativum, Oryza sativa, Hordeum vulgare, Triticum aestivum, Zea mays, Secale cereale, Avena sativa, Beta vulgaris, , Pastinaca sativa, Vigna angularis, Vigna radiata, Arachis hypogaea, Lens culinaris, Phaseolus vulgaris, Cicer arietinum, Medicago sativa, Eruca vesicaria, Sinapis alba, Brassica juncea, Brassica nigra, Lactuca sativa, Solanum tuberosum, Ipomoea batatas, Manihot esculenta, triticale, Triticum spelta, and legume plants.
  • the transgenic plant, plant cell, plant part, or plant tissue is selected from the group consisting of: Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum L., Oryza sativa, Hordeum vulgare, Triticum aestivum, Zea mays, Secale cereale, Avena sativa, Beta vulgaris, Pastinaca sativa, Eruca vesicaria, Sinapis alba, Brassica juncea, Brassica nigra, Lactuca sativa, Solanum tuberosum, Ipomoea batatas, Manihot esculenta, triticale, Triticum spelta, and legume plants.
  • the transgenic plant is a transgenic legume plant, plant cell, plant part, or plant tissue selected from the group consisting of: Phaseolus, Pisum, Vigna, Cajanus, Lens, Cicer, Vicia, Arachis, Glycine, Macrotyloma, Mucuna, Lupinus, Ceratonia, Canavalia, Cyamopsis, Lablab, Psophocarpus, Clitoria, Lathryus, Trifolium, Medicago, Melilotus, and Tamarindus.
  • the transgenic plant is a transgenic legume plant, plant cell, plant part, or plant tissue selected from the group consisting of: Phaseolus vulgaris, Phaseolus lunatus, Phaseolus coccineus, Phaseolus acutifolius, Pisum sativum, Vigna radiata, Vigna mungo, Vigna uniguiculata, Vigna aconitifoilia, Vigna angularis, Cajanus cajan, Lens culinaris, Cicer arietinum, Vicia faba, Vicia ervilia, Vicia sativa, Arachis hypogaea, Glycine max, Macrotyloma uniflorum, Mucuna pruriens, Lupinus albus, Lupinus mutabilis, Ceratonia siliqua, Canavalia gladiata, Canavalia ensiformis, Cyamopsis tetragonoloba, Lablab purpureus, Psosphocarpus te
  • a transgenic plant, plant cell, plant part, or plant tissue comprising a nucleic acid encoding for a recombinant myofibrillar protein, wherein said nucleic acid is operatively linked to at least one regulatory element selected from the group consisting of: a promoter, a terminator, an enhancer, a storage signal to chloroplasts, a storage signal to vacuoles, and combinations thereof.
  • said recombinant myofibrillar protein is selected from the group consisting of actin (e.g., beta- actin, gamma-actin), myosin (e.g., non-muscle and muscle myosin II), actinin (e.g., alpha-actinin), nebulin, Cap Z, m-protein, c-protein, M-protein, tropomodulin, troponin (e.g., troponin T, troponin I, and troponin C), tropomyosin, keratin and the fragments thereof.
  • actin e.g., beta- actin, gamma-actin
  • myosin e.g., non-muscle and muscle myosin II
  • actinin e.g., alpha-actinin
  • nebulin ebulin
  • Cap Z m-protein, c-protein, M-protein
  • tropomodulin troponin (e.g.
  • the recombinant myofibrillar protein is actin, and wherein the actin is selected from beta-actin or gamma-actin.
  • the recombinant myofibrillar protein is myosin, and wherein the myosin is selected from non-muscle myosin or muscle myosin II.
  • the recombinant myofibrillar protein is actinin, and wherein the actinin is alpha-actinin.
  • the recombinant myofibrillar protein is troponin, and wherein the troponin is selected from troponin T, troponin I, and troponin C.
  • the transgenic plant cell is selected from the group consisting of: Arabidopsis, Nicotiana, Glycine, Pisum, Oryza, Hordeum, Triticum, Zea, Secal, Avena, Beta, Pastinaca, Vigna, Arachis, Lens, Phaseolus, Cicer, Medicago, Eruca, Sinapis, Brassica, Lactuca, Solanum, Ipomoea, Manihot, and triticale.
  • the transgenic plant cell is selected from the group consisting of: Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum L., Glycine max, Pisum sativum, Oryza sativa, Hordeum vulgare, Triticum aestivum, Zea mays, Secale cereale, Avena sativa, Beta vulgaris, , Pastinaca sativa, Vigna angularis, Vigna radiata, Arachis hypogaea, Lens culinaris, Phaseolus vulgaris, Cicer arietinum, Medicago sativa, Eruca vesicaria, Sinapis alba, Brassica juncea, Brassica nigra , Lactuca sativa, Solanum tuberosum, Ipomoea batatas, Manihot esculenta, triticale and Triticum spelta.
  • the promoter comprises or is selected from the group consisting of: 35S promoter, 2x35S promoter, beta-conglycinin alpha subunit of the 7S storage (7s) promoter from soybean, the beta-phaseolin (Phas) promoter from common bean, USP promoter from Vicia faba, SBP promoter from Vicia faba, Legumin B4 promoter from Vicia faba, Napin promoter from Brassica napus, Vicilin promoter from Pisum sativum, a-globulin promoter from cotton, y-zein promoter from maize, glutenin promoter from wheat, VVPVPE promoter from Vitis spp, Groundnut seed promoter (GSP) from peanut, 7aP promoter from soybean, AtLAC15 promoter from Arabidopsis thaliana, SSPs promoter from chickpea, Lectin promoter from soybean, Oleosin promoter from Brassica napus, AhLEClA promoter from peanut, Glu
  • GSP Groundnut seed
  • the terminator comprises or is selected from the group consisting of: Extensin terminator from tobacco, UblO terminator from Arabidopsis lhaliana, Hsp70 terminator from Arabidopsis thaliana, Hspl 8.2 terminator from Arabidopsis lhaliana, Act2 terminator from Arabidopsis lhaliana. G7 terminator from Arabidopsis lhaliana. 3g24240 terminator from Arabidopsis thaliana, NOS terminator from Agrobacterium lumefaciens, Ocs terminator from Agrobacterium lumefaciens, Mas terminator from Agrobacterium lumefaciens.
  • the plant expression vector comprises a translational or transcriptional enhancer sequence selected from the group consisting of: 5’ UTR TEV (Tobacco Etch Virus Translational Enhancer) and a matrix attached region RB7 MAR.
  • 5’ UTR TEV tobacco Etch Virus Translational Enhancer
  • RB7 MAR matrix attached region
  • the plant expression vector comprises a signal sequence to the Endoplasmic Reticule and Golgi Apparatus, wherein the signal sequence is a KDEL sequence.
  • the transgenic plant comprises a nucleic acid encoding for a myofibrillar protein comprising a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences selected from the group consisting of SEQ ID NOs: 32 to 73, 92-93 and/or 98.
  • a plant expression vector comprising a nucleic acid encoding for a recombinant protein.
  • the recombinant protein is a myofibrillar protein.
  • the myofibrillar protein is selected from the group consisting of tropomyosin, myosin, actin, troponin, a-actinin, elastin, collagen (for example type I, II and/or III), keratin, laminin (B, 2, 10), fibronectin, fibrinogen, and thrombin and the fragments thereof.
  • the myofibrillar protein is tropomyosin or myosin.
  • the nucleic acid encoding for the myofibrillar protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences selected from the group consisting of: SEQ ID NOs: 32 to 73, 92-93 and/or 98.
  • the plant expression vector comprising the nucleic acid encoding for the myofibrillar protein is operatively linked to at least one regulatory element selected of the group consisting of: a promoter, a terminator, an enhancer, a storage signal to chloroplasts, a storage signal to vacuoles, and the combination thereof.
  • the expression vector comprises a promoter operatively linked to the 5’ end of the nucleic acid encoding for the recombinant myofibrillar protein, a terminator located at the 3’ end of the nucleic acid encoding for the recombinant myofibrillar protein followed by a region attachment sequence.
  • the expression vector comprises a promoter and a storage signal to vacuoles operatively linked to the 5’ end of the nucleic acid encoding for the recombinant myofibrillar protein, a KDEL fused to a terminator located at the 3’ end of the nucleic acid encoding for the recombinant myofibrillar protein followed by a region attachment sequence.
  • the expression vector comprises a promoter and a storage signal to chloroplasts operatively linked to the 5’ end of the nucleic acid encoding for the recombinant myofibrillar protein, a terminator located at the 3’ end of the nucleic acid encoding for the recombinant myofibrillar protein followed by a region attachment sequence.
  • the expression vector comprises a nucleic acid sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences selected from the group consisting of: 83 to 91, 95-97, and/or 99-104.
  • a plant expression vector comprising a nucleic acid encoding for a recombinant protein, wherein said nucleic acid is operatively linked to at least one regulatory element selected of the group consisting of: a promoter, a terminator, an enhancer, a storage signal to chloroplasts, a storage signal to vacuoles, and the combination thereof.
  • the plant expression vector comprises a promoter operatively linked to the 5’ end of the nucleic acid encoding for the recombinant protein, a terminator located at the 3’ end of the nucleic acid encoding for the recombinant protein followed by a region attachment sequence.
  • the plant expression vector comprises a promoter and a storage signal to vacuoles operatively linked to the 5’ end of the nucleic acid encoding for the recombinant protein, a KDEL fused to a terminator located at the 3’ end of the nucleic acid encoding for the recombinant protein followed by a region attachment sequence.
  • the plant expression vector comprises a promoter and a storage signal to chloroplasts operatively linked to the 5’ end of the nucleic acid encoding for the recombinant protein, a terminator located at the 3’ end of the nucleic acid encoding for the recombinant protein followed by a region attachment sequence.
  • the promoter is phas
  • the terminator is Arc5
  • the region attachment sequence is Rb7Mar
  • the chloroplast storage signal sequence is a 168-bp chloroplast signal from the ribulose 1,5 -bisphosphate carboxylase gene from Pisum sativum
  • the vacuole storage signal sequence is an osmotin Gene AP24 from Nicotiana tabacum.
  • the recombinant protein is selected form the groups consisting of extracellular fibrillar proteins, milk proteins, egg proteins, heme proteins, myofibrillar proteins, enzymes, antigens and cytokines.
  • nucleic acid encoding for a recombinant myofibril protein, wherein said nucleic acid comprises a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the following sequences: SEQ ID NOs: 32 to 73, 92-93 and/or 98.
  • a food product comprising a transgenic plant, plant cell, plant part, or plant tissue, wherein said transgenic plant, plant cell, plant part, or plant tissue comprises a nucleic acid encoding for a recombinant myofibrillar protein.
  • said recombinant myofibrillar protein is selected from the group consisting of actin (e.g., beta- actin, gamma-actin), myosin (e.g., non-muscle and muscle myosin II), actinin (e.g., alpha-actinin), nebulin, Cap Z, m-protein, c-protein, M-protein, tropomodulin, troponin (e.g., troponin T, troponin I, and troponin C), tropomyosin, keratin and the fragments thereof.
  • actin e.g., beta- actin, gamma-actin
  • myosin e.g., non-muscle and muscle myosin II
  • actinin e.g., alpha-actinin
  • nebulin ebulin
  • Cap Z m-protein, c-protein, M-protein
  • tropomodulin troponin (e.g.
  • a food product comprising a transgenic plant, plant cell, plant part, or plant tissue
  • said transgenic plant, plant cell, plant part, or plant tissue comprises a nucleic acid encoding for a recombinant protein
  • said recombinant protein is selected from the group consisting of myofibrillar proteins, extracellular fibrillar proteins (such as collagen, keratin, gelatin, elastin), milk proteins (casein, lactoalbumin, and lactoglobulin), ovo proteins (such as ovalbumin, ovotransferrin, and ovomucoid), wherein said nucleic acid encoding for a recombinant protein is operatively linked to a promoter at the 5’ end, a terminator located at the 3’ end followed by a region attachment sequence.
  • the promoter is phas
  • the terminator is Arc5
  • the region attachment sequence is Rb7Mar.
  • said nucleic acid encoding for the recombinant protein is also linked to a storage signal sequence selected from the group consisting of a 168-bp chloroplast signal from the ribulose 1,5-bisphosphate carboxylase gene from Pisum sativum and an osmotin Gene AP24 from Nicotiana tabacum.
  • a food product comprising a transgenic seed comprising a nucleic acid encoding for a recombinant myofibrillar protein.
  • said recombinant myofibrillar protein is selected from the group consisting of actin (e.g., beta- actin, gamma-actin), myosin (e.g., non-muscle and muscle myosin II), actinin (e.g., alpha-actinin), nebulin, Cap Z, m-protein, c-protein, M-protein, tropomodulin, troponin (e.g., troponin T, troponin I, and troponin C), tropomyosin, keratin and the fragments thereof.
  • actin e.g., beta- actin, gamma-actin
  • myosin e.g., non-muscle and muscle myosin II
  • actinin e.g., alpha-actinin
  • a food product comprising a transgenic seed comprising a nucleic acid encoding for a recombinant protein, wherein said recombinant protein is selected from the group consisting of myofibrillar proteins, extracellular fibrillar proteins (such as collagen, keratin, gelatin, elastin), milk proteins (casein, lactoalbumin, and lactoglobulin), ovo proteins (such as ovalbumin, ovotransferrin, and ovomucoid), wherein said nucleic acid encoding for a recombinant protein is operatively linked to a promoter at the 5’ end, a terminator located at the 3’ end followed by a region attachment sequence.
  • myofibrillar proteins such as collagen, keratin, gelatin, elastin
  • milk proteins casein, lactoalbumin, and lactoglobulin
  • ovo proteins such as ovalbumin, ovotransferrin, and ovomucoid
  • the promoter is phas
  • the terminator is Arc5
  • the region attachment sequence is Rb7Mar.
  • said nucleic acid encoding for the recombinant protein is also linked to a storage signal sequence selected from the group consisting of a 168-bp chloroplast signal from the ribulose 1,5-bisphosphate carboxylase gene from Pisum sativum and an osmotin Gene AP24 from Nicotiana tabacum.
  • FIGURE 1 (FIG. 1).
  • Figure 1 depicts the pRG-2x35S+bMII+TNOS binary vector. This plasmid allows the expression of the bovine myosin II gene driven by the constitutive promoter 2x CaMV35S, and includes the Nopaline synthase terminator (TNOS). The myosin consists of a dicot codon-optimized sequence.
  • TNOS Nopaline synthase terminator
  • FIGURE 2 (FIG. 2).
  • Figure 2 shows the pRG-2x35S+ HMM+TNOS binary vector.
  • This plasmid allows the expression of the bovine HMM (Heavy Meromyosin Protein) gene driven by constitutive promoter 2x CaMV35S, and includes the Nopaline synthase terminator (TNOS).
  • the HMM consists of a dicot codon-optimized sequence.
  • FIGURE 3 (FIG. 3).
  • Figure 3 illustrates the pRG-2x35S+TPMl-LL-
  • TPM2+TNOS binary vector This plasmid allows the expression of the tropomyosin (TPM1-LL-TPM2) gene driven by constitutive promoter CaMV35S, and includes the Nopaline synthase terminator (TNOS).
  • the tropomyosin gene consists of the TPM1 (alpha version) and TMP2 (beta version) linked via a long linker (LL) of 63 bp.
  • the TPM1-LL-TPM2 consists of a dicot codon-optimized sequence.
  • FIGURE 4 (FIG. 4).
  • Figure 4 illustrates a western blot to detect the recombinant protein TPM1-LL-TPM2, from the extract collected from the transient expression in Nicotiana benthamiana.
  • FIGURE 5 (FIG. 5).
  • Figure 5 represents a pM04 expression vector for the expression of the Green Fluorescent Protein (GFP).
  • GFP Green Fluorescent Protein
  • FIGURE 6 (FIG. 6).
  • Figure 6 represents a pM05 expression vector for the expression of GFP and its storage in the vacuoles.
  • FIGURE 7 (FIG. 7).
  • Figure 7 represents a pM06 expression vector for the expression of GFP and its storage in the chloroplasts.
  • FIGURE 8 (FIG. 8).
  • Figure 8 represents a pMIO expression vector for the expression of tropomyosin.
  • FIGURE 9 (FIG. 9).
  • Figure 9 represents a pMl 1 expression vector for the expression of Tropomyosin and its storage in the vacuoles.
  • FIGURE 10 (FIG. 10).
  • Figure 10 represents a pM12 expression vector for the expression of Tropomyosin and its storage in the chloroplasts.
  • FIGURE 11 (FIG. 11).
  • Figure 11 represents a pM13 expression vector for the expression of keratin.
  • FIGURE 12 (FIG. 12).
  • Figure 12 represents a pM14 expression vector for the expression of keratin and its storage in vacuoles.
  • FIGURE 13 (FIG. 13).
  • Figure 13 represents a pM15 expression vector for the expression of keratin and its storage in chloroplasts.
  • FIGURE 14 (FIG. 14).
  • Figure 14 represents a pM16 expression vector for the expression of actin.
  • FIGURE 15 (FIG. 15).
  • Figure 15 represents a pM17 expression vector for the expression of actin and its storage in vacuoles.
  • FIGURE 16 (FIG. 16).
  • Figure 16 represents a pM18 expression vector for the expression of actin and its storage in chloroplasts.
  • a or “an” entity refers to one or more of that entity; for example, “a nucleic acid sequence,” is understood to represent one or more nucleic acid sequences, unless stated otherwise.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • the term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context.
  • the number of nucleotides in a nucleic acid molecule must be an integer.
  • “at least 18 nucleotides of a 21- nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property.
  • At least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.
  • “At least” is also not limited to integers (e.g., “at least 5%” includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).
  • Percent identity refers to the extent of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity can be determined by aligning two sequences, introducing gaps to maximize identity between the sequences. Alignments can be generated using programs known in the art.
  • nucleotide sequences can be performed with the blastn program set at default parameters, and alignment of amino acid sequences can be performed with the blastp program set at default parameters (see National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).
  • NCBI National Center for Biotechnology Information
  • a myofibril is a striated organelle component of the skeletal muscle that is widely present in different animal species.
  • Myofibrils are made up of thick and thin myofilaments composed of proteins (myofibrillar proteins) such as myosin, actin, tropomyosin, and troponin.
  • a myofibril is formed of a sequence of repeating units, known as sarcomeres. The sarcomere, the basic unit of contraction in muscle, consists of repeating units of thick (myosin) and thin (actin) filaments.
  • Myofibrillar proteins have multiple functionalities such as gelation, emulsification, water binding, and adhesion, which are important for the meat industry. These functionalities are useful for the development of texturized and restructured meat products (Xiong, Y. L. (2014). Encyclopedia of Meat Sciences, 267-273).
  • the invention disclosed herein is directed to the production of recombinant myofibrillar proteins.
  • the myofibrillar proteins disclosed herein comprise actin (e.g., beta-actin, gamma-actin), myosin (e.g., non-muscle and muscle myosin II), actinin (e.g., alphaactinin), nebulin, Cap Z, m-protein, c-protein, M-protein, tropomodulin, troponin (e.g., troponin T, troponin I, and troponin C), tropomyosin, keratin and the fragments thereof.
  • actin e.g., beta-actin, gamma-actin
  • myosin e.g., non-muscle and muscle myosin II
  • actinin e.g., alphaactinin
  • nebulin ebulin
  • Cap Z m-protein, c-protein, M-protein
  • tropomodulin troponin (e.g., troponin T, troponin I,
  • the myosin protein a fibrous and most abundant protein in muscle, is helpful in the formation of thick myofilaments that results in the formation of a strong skeletal muscle, as well as in the formation of the cardiac layers (epicardium, myocardium and endocardium) (Khalid et al. (2022), Int. J. Food Prop. 25(I):375).
  • Myosin is a hexameric macromolecule composed of two heavy chains (MHC) and two pairs of light chains (MLC).
  • myosin isoforms There are several myosin isoforms that show different functions and spatial localization among animal tissues.
  • myosin II (520 kDa) is expressed at levels that far exceed all other myosin isoforms.
  • the light chains of the myosin II are of two types, either essential or regulatory and both are involved in the physiological effects of the motor protein.
  • the N-terminal region is the major functional unit of the MHC, where the actin and ATP binding sites are associated along with the essential and regulatory light chains (Chardulo et al. (2021), Anim. Biotechnol. 32(3): 300).
  • the hexameric protein is also divided into two parts, heavy meromyosin (HMM) and light meromyosin (LMM). These two parts are the results of the chymotrypsin mediated digestion.
  • the Myosin II and the HMM region itself are capable of forming high order polymeric aggregates (Gollapudi et al., (2021), J. Biol. Chem. 296).
  • the tail-tail interactions between the adjacent myosin II molecules results in forming a thick filament consisting of coiled-coil tails and head domains projected on either side of the bundle.
  • These head domains in the presence of Ca +2 ions, ATP molecules, and adjacent thin actin filaments, confer a sliding movement contracting the interlinked actin filaments in muscle (Hellerschmied & Clausen, (2014), Curr. Opin. Struct. Biol. 25: 9).
  • the Myosin II and HMM proteins also show water holding capacity in meat.
  • the water holding capacity of myosin is due to its ability to form polymeric gels (gelation capacity).
  • gelation capacity polymeric gels
  • the interactions between the adjacent myosin heads (Head - Head) or tails (Tail - Tail or Tail - Head) result in a gel matrix.
  • Tropomyosins are the components, along with actin, myosin, and troponin, involved in the sarcomere contractile apparatus in muscle cells.
  • the monomeric subunits of tropomyosin form a-helical coiled-coil that polymerizes in a head-to-tail fashion along actin filaments.
  • the tropomyosin, arranged in dimers, is found in striated muscle and is associated with actin and troponin to form the functional thin filament.
  • tropomyosin is a polymer of approximately 50 glutamyl amino acid residues, and consists of 9 y-glutamyl(E) - leucine (L) dimers and one y-EE-Valine tripeptide which could add the umami and kokumi flavor as an ingredient upon disintegration (Yamamoto, T., (2019), Koku in Food Science and Physiology: Recent Research on a Key Concept in Palatability 17-31).
  • Mammalian cells can produce over 40 different splice variants using four different tropomyosin genes, TPM1 (alpha version), TMP2 (beta version), TPM3 (alpha version), TPM4 (alpha version).
  • TPM1 alpha version
  • TMP2 beta version
  • TPM3 alpha version
  • TPM4 alpha version
  • TPM-1 or a-TM
  • TPM-2 or P-TM
  • these two isoforms form dimers.
  • Keratins are fibrous structural proteins often found in epithelial cells. Keratin serves important structural and protective functions, and also regulates key cellular activities such as cell growth and protein synthesis.
  • the keratin is an alpha-keratin.
  • the keratin can be used as a scaffold protein for tissue engineering.
  • keratin, as a non-burning hydrophilic, and biodegradable compound, can be used for producing bioplastics.
  • the recombinant myofibrillar protein is derived from any of the following animals: bovine, swine, poultry, ovine, and cam elids.
  • the animals are selected from the group consisting of: Bos taurus, Sus scrofa, Gallus gallus, Camelus dromedarius, Camelus bactrianus, Camelus ferus, Lama glama, Lama guanicoe, Vicugna pacos, Vicugna vicugna, Ovis aries, and Capra aegagrus hircus.
  • the recombinant myofibrillar proteins are selected from the group consisting of: myosin, tropomyosin, troponin, and actin.
  • the invention comprises fragments of the recombinant myofibrillar proteins.
  • the recombinant myofibrillar protein sequences comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequences selected from the group consisting of: SEQ ID NOs: 1 to 31.
  • a recombinant nucleic acid encoding for the myofibrillar proteins.
  • the recombinant nucleic acid has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 32 to 73.
  • the myofibrillar protein is heterologously expressed.
  • the heterologously expressed myofibrillar protein is the bovine myosin heavy chain II (MyHC II, SEQ ID NO: 1) (NCBI accession NM 001166227.1).
  • MyHC II bovine myosin heavy chain II
  • the nucleic acid encoding for the MyHC II is 5823 nucleotides (1940 amino acids) long, SEQ ID NO: 32.
  • the heterologously expressed myofibrillar protein is a functional fragment of the Myosin heavy chain II such as the bovine HMM (Heavy Meromyosin Protein, SEQ ID NO: 2).
  • a method to determine the size in nucleotides and amino acids of the bovine HMM is the digestion of the bovine myosin protein by chymotrypsin and pepsin.
  • a smaller and functional fragment of MyHC II is used for expression in a recombinant system.
  • the heterologously expressed myofibrillar protein is a bovine tropomyosin, selected from the group consisting of: TPM-1 (or a- TPM, SEQ ID No 8 and 41) and TPM-2 (or P-TPM, SEQ ID NOs: 9 and 42).
  • TPM-1 or a- TPM, SEQ ID No 8 and 41
  • TPM-2 or P-TPM, SEQ ID NOs: 9 and 42.
  • Nucleic acids encoding for TPM-1 (NCBI accession AB 198070.1) and TPM-2 (NCBI accession AB 198071.1) are 855 nucleotides (284 amino acids) long each.
  • both genes are linked in an expression cassette via a linker sequence (SEQ ID NO: 45 - linker-, SEQ ID NO: 46 - a-TPM+linker+P-TPM).
  • TPM-1 and TPM-2 genes are expressed separately, each of them individually cloned into an expression cassette.
  • the hosts suitable for the expression of the recombinant myofibrillar proteins are bacteria, yeasts, multicellular eukaryotic hosts, such as filament fungi, and plants.
  • the host for the recombinant expression of myofibrillar protein is a eukaryotic organism, such as fungi and plants.
  • the host is a plant.
  • the plant is a dicotyledon plant.
  • the nucleic acids encoding for the myosin gene can be codon optimized for the expression in several hosts.
  • the cDNA encoding for the bovine MyHC II can be optimized for the expression in dicotyledon plants; SEQ ID NO: 33 is a nonlimiting example of codon optimized bovine MyHC II for the expression in dicotyledon plants (e.g., tobacco and soy).
  • SEQ ID NO: 33 is a nonlimiting example of codon optimized bovine MyHC II for the expression in dicotyledon plants (e.g., tobacco and soy).
  • the heterologous expression of large myofibrillar proteins such as the myosin or a functional fragment of the MyHC II can be expressed recombinantly.
  • the HMM fragment SEQ ID NO: 35 was expressed in tobacco.
  • the heterologously expressed myofibrillar protein is a Sus scrofa myosin heavy chain (SEQ ID NO: 3), wherein the nucleic acid encoding for the expression of the myosin heavy chain has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36.
  • the heterologously expressed myofibrillar protein is a Gallus gallus myosin heavy chain (SEQ ID NO: 4), wherein the nucleic acid encoding for the expression of the myosin heavy chain has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37.
  • the heterologously expressed myofibrillar protein is a Camelus dromedarius myosin heavy chain (SEQ ID NO: 5), wherein the nucleic acid encoding for the expression of the myosin heavy chain has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.
  • the heterologously expressed myofibrillar protein is a Ovis aries myosin heavy chain (SEQ ID NO: 6), wherein the nucleic acid encoding for the expression of the myosin heavy chain has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39.
  • the heterologously expressed myofibrillar protein is a Capra aegagrus hircus myosin heavy chain (SEQ ID NO: 7), wherein the nucleic acid encoding for the expression of the myosin heavy chain has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.
  • the nucleic acids encoding for the tropomyosin can be optimized for the expression in several hosts.
  • the cDNA encoding for the bovine TPM-1 and TPM-2 (SEQ ID NO: 41 and SEQ ID NO: 42, respectively) can be optimized for the expression in dicotyledon plants (e.g., tobacco and soy).
  • dicotyledon plants e.g., tobacco and soy.
  • Non-limiting examples of tropomyosin codon-optimized sequences for the expression in dicotyledon plants such as tobacco and soy are SEQ ID NO: 43 (TPM-1) and SEQ ID No 44 (TPM-2).
  • the heterologously expressed myofibrillar protein is a Sus scrofa TPM1, (SEQ ID NO: 10), wherein the nucleic acid encoding for the expression of the TPM1 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 47.
  • the heterologously expressed myofibrillar protein is a Sus scrofa TPM2, (SEQ ID NO: 11), wherein the nucleic acid encoding for the expression of the TPM2 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48.
  • the nucleic acid encoding for the expression of Sus scrofa TPM1 and TPM1 is codon optimized for expression in legume plants such as soybean.
  • the codon optimized sequences of Sus scrofa for TPM1 and TPM2 comprises a sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71 and SEQ ID NO: 72 respectively.
  • SEQ ID NO: 71 and SEQ ID NO: 72 are fused through a linker sequences, resulting in a single polynucleotide chain with a sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73.
  • the heterologously expressed myofibrillar protein is a Gallus gallus TPM1, (SEQ ID NO: 12), wherein the nucleic acid encoding for the expression of the TPM1 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49.
  • the heterologously expressed myofibrillar protein is a Gallus gallus TPM2, (SEQ ID NO: 13), wherein the nucleic acid encoding for the expression of the TPM2 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50.
  • the heterologously expressed myofibrillar protein is a Camelus dromedarius TPM1, (SEQ ID NO: 14), wherein the nucleic acid encoding for the expression of the TPM1 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:
  • the heterologously expressed myofibrillar protein is a Camelus dromedarius TPM2, (SEQ ID NO: 15), wherein the nucleic acid encoding for the expression of the TPM2 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:
  • the heterologously expressed myofibrillar protein is a Ovis aries TPM1, (SEQ ID NO: 16), wherein the nucleic acid encoding for the expression of the TPM1 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53.
  • the heterologously expressed myofibrillar protein is a Ovis aries TPM2, (SEQ ID NO: 17), wherein the nucleic acid encoding for the expression of the TPM2 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 54.
  • the heterologously expressed myofibrillar protein is a Capra aegagrus hircus TPM1, (SEQ ID NO: 18), wherein the nucleic acid encoding for the expression of the TPM1 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55.
  • the heterologously expressed myofibrillar protein is a Capra aegagrus hircus TPM2, (SEQ ID NO: 19), wherein the nucleic acid encoding for the expression of the TPM2 has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 56.
  • the heterologously expressed myofibrillar protein is a. Bos taurus troponin, (SEQ ID NO: 20), wherein the nucleic acid encoding for the expression of the troponin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of SEQ ID NO: 57, or SEQ ID NO: 58.
  • the heterologously expressed myofibrillar protein is a Sus scrofa troponin (SEQ ID NO: 21), wherein the nucleic acid encoding for the expression of the troponin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59.
  • the heterologously expressed myofibrillar protein is a Gallus gallus troponin, (SEQ ID NO: 22), wherein the nucleic acid encoding for the expression of the troponin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60.
  • the heterologously expressed myofibrillar protein is a Camelus dromedarius troponin, (SEQ ID NO: 23), wherein the nucleic acid encoding for the expression of the troponin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 61.
  • the heterologously expressed myofibrillar protein is a Ovis aries troponin, (SEQ ID NO: 24), wherein the nucleic acid encoding for the expression of the troponin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 62.
  • the heterologously expressed myofibrillar protein is a Capra aegagrus hircus troponin, (SEQ ID NO: 25), wherein the nucleic acid encoding for the expression of the troponin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63.
  • the heterologously expressed myofibrillar protein is Bos taurus actin, (SEQ ID NO: 26), wherein the nucleic acid encoding for the expression of the actin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the following sequences SEQ ID NO: 64, or SEQ ID NO: 65.
  • the heterologously expressed myofibrillar protein is a Sus scrofa actin, (SEQ ID NO: 27), wherein the nucleic acid encoding for the expression of the actin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66 and/or SEQ ID NO: 98.
  • the heterologously expressed myofibrillar protein is a Gallus gallus actin, (SEQ ID NO: 28), wherein the nucleic acid encoding for the expression of the actin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67.
  • the heterologously expressed myofibrillar protein is a Camelus dromedarius actin, (SEQ ID NO: 29), wherein the nucleic acid encoding for the expression of the actin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68.
  • the heterologously expressed myofibrillar protein is a Ovis aries actin, (SEQ ID NO: 30), wherein the nucleic acid encoding for the expression of the actin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69.
  • the heterologously expressed myofibrillar protein is a Capra aegagrus hircus actin, (SEQ ID NO: 31), wherein the nucleic acid encoding for the expression of the actin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70.
  • the heterologously expressed myofibrillar protein is a Sus scrofa keratin 2 (KRT2), (SEQ ID NO: 94), wherein the nucleic acid encoding for the expression of keratin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 92 and/or SEQ ID NO: 93.
  • the recombinant myofibrillar protein is a fragment of the native myofibrillar protein.
  • the fragment of the myofibrillar protein comprises a functional domain of the native myofibrillar protein.
  • the fragment of the myofibrillar protein comprises a portion of the native myofibrillar protein with high biological value, and/or with high digestibility.
  • the myofibrillar protein comprises more than one polypeptide chain.
  • a linker is inserted between both nucleic acids encoding for the polypeptide chains.
  • the linker is a short or long or rigid or flexible or cleavable linker (Chen, X., Zaro, J. L., & Shen, W. C., Fusion protein linkers: property, design and functionality, Advanced drug delivery reviews, 65(10), 1357-1369 (2013)).
  • the recombinant myofibrillar protein is a fusion protein of at least two polypeptides, wherein said polypeptides code for a single myofibrillar protein or a fragment thereof. In some aspects, the at least two polypeptides code for at least two different myofibrillar proteins or the fragments thereof.
  • Plants as hosts for expression of myofibrillar proteins Plants as hosts for expression of myofibrillar proteins.
  • plant includes reference to whole plants, plant organs, plant tissues, and plant cells, and progeny of the same and includes all monocotyledon and dicotyledon. In some aspects, plant also includes seeds, plant progeny, propagules whether sexually or asexually, descendants of these, such as cuttings or seed, as well as pre-harvest and post-harvest tissues and organs.
  • plant part refers to any part of a plant including but not limited to the embryo, shoot, root, stem, seed, stipule, leaf, petal, flower bud, flower, ovule, bract, trichome, branch, petiole, internode, bark, pubescence, tiller, rhizome, frond, blade, ovule, pollen, stamen, and the like.
  • the two main parts of plants grown in some sort of media, such as soil or vermiculite are often referred to as the “above-ground” part, also often referred to as the “shoots”, and the “below-ground” part, also often referred to as the “roots”.
  • plant tissue refers to any part of a plant, such as a plant organ.
  • plant organs include, but are not limited to the leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen, and leaf sheath.
  • seed is meant to encompass the whole seed and/or all seed components, including, for example, the coleoptile and leaves, radicle and coleorhiza, scutellum, starchy endosperm, aleurone layer, pericarp and/or testa, either during seed maturation and seed germination.
  • the plant is a legume plant.
  • the genetically engineered plant is selected from the group consisting of: beans, soybeans, chickpeas, peanuts, lentils, lupins, grass peas, mesquite, carob, tamarind, alfalfa, and clover.
  • the legume plants can be selected from the genus Phaseolus, Pisum, Vigna, Cajanus, Lens, Cicer, Vicia, Arachis, Glycine, Macrotyloma, Mucuna, Lupinus, Ceratonia, Canavalia, Cyamopsis, Lablab, Psophocarpus, Clitoria, Lathryus, Trifolium, Medicago, Melilotus, and Tamarindus.
  • the bean can be selected from the species selected from the group consisting of: Phaseolus vulgaris, Phaseolus lunatus, Phaseolus coccineus, Phaseolus acutifolius, Pisum sativum, Vigna radiata, Vigna mungo, Vigna uniguiculata, Vigna aconitifoilia, Vigna angularis, Cajanus cajan, Lens culinaris, Cicer arietinum, Vicia faba, Vicia ervilia, Vicia sativa, Arachis hypogaea, Glycine max, Macrotyloma uniflorum, Mucuna pruriens, Lupinus albus, Lupinus mutabilis, Ceratonia siliqua, Canavalia gladiata, Canavalia ensiformis, Cyamopsis tetragonoloba, Lablab purpureus, Psosphocarpus tetranoglobulus, Clitoria ternatea
  • the plant is selected from the group consisting of: Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum L., Oryza sativa, Hordeum vulgare, Triticum aestivum, Zea mays, Secale cereale, Avena sativa, Beta vulgaris, Pastinaca sativa, Eruca vesicaria, Sinapis alba, Brassica juncea, Brassica nigra, Lactuca sativa, Solanum tuberosum, Ipomoea batatas, Manihot esculenta, triticale and Triticum spelta.
  • transgenic plant means a plant that has been transformed with one or more exogenous nucleic acids (recombinant sequences).
  • transformation refers to a process by which a recombinant sequence is introduced and expressed in a plant cell.
  • plant stable transformation the foreign DNA is fully integrated into the host genome and expressed in later generations of the plant.
  • plant transient transformation the foreign DNA is not integrated into the host genome and it is not expressed in later generations of the plant. Transformation may occur through Agrobacterium-inoculation, viral infection, electroporation, heat shock, lipofection, polyethylene glycol treatment, microinjection, and particle bombardment methods.
  • “Stably integrated” refers to the permanent, or non-transient retention and/or expression of a polynucleotide in and by a cell genome.
  • a stably integrated polynucleotide is one that is a fixture within a transformed cell genome and can be replicated and propagated through successive progeny of the cell or resultant transformed plant. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of nucleic acid sequences into a prokaryotic or eukaryotic host cell, including Agrobacterium-mediated transformation protocols, viral infection, whiskers, electroporation, heat shock, lipofection, polyethylene glycol treatment, micro-injection, and particle bombardment.
  • stably expressed or “stable expression” refer to expression and accumulation of a protein in a plant cell.
  • a protein may accumulate because it is not degraded by endogenous plant proteases.
  • a protein is considered to be stably expressed in a plant if it is present in the plant in an amount of 1% or higher per total protein weight of soluble protein extractable from the plant.
  • gene regulatory elements or a “transcription regulatory region” refers to nucleic acid sequences that influence and/or promote initiation of transcription. Transcription regulatory regions are typically considered to include regulatory regions, such as enhancer or inducer elements. The gene regulatory elements will generally be appropriate to the host cell in which the target gene is being expressed. The regulatory elements, together with other transcriptional and translational regulatory nucleic acid sequences (also termed “control sequences”), is necessary to express any given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • signal peptide also known as “signal sequence”, “targeting signal”, “localization signal”, “localization sequence”, “transit peptide”, “leader sequence”, or “leader peptide”, is used herein to refer to an N-terminal peptide which directs a newly synthesized protein to a specific cellular location or pathway. Signal peptides are often cleaved from a protein during translation or transport and are therefore not typically present in a mature protein.
  • expression cassettes or vectors for the expression of recombinant proteins in plants, to produce a recombinant protein.
  • the expression cassette or vector may comprise, for example, a promoter, a 5' untranslated region (UTR), a sequence encoding one or more heterologous proteins, and a terminator.
  • the expression cassette or vector may further comprise a selectable marker, a retention signal and/or a signal peptide to plastid or vacuole storage.
  • the transgenic plant is selected from the group consisting of: Arabidopsis thaliana, Nicotiana benthamiana, Nicotiana tabacum L., Glycine max, Pisum sativum, Oryza sativa, Hordeum vulgare, Triticum aestivum, Zea mays, Secale cereale, Avena sativa, Beta vulgaris, , Pastinaca sativa, Vigna angularis, Vigna radiata, Arachis hypogaea, Lens culinaris, Phaseolus vulgaris, Cicer arietinum, Medicago sativa, Eruca vesicaria, Sinapis alba, Brassica juncea, Brassica nigra, Lactuca sativa, Solanum tuberosum, Ipomoea batatas, Manihot esculenta, triticale and Triticum spelta.
  • the term “recombinant protein” or “recombinant myofibrillar protein” refers to a recombinant protein, where the recombinant protein is codified by foreign cDNA encoding for a myofibrillar protein.
  • the term “exogenous nucleic acid” means a cDNA coding for the recombinant myofibrillar protein; also, the term “exogenous nucleic acid” is used herein interchangeably with “recombinant nucleic acid”.
  • the transcription and further translation of said “exogenous nucleic acid” by the transgenic host, is known as “heterologous expression”.
  • the recombinant nucleotide sequence comprises a sequence named “promoter” that refers to nucleic acid sequences that promote initiation of transcription.
  • the promoter may be a constitutive promoter.
  • a constitutive promoter is capable of initiating transcription in a cell (e.g., a plant cell) under any circumstances and its activity is not affected by environmental conditions.
  • the expression cassette comprises a nucleic acid sequence encoding a recombinant protein, which is operably linked to a promoter.
  • the nucleic acid comprises, in order from 5' to 3', a promoter, optionally a 5' untranslated region (UTR), a sequence encoding a heterologous protein, a terminator and optionally a translation enhancer sequence.
  • promoters are tissue specific because these promoters preferentially initiate transcription in certain organs.
  • Other promoters are inducible, modulated by external stimuli such as different chemical, biotic and abiotic environmental factors.
  • the promoter of the present disclosure is a “plant promoter”, preferably a “legume plant promoter”.
  • promoters under developmental control include promoters that preferentially initiate transcription in certain organs, such as leaves, roots, flowers, seeds and tissues such as fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue-preferred.” Promoters which initiate transcription only in certain tissue are referred to as “tissue-specific.”
  • a “celltype” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in leaves, roots, flowers, or seeds.
  • An “inducible” promoter is a promoter which is under environmental control.
  • inducible promoters examples include anaerobic conditions or the presence of light.
  • Tissue-specific, tissue-preferred, cell-type specific, and inducible promoters constitute the class of “non-constitutive” promoters.
  • a “constitutive” promoter is a promoter which is active under most environmental conditions.
  • the promoter when the host is a plant, is a constitutive promoter such as the 2x35 S promoter derived from the cauliflower mosaic virus (CaMV).
  • the promoter is a tissue specific promoter.
  • the tissue specific promoter could be the beta-conglycinin alpha’ subunit of the 7S storage protein (7s) promoter from soybean (Zakharov et al. (2004), J. Exp. Bot. 55: 1463), beta-phaseolin (phas) promoter from common bean (Zakharov et al. (2004), J. Exp. Bot. 55: 1463), USP promoter from Vicia faba (Zakharov et al. (2004), J. Exp. Bot.
  • Biotechnol.172: 1763 a-globulin promoter from cotton (Sunilkumar et al. (2002), Transgenic Res. 11 :347), y-zein promoter from maize (Marzabal et al. (1998), Plant J. 16:41), Glutenin promoter from wheat (Lamacchia et al. (2001), J. Exp. Bot. 52:243), VVPVPE promoter from Vitis spp (Gong et al. (2019), Planta, 250:657), Groundnut seed promoter (GSP) from peanut (Sunkara et al. (2014), Appl. Biochem. Biotechnol. 172: 325), 7aP promoter from soybean (Fu et al.
  • AtLAC15 promoter from Arabidopsis thaliana (El-Mezawy et al. (2009), Biotechnol. Lett. 31 : 1961), SSPs promoter from chickpea (Verma & Bhatia, (2019), Funct. Integr. Genomics, 19:373), Lectin promoter from soybean (Ma et al. (2008), J. Plant Growth Regul. 27:68), Oleosin promoter from Brassica napus (Keddie et al. (1994) Plant Mol. Biol. 24:327), AhLEClA promoter from peanut (Tang et al.
  • the recombinant sequences comprise a sequence named “terminator” that refers to nucleic acid sequences that define the end of a gene.
  • Useful terminators include the following, but not limited to: Extensin terminator from tobacco (Rosenthal et al. (2016), Plant Mol. Biol. 96:429), UblO terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022: 1), Hsp70 terminator from Arabidopsis thaliana, (Tian et al. 2002, BIO-DES MANUF.
  • Hspl8.2 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022: 1), Act2 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022: 1), G7 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022: 1), 3g24240 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022: 1), NOS terminator from Agrobacterium tumefaciens (Tian et al. 2002, BIO-DES MANUF.
  • the translational or transcriptional enhancer sequence of the present disclosure is a “plant translational or transcriptional enhancer”.
  • a plant translation enhancer is the 5’ UTR TEV (Tobacco Etch Virus Translational Enhancer) (SEQ ID NO: 74).
  • the 5' leader of the tobacco etch virus (TEV) is one of the better-studied potyvirus translational enhancers, it contains two cap-independent regulatory elements (CIREs) that fold into pseudoknots, which can independently enhance translation of the downstream transgene (Carrington & Freed, (1993) J. Virol., 64: 1590).
  • the recombinant sequences include a matrix attached region (MAR) as enhancers.
  • MAR matrix attached region
  • the Rb7 MAR (SEQ ID NO:75) is a DNA element shown to increase transgene expression in plants.
  • the addition of the Rb7 MAR has been shown to strongly enhance protein production when added to most transcriptional terminators (Diamos & Mason, (2016), Plant Bi otechnol. J. 16: 1971).
  • MARs can further improve the stability of transgene expression levels and may confer protection against transgene silencing (Vain et al. (1999), Plant J. 18:233).
  • the arc5 terminator is fused to the Rb7 Matrix Attachment Region (MAR) that increases the likelihood and magnitude of transgene expression.
  • MAR Rb7 Matrix Attachment Region
  • the Rb7MAR enhancer comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 75.
  • signal peptide also known as “signal sequence”, “targeting signal”, “localization signal”, “localization sequence”, “transit peptide”, “leader sequence”, or “leader peptide”, refers to an N-terminal peptide which directs a newly synthesized protein to a specific cellular location or pathway. Signal peptides are often cleaved from a protein during translation or transport and are therefore not typically present in a mature protein. In some aspects, the signal peptide signal sequences of the present disclosure are “plant signal peptide signal sequence”.
  • the signal peptide signal sequence is the 168-bp chloroplast signal from pea (Pisum sativum) from the ribulose 1,5 -bisphosphate carboxylase gene (GenBank X04334.1) (SEQ ID NO: 76). In some aspects, the signal peptide signal sequence is the signal peptide of Nicotiana tabacum osmotin Gene AP24 (SEQ ID NO: 77).
  • the 168-bp chloroplast signal from pea (Pisum sativum) from the ribulose 1,5-bisphosphate carboxylase gene encoding for the signal peptide comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 76.
  • the signal peptide of Nicotiana tabacum osmotin Gene AP24 comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 77.
  • the expression cassette also contains a signal peptide to direct the recombinant proteins through the ER and Golgi apparatus for functional folding, preferably this signal peptide is KDEL (SEQ ID NO: 78).
  • this signal peptide is KDEL (SEQ ID NO: 78).
  • the KDEL signal peptide comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 78.
  • the KDEL signal peptide comprises a two-amino-acid linker, to operatively link the KDEL to the gene encoding the recombinant protein of interest.
  • the two amino acid sequence linker comprises the sequence SEQ ID NO:79.
  • the KDEL signal peptide also contains a stop codon.
  • the expression cassette comprises a seed-specific promoter such the 7S or phas, a terminator such arc5 fused to the Rb7MAR enhancer, and the optional inclusion of signal peptides such as a vacuole storage signal peptide, a chloroplast storage signal peptide or a KDEL signal peptide.
  • GOI refers to the gene of interest coding for the recombinant protein of interest
  • SPChl refers to the signal peptide to chloroplast storage
  • SPV refers to the signal peptide to vacuole storage.
  • the expression cassette comprises a Phas promoter operatively linked to the 5’ end of the GOI, an arc5 terminator located at the 3’ end of the GOI, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette comprises a Phas promoter operatively and a signal peptide to vacuole storage linked to the 5’ end of the GOI, an arc5 terminator located at the 3’ end of the GOI, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette comprises a Phas promoter and a signal peptide to chloroplast storage are operatively linked to the 5’ end of the GOI, an arc5 terminator located at the 3’ end of the GOI, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette comprises a Phas promoter operatively linked to the 5’ end of the tropomyosin coding gene, an arc5 terminator located at the 3’ end of the tropomyosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the cassette expression vector for tropomyosin expression comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 83.
  • the expression cassette comprises a Phas promoter and a signal peptide to vacuole storage operatively linked to the 5’ end of the tropomyosin coding gene, a KDEL and an arc5 terminator located at the 3’ end of the tropomyosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing Tropomyosin, wherein the Tropomyosin expression directed to vacuole storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 84.
  • the expression cassette comprises a Phas promoter and a signal peptide to chloroplast storage operatively linked to the 5’ end of the tropomyosin coding gene, an arc5 terminator located at the 3’ end of the tropomyosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing Tropomyosin, wherein the Tropomyosin expression directed to chloroplast storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 85.
  • the expression cassette comprises a Phas promoter operatively linked to the 5’ end of the Myosin coding gene, an arc5 terminator located at the 3’ end of the Myosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the cassette expression vector for Myosin expression comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 86.
  • the expression cassette comprises a Phas promoter and a signal peptide to vacuole storage operatively linked to the 5’ end of the Myosin coding gene, a KDEL and an arc5 terminator located at the 3’ end of the Myosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing Myosin, wherein the Myosin expression directed to vacuole storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 87.
  • the expression cassette comprises a Phas promoter and a signal peptide to chloroplast storage operatively linked to the 5’ end of the Myosin coding gene, an arc5 terminator located at the 3’ end of the Myosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing Myosin, wherein the Myosin expression directed to chloroplast storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 88.
  • the expression cassette comprises a Phas promoter operatively linked to the 5’ end of the Heavy Mero Myosin coding gene, an arc5 terminator located at the 3’ end of the Heavy Mero Myosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the cassette expression vector for Heavy Mero Myosin expression comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 89.
  • the expression cassette comprises a Phas promoter and a signal peptide to vacuole storage operatively linked to the 5’ end of the Heavy Mero Myosin coding gene, a KDEL and an arc5 terminator located at the 3’ end of the Heavy Mero Myosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing Heavy Mero Myosin, wherein the Myosin expression directed to vacuole storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 90.
  • the expression cassette comprises a Phas promoter and a signal peptide to chloroplast storage operatively linked to the 5’ end of the Heavy Mero Myosin coding gene, an arc5 terminator located at the 3’ end of the Heavy Mero Myosin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing Heavy Mero Myosin, wherein the Myosin expression directed to chloroplast storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 91.
  • the expression cassette comprises a Phas promoter operatively linked to the 5’ end of actin coding gene, an arc5 terminator located at the 3’ end of actin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the cassette expression vector for actin expression comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the expression cassette comprises a Phas promoter and a signal peptide to vacuole storage operatively linked to the 5’ end of actin coding gene, a KDEL and an arc5 terminator located at the 3’ end of actin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing actin, wherein the actin expression directed to vacuole storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 100.
  • the expression cassette comprises a Phas promoter and a signal peptide to chloroplast storage operatively linked to the 5’ end of actin coding gene, an arc5 terminator located at the 3’ end of the actin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing actin, wherein the actin expression directed to chloroplast storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 101.
  • the expression cassette comprises a 2x CaMV35S promoter, a Nopaline synthase terminator (TNOS), and a bovine myosin II coding gene.
  • the expression cassette for producing myosin II comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 102.
  • the expression cassette comprises a 2x CaMV35S promoter, a Nopaline synthase terminator (TNOS), and a bovine HMM (Heavy Meromyosin Protein) coding gene.
  • the expression cassette for producing HMM comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 103.
  • the expression cassette comprises a CaMV35S promoter, a Nopaline synthase terminator (TNOS), and a tropomyosin (TPM1-LL-TPM2) coding gene.
  • the expression cassette for producing tropomyosin comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 104.
  • the expression cassette disclosed herein can be used for the expression of several recombinant proteins.
  • the recombinant proteins are extracellular fibrillar proteins such as collagen, gelatin, keratin and elastin.
  • the recombinant proteins are milk proteins such as casein, lactalbumins, and lactoglobulin.
  • the recombinant proteins are egg proteins such as ovalbumin, ovotransferrin, and ovomucoid.
  • the recombinant proteins are enzymes useful for the food industry.
  • the expression cassette comprises a Phas promoter operatively linked to the 5’ end of the keratin coding gene, an arc5 terminator located at the 3’ end of the keratin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the cassette expression vector for keratin expression comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 95.
  • the expression cassette comprises a Phas promoter and a signal peptide to vacuole storage operatively linked to the 5’ end of Keratin coding gene, a KDEL and an arc5 terminator located at the 3’ end of keratin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing Keratin, wherein the Keratin expression directed to vacuole storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 96.
  • the expression cassette comprises a Phas promoter and a signal peptide to chloroplast storage operatively linked to the 5’ end of Keratin coding gene, an arc5 terminator located at the 3’ end of the Keratin coding gene, and following the arc5, a Rb7Mar region attachment.
  • the expression cassette is for producing Keratin, wherein the Keratin expression directed to chloroplast storage comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 97.
  • the expression cassette disclosed herein can be used for the expression of recombinant proteins useful in therapeutics, such as, cytokines (like interferons), hormones (like insulin and glucagon), blood proteins (like hemoglobin, albumin, immunoglobulins, coagulation factors - factor VIII and factor IX-, antithrombin and al antitrypsin), antigens for vaccine production.
  • cytokines like interferons
  • hormones like insulin and glucagon
  • blood proteins like hemoglobin, albumin, immunoglobulins, coagulation factors - factor VIII and factor IX-, antithrombin and al antitrypsin
  • antigens for vaccine production such as, cytokines (like interferons), hormones (like insulin and glucagon), blood proteins (like hemoglobin, albumin, immunoglobulins, coagulation factors - factor VIII and factor IX-, antithrombin and al antitrypsin), antigens for vaccine production.
  • the myofibrillar proteins are animal derived myofibrillar proteins.
  • the myofibrillar proteins are derived from red meat (e.g., beef, pork, goat, and lamb), poultry (e.g., chicken and turkey), and seafood (e.g., fish, crustaceans, and molluscus).
  • the animal derived myofibrillar protein is myosin II, the HMM (Heavy Meromyosin Protein), and/or the tropomyosin.
  • the animal derived myofibrillar protein comprises actin, troponin, a- actinin, elastin, collagen (I, II and III), keratin, laminin (B, 2, 10), fibronectin, fibrinogen, and/or thrombin and the fragments thereof.
  • TSP total soluble protein
  • the expression level in TSP refers to an amount of a protein of interest relative to the total amount of protein that may reasonably be extracted from a plant using standard methods. Methods for extracting total protein from plant tissues such as seeds are known in the art (Cunha, N., et al., Expression of functional recombinant human growth hormone in transgenic soybean seeds, Transgenic Res. 20:811-826 (2011); Cunha, N., et al., Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds, Transgenic Res. 20:841-855 (2011); Ding, S.
  • a transgenic plant, plant tissue, or plant cell comprising an expression cassette comprising an exogenous nucleic acid encoding for an animal myofibrillar protein.
  • said nucleic acid is operatively linked to a seed-specific promoter and a transcription terminator.
  • said animal myofibrillar protein is expressed in a seed in an amount of about 5% TSP.
  • said animal myofibrillar protein is expressed in a seed in an amount of about 6% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 7% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 8% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 9% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 10% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 11% TSP.
  • said animal myofibrillar protein is expressed in a seed in an amount of about 12% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 13% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 14% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 15% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 18% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 20% TSP.
  • said animal myofibrillar protein is expressed in a seed in an amount of about 25% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount of about 30% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount between about 5% TSP and about 35% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount between about 8% TSP and about 35% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount between about 10% TSP and about 35% TSP.
  • said animal myofibrillar protein is expressed in a seed in an amount between about 12% TSP and about 35% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount between about 5% TSP and about 30% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount between about 5% TSP and about 29% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount between about 5% TSP and about 28% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount between about 10% TSP and about 30% TSP.
  • said animal myofibrillar protein is expressed in a seed in an amount between about 8% TSP and about 30% TSP. In some aspects, said animal myofibrillar protein is expressed in a seed in an amount between about 6% TSP and about 28% TSP.
  • the myofibrillar proteins and transgenic plants, plant cell, plant part, and plant tissues described herein may be used to prepare food compositions.
  • the recombinant myofibrillar proteins produced by the transgenic plants may be used in its entirety, fractions and modifications thereof including solubilized, precipitated, partially or fully hydrolyzed, crosslinked, emulsified, texturized, cooked, extruded, reacted, structured versions to prepare meat-like (meat analogs) food stuffs including comminuted meats such as minced meat, meat strips, cubes and steaks; reconstituted and formed meat-like products including burgers, fillets, balls, sticks, slabs; reconstituted and stuffed/filled meat-like (meat analog) products including sausages, hamlike products, spreadables, reconstituted and coated meat-like products including nuggets, patties, strips, poppers, rings and more.
  • the recombinant myofibrillar proteins may also be used to prepare food compositions
  • the food composition is prepared using the seed of the transgenic plant expressing the recombinant myofibrillar protein. In some aspects, the food composition is prepared using the recombinant myofibrillar protein extracted and purified from the seed.
  • the bovine Myosin II (bMII), the bovine HMM (Heavy Meromyosin Protein), and the bovine tropomyosin (TPM1-LL-TPM2) genes were synthesized and cloned into transformation vectors.
  • the myosin II gene sequence was obtained from the NCBI accession NM_001166227.1 vector (SEQ ID NO: 32).
  • the HMM gene size and sequence was determined via BLAST homology between the alignment of the bMII (NCBI accession NM 001166227.1) against the HMM from Gallus gallus (chicken) (Walcott et. Al 2009; Wendt et al 1999).
  • the resulting HMM gene protein size is 1169 aa (SEQ ID NO: 2) protein.
  • the tropomyosin gene consists of the TPM1 (alpha version; SEQ ID NO: 41) and TPM2 (beta version; SEQ ID NO: 42) and were obtained from the NCBI accessions AB198070.1 and AB198071.1, respectively.
  • the TPM1 and TPM2 were linked via a long linker (LL) of 63 bp (SEQ ID NO: 45) for a final sequence length of 1770 bp (SEQ ID NO: 46). All codon- optimized genes were synthesized by Genscript.
  • the bMII, HMM, and TPM1-LL-TPM2 genes were independently cloned into the pRG-2x35S-TNOS vector using the Ncol and Xbal restriction sites.
  • the bMII, HMM, and TPM1-LL-TPM2 genes were cloned in between the 35S promoter and NOS terminator to create the pRG-2x35S+bMII+TNOS vector (FIG. 1 - pMOl - SEQ ID No 102), pRG-2x35S+ HMM+TNOS vector (FIG. 2 - pM02 - SEQ ID No 103), and pRG- 2x35S+TPMl-LL-TPM2+TNOS vector (FIG. 3 - pM03 - SEQ ID No 104), respectively.
  • Agrobacterium cultures transformed with either pRG-x35S+bMII+TNOS, pRG- 2x35 S+ HMM+TNOS, and pRG-2x35S+TPMl-LL-TPM2+TNOS vectors were collected by centrifugation at 3000 rpm for 30 min.
  • the Agrobacterium pellets were then suspended in infiltration medium (10 mM MgC12, 5 mM MES-KOH (pH 5.6) and 1.6 mM AS) and adjusted to a final OD600 of 1.0.
  • Agrobacterium cultures were then incubated at room temperature for 3 hours.
  • Agrobacterium cultures were diluted on a 1 : 1 ratio with infiltration medium, just before infiltration. Cultures were introduced into the leaves of N. benthamiana by loading 1-mL needleless syringe with the mixture. A nick was made on the underside of the lower two leaves of the plant using the comer of a razor blade. The mouth of the syringe was placed on the nick and the Agrobacterium culture was injected into the leaf.
  • Example 3 Transient expression analysis.
  • Protein extraction Approximately 1 g of frozen leaf tissue was ground by hand in a pre-chilled mortar and pestle containing 3 mL of urea protein extraction buffer.
  • the protein extraction buffer consisted of 7 M urea, 2 M thiourea, 40 mM Tris base, 1% (v/v) Triton-xlOO, 2 mg bromophenol blue, as well as 2 mm DTT added immediately before use.
  • the solution was vortexed for 10 seconds prior to three 15-second rounds of ultrasonic processing with a GEX 130 ultrasonic processor (Cole Parmer, Vernon Hills, IL) on ice at 20,000 hz, pausing 30 seconds between each round to avoid excessive sample heating.
  • Samples were centrifuged at 4°C for 20 minutes at 14,000 rpm; the supernatant was collected and mixed with lOOx protease inhibitor cocktail (Sigma- Aldrich, St. Louis, MO). Sample protein quantity was determined spectrophotometrically at 750 nm with the Bio Rad RC DC protein assay kit (Bio-Rad Laboratories, Hercules, CA).
  • Blots were rinsed several times in TBS and blocked for 2 hours at room temperature in blocking buffer (25 mM Tris-HCl, pH 7.2; 150 mM NaCl; 0.3% [v/v] Tween 20; 0.2% [w/v] bovine serum albumin).
  • blocking buffer 25 mM Tris-HCl, pH 7.2; 150 mM NaCl; 0.3% [v/v] Tween 20; 0.2% [w/v] bovine serum albumin).
  • Protein TPM1-LL-TPM2 was detected from extracts transformed with pM03:Tropomyosin_Transient (FIG. 4), respectively.
  • pM03:Tropomyosin_Transient FIG. 4
  • the heterologous tropomyosin 78-80 kDa
  • shows a higher molecular mass than expected 66.8 kDa
  • Protein sequencing The protein extract from pM03 :Tropomyosin_Transient was run on a gel and the expected ban was excised and subsequently sequenced via Thermo Orbitrap Fusion mass spectrometry at the TIMPL laboratory at Washington State University. Sequencing results show a 100% homology between the extracted tropomyosin protein and the expected sequence.
  • the regulatory elements promoter, signal peptide, Tag, terminator and Matrix Attachment region; see Table 1
  • GOI HMM or tropomyosin; see Table 1
  • Table 1 Gene of interest and regulatory elements per vector for stable transformation of legumes.
  • the regulatory elements promoter, signal peptide, Tag, terminator and Matrix Attachment region; see Table 2) and GOI (Keratin or Actin; see Table 2) were incorporated into the in-house pMoolecl plasmid.
  • the Keratin and Actin genes (from porcine origin) sequences were codon-optimized for dicotyledon plant expression (SEQ ID NO: XX and XX, respectively).
  • Table 2 Gene of interest and regulatory elements per vector for stable transformation of A. thaliana.
  • Example 6 Total Soluble Protein Production in Soybean Transgenic events carrying the myofibrillar proteins gene.
  • the transgenic TO plants will be transformed with pM04, pM05, pM06, pMIO, pMl l pM12, pM13, pM14, pM15, pM16, pM17, pM18.
  • the plants will be cultivated and propagated up to T2 seeds.
  • T1 and/or T2 seeds will be screened for the presence of the myofibrillar gene via PCR; a small section of the seed will be excised for PCR purposes.
  • Protein extraction will be performed for positive seeds per transgenic event.
  • Seeds will be ground in extraction buffer (5% w/v SDS, 175 mM Tris-HCl, pH 8.0, 0.4% v/v beta-mercaptoethanol) with Omni ceramic beads (1.4 mm); the extracts will be heated to 65 °C for 25 minutes, centrifuged and the supernatants will be transferred to fresh tubes.
  • Myofibrillar gene protein quantitation from seed extracts will be done using company- manufactured ELISA kits. All samples will be normalized to 50 pg/mL total soluble protein (TSP) and tested for myofibrillar gene protein content according to the manufacturer’s protocols. The protein concentration will be determined by reference to the standard curve. Twenty pL (1 pg) will be tested for each sample.

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Abstract

Certains aspects de la présente invention concernent l'expression hétérologue de protéines myofibrillaires dans des plantes. Certains aspects de la présente invention concernent des procédés et des acides nucléiques pour exprimer les protéines myofibrillaires recombinantes dans des plantes.
PCT/IB2024/052721 2023-03-21 2024-03-21 Expression de protéines myofibrillaires animales dans des plantes Pending WO2024194831A1 (fr)

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WO2021138674A1 (fr) 2020-01-02 2021-07-08 Fybraworks Foods, Inc. Nouveau procédé de fabrication de viande synthétique
WO2021226690A1 (fr) 2020-05-13 2021-11-18 De Leao Rosenmann Bernardo Composition nutritionnelle pour chiens ou chats, constituée d'une biomasse d'un organisme génétiquement modifié, exprimant des protéines fibrillaires du muscle animal, associée à d'autres sources nutritionnelles provenant de résidus agro-industriels, et son procédé d'obtention
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WO2021138674A1 (fr) 2020-01-02 2021-07-08 Fybraworks Foods, Inc. Nouveau procédé de fabrication de viande synthétique
WO2021226690A1 (fr) 2020-05-13 2021-11-18 De Leao Rosenmann Bernardo Composition nutritionnelle pour chiens ou chats, constituée d'une biomasse d'un organisme génétiquement modifié, exprimant des protéines fibrillaires du muscle animal, associée à d'autres sources nutritionnelles provenant de résidus agro-industriels, et son procédé d'obtention
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