High expression of animal heme proteins in plants

Abstract

The present disclosure provides methods of producing heme proteins in transgenic plants, plant tissues, or plant cells, and methods of describing the expression of these heme proteins in seeds. In addition, the present disclosure provides transgenic plants that express the heme proteins, myoglobin and hemoglobin by introducing and integrating recombinant DNA constructs into the host genetic material of the subject plant. Particular combinations of regulatory elements disclosed herein allow for high levels of heme protein expression in seeds.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 367,299, filed on June 29, 2022, which is hereby incorporated by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] The contents of the electronically submitted sequence listing in XML format submitted with the application (name: 5061_002PC01_Seqlisting_ST26.xml; size: 43,109 bytes; and creation date: June 16, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to the production of animal heme proteins in transgenic plants. The present disclosure also relates to food compositions including recombinant heme proteins produced in genetically engineered plants. The present disclosure also relates to improved expression cassettes for producing animal heme proteins in transgenic plants and transgenic seeds, the transgenic seeds comprising selected regulatory elements and codon-optimized protein coding sequences that result in significantly high (e.g., >5%, >8%, and >10% total soluble protein [tsp]) expression levels of the recombinant protein in the plant seeds. Background Art

[0006] Climate change and the expected growth of the global population to 9.7 billion by 2050 require people to adopt more sustainable lifestyle practices. Livestock production supplies the majority of dietary protein; however, livestock accounts for approximately 18% of global greenhouse gas emissions (Stehfest et al., (2009), Clim. Change 95:83). These greenhouse emissions from livestock production are expected to increase by 80% by 2050 (Tilman and Clark, (2014), Nature 515:518).

[0007] Meat production faces other problems, such as high resource intake, the presence of antibiotic residues in meat, zoonotic diseases, and ethical issues related to the exploitation of animals. Public health issues such as type 2 diabetes, cardiovascular disease, and cancer are also associated with meat consumption (Zhang et al., (2022), Curr. Opin. Food Sci. 43:43). Despite these problems, meat still has a special place in the human diet, and demand continues to grow unprecedentedly. In the past two decades, global meat demand has increased by 58%. Studies predict that by 2050, total meat consumption will increase by 62-144% (Alexandratos and Bruinsma, (2012), Agricultural Development Economics Division; Valin et al., (2014), Journal of Agricultural Economics (J. Agric. Econ.) 45:51).

[0008] Proposed efforts to reduce livestock production and consumption include a shift to plant-based protein diets. The demand for plant-based proteins is rising due to their health benefits, environmentally friendly production, animal welfare, and curiosity based on consumer tastes (Johansson, (2019) Master's thesis Chalmers University of Technology; World Health Organization, (2015), www.who.int / en / news-room / fact-sheets / detail / obesity-and-overweight). A review of 91 articles found that consumers had the highest acceptance of plant-based protein meat substitutes, followed by cultured meat (Onwezen et al., (2021), Appetite, 159: 105058). However, the production of plant-based meat substitutes still faces some challenges, such as the reconstruction of meat-like color, flavor, nutritional value and structure (Zhang et al., (2022), Current Reviews in Food Science 43: 43).

[0009] Genetic engineering represents a convenient strategy for upgrading plant-based recombinant protein products. These recombinant proteins in plants can help replace animal-based proteins in the human diet and provide desired organoleptic properties when used as ingredients or used whole with original plant parts. The expression of recombinant proteins in plants can help upgrade their color, flavor, nutritional value and structure in their native form or when used as ingredients. The present disclosure provides a solution for producing key animal heme proteins at high expression levels in transgenic plants and their seeds.

[0010] Some efforts have been made to express plant-derived heme proteins in plants to further produce food products. For example, US Patent Application Publication US2019292555 discloses transgenic rice and Arabidopsis plants expressing soybean leghemoglobin Lbc2 under the control of an alcohol-inducible promoter. The patent application disclosed in this disclosure does not show any data related to the expression level of recombinant leghemoglobin.

[0011] US Patent Application Publication US2019292217 describes transgenic Arabidopsis thaliana plants that overexpress enzymes of the world heme biosynthesis pathway (glutamate-tRNA reductase (GluTR) binding protein) and expression of soybean leghemoglobin. The document does not provide any data on the expression level of leghemoglobin.

[0012] International application publication WO2022072846 discloses information on transgenic plants expressing heme proteins with altered fatty acid profiles and upregulated heme biosynthesis, but without experiments.

[0013] International Application Publication WO9902687 discloses a method for increasing the iron content in transgenic rice plants by expressing rice hemoglobin or Arabidopsis hemoglobin; however, the transgenic plants showed low hemoglobin expression levels.

[0014] Research discloses the production of human myoglobin in Nicotiana benthamiana (Carlsson et al., (2020), Sci. Rep. 10:1). This document does not show data on the loading of heme into the recombinant myoglobin, nor does it show the function or correct structural folding of this recombinant protein, nor does it show data on the incorporation of the recombinant protein into food products.

[0015] However, these attempts to produce heme proteins in plants have not resulted in high levels of recombinant heme protein expression in plant seeds (e.g., >5% tsp). Molecular farming studies have focused on expressing genes through nuclear transformation, with average expression levels of recombinant proteins in stably transformed plants ranging from 0.5%-2% tsp (Fischer and Emans, (2000), Transgenic Res. 9:279; Shanmugaraj et al., (2020), Plants, 9:842). These recombinant proteins produced in plants are primarily pharmaceutical proteins, proteins used in diagnostics, research, and the cosmetics industry. In chloroplasts, researchers have achieved higher yields of recombinant protein expression, ranging from 3-46% tsp from plants (Dhingra and Daniell, (2006), Arabidopsis protocols, 245; Shanmugaraj et al., (2020), Plants, 9:842). The aforementioned recombinant proteins expressed in chloroplasts are mainly drugs, but also contain herbicide resistance genes. In seeds, the recombinant proteins accumulate to lower average concentrations (0.05-1% tsp) (Jaeger et al., (2002), "Nature Biotechnology (Nat. Biotechnol.)" 20: 1265; Shanmugaraj et al., (2020), "Plants", 9: 842). However, independent studies have identified regulatory elements that produce significantly higher levels of the protein in plant seeds (Jaeger et al., (2002), Nature Biotechnology 20:1265; Ishimoto et al., (2012), Biosci. Biotechnol. Biochem. 76:2142; Wadahama et al., (2012), Plant Physiol. 158:1395; Goossens et al., (1999), Plant Physiol. 120:1095; Diamos and Mason, (2018), Plant Biotechnol. J. 16:1971). These studies have reported up to 15-36% tsp in seeds of model species such as Arabidopsis and tobacco (Jaeger et al., (2002), Nature Biotechnology 20:1265; Goossens et al., (1999), Plant Physiology 120:1095). However, none of these independent studies focused on complex proteins (such as the heme proteins in the present disclosure) and have only been validated in model plant species.The combination of regulatory elements described herein has the potential to produce stable seed proteins exceeding 5%, 8% or 10% tsp in commercially important seed crops such as legumes.

[0016] Therefore, the prior art still does not provide a solution for producing heme proteins at high expression levels in seeds of transgenic plants. Summary of the invention

[0017] In some aspects, a transgenic plant, plant tissue or plant cell is provided herein, comprising an exogenous nucleic acid encoding a heme protein. In some aspects, the nucleic acid is operably linked to a seed-specific promoter and a transcription terminator. In some aspects, the heme protein is expressed in a seed at an amount of at least about 5% total soluble protein (TSP).

[0018] In some aspects, the nucleic acid is operably linked to a transcriptional or translational enhancer.

[0019] In some aspects, the heme protein is expressed in the seed in an amount of at least about 8% TSP.

[0020] In some aspects, the heme protein is expressed in the seed in an amount of at least about 10% TSP.

[0021] In some aspects, the heme protein comprises a plant-derived heme protein, a microbial-derived heme protein, or an animal-derived heme protein, or a synthetic protein designed based on a natural heme protein.

[0022] In some aspects, the heme protein includes a heme protein involved in oxygen transport, an enzyme having a prosthetic heme group, or a heme protein involved in the electron transport chain.

[0023] In some aspects, the heme protein comprises hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, or cytochrome c.

[0024] In some aspects, the heme protein is an animal-derived heme protein selected from the group consisting of hemoglobin and myoglobin.

[0025] In some aspects, the seed-specific promoter includes a β-conglycinin α subunit from the 7S storage (7s) promoter of soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from Vicia faba, a SBP promoter from Vicia faba, a legumin B4 promoter from Vicia faba, a napin (Napin) promoter from Brassicanapus, a vicilin promoter from Pisum sativum, an α-globulin promoter from cotton, a γ-zein promoter from corn, a glutenin promoter from wheat, a VvβVPE promoter from grapes (Vitis spp), a groundnut seed promoter (GSP) from peanut, a 7αP promoter from soybean, a 7αP promoter from Arabidopsis thaliana thaliana), the SSPs promoter from chickpea, the lectin promoter from soybean, the oleosin promoter from Brassica napus, the AhLEClA promoter from peanut, the Glu-ID-1 promoter from wheat, the sesame 2S albumin (2Salb) promoter from sesame, or the 8SGα promoter from mung bean.

[0026] In some aspects, the transgenic plant, plant tissue or plant cell further comprises a terminator sequence. In some aspects, the terminator sequence comprises an extensin terminator from tobacco, an Ub10 terminator from Arabidopsis thaliana, an Hsp70 terminator from Arabidopsis thaliana, an Hsp18.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana, a G7 terminator from Arabidopsis thaliana, a 3g24240 terminator from Arabidopsis thaliana, a NOS terminator from Agrobacterium tumefaciens, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from Agrobacterium tumefaciens, a Cauliflower Mosaic Virus, a Virus), 35s terminator from Rbc terminator from Chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3′utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from potato (Solanum tuberosum), tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens, or Arc5 terminator from Phaseolus vulgaris.

[0027] In some aspects, the transgenic plant, plant tissue or plant cell further comprises a transcriptional or translational enhancer selected from the group consisting of: a 5′ untranslated region (UTR) from Tobacco Etch Virus (TEV) as part of a transcriptional terminator and a Rb7Mar 3′ matrix junction region.

[0028] In some aspects, the exogenous nucleic acid is operably linked to the β-conglycinin alpha subunit from the 7S storage protein (7s) promoter and the NOS terminator from soybean.

[0029] In some aspects, the exogenous nucleic acid is operably linked to the β-conglycinin alpha subunit from a 7S storage protein (7S) promoter of soybean and an Arc5 terminator and Rb7MAR fused to the Arc5 terminator.

[0030] In some aspects, the exogenous nucleic acid is operably linked to the β-conglycinin alpha subunit from the 7S storage protein (7S) promoter of soybean, the 5′UTR TEV enhancer, and the Arc5 terminator and Rb7MAR fused to arc5.

[0031] In some aspects, the exogenous nucleic acid is operably linked to a beta-phaseolin (Phas) promoter from common bean and a NOS terminator.

[0032] In some aspects, the exogenous nucleic acid is operably linked to a beta-phaseolin (Phas) promoter from common bean and an Arc5 terminator fused to the Rb7 MAR region.

[0033] In some aspects, the exogenous nucleic acid is operably linked to a beta-phaseolin (Phas) promoter from common bean, a 5' UTRTEV enhancer, and an Arc5 terminator fused to the Rb7 MAR region.

[0034] In some aspects, the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1.

[0035] In some aspects, the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:2.

[0036] In some aspects, the transgenic plant, plant tissue or plant cell is derived from soybean (Glycine max), rice (Oryza sativa), barley (Hordeum vulgare), corn (Zea mays), rye (Secale cereale), oats (Avena sativa), sugar beet (Beta vulgaris), cultivated sugar beet subsp. vulgaris (Beta vulgaris subsp. vulgaris), parsnip (Pastinaca sativa), bean, pea, cowpea (Vigna angularis), mung bean (Vigna radiata), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentil (Lens culinaris), Medicago sativa, rocket (Eruca vesicaria), mustard (Brassica juncea), lettuce (Lactuca sativa), Brassica, potato, sweet potato (Ipomoea batatas), cassava (Manihot esculenta), esculenta), wheat (Triticum aestivum) or spelt (Triticum spelta).

[0037] In some aspects, the present invention provides a method for obtaining a recombinant heme protein. In some aspects, the method comprises: i) providing a transgenic plant capable of expressing a heme protein of at least about 5% TSP in seeds; ii) cultivating the transgenic plant; iii) collecting the transgenic plant; and iv) isolating and purifying the animal heme protein from the collected plant.

[0038] In some aspects, the collecting comprises collecting the seeds of the transgenic plant.

[0039] In some aspects, provided herein is a transgenic seed comprising a recombinant heme protein of at least about 5% TSP.

[0040] In some aspects, provided herein is a transgenic seed comprising a recombinant heme protein of at least about 8% TSP.

[0041] In some aspects, provided herein is a transgenic seed comprising a recombinant heme protein of at least about 10% TSP.

[0042] In some aspects, the transgenic seed is from a species selected from the group consisting of soybean, rice, barley, corn, rye, oats, sugar beet, Beta vulgaris, parsnips, beans, peas, cowpeas, mung beans, chickpeas, peanuts, lentils, clover, rocket, mustard, lettuce, Brassica, potato, sweet potato, cassava, wheat, or spelt.

[0043] In some aspects, the recombinant heme protein is an animal heme protein.

[0044] In some aspects, the recombinant heme protein is myoglobin.

[0045] In some aspects, the recombinant heme protein is hemoglobin.

[0046] In some aspects, provided herein is a food composition comprising any of the transgenic seeds disclosed herein.

[0047] In some aspects, provided herein is a food composition comprising a heme protein of any of the plants, plant tissues, or plant cells disclosed herein.

[0048] In some aspects, provided herein is a meat analog food composition comprising any of the transgenic seeds disclosed herein.

[0049] In some aspects, provided herein is a meat analog food composition comprising a heme protein of any one of the plants, plant tissues, or plant cells disclosed herein.

[0050] In some aspects, the present disclosure provides a polynucleotide comprising a nucleic acid encoding a hemoglobin protein, wherein the nucleic acid is operably linked to a seed-specific promoter selected from the group consisting of: a β-conglycinin alpha subunit from a 7S storage (7s) promoter of soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from broad bean, a SBP promoter from broad bean, a legumin B4 promoter from broad bean, a napus protein promoter from Brassica napus, a vicilin promoter from pea, an α-globulin promoter from cotton, γ-zein promoter from corn, glutenin promoter from wheat, VvβVPE promoter from grape, peanut seed promoter (GSP) from peanut, 7αP promoter from soybean, AtLAC15 promoter from Arabidopsis, SSPs promoter from chickpea, lectin promoter from soybean, oleosin promoter from Brassica napus, AhLEC1A promoter from peanut, Glu-1D-1 promoter from wheat, sesame 2S albumin (2Salb) promoter from sesame and 8SGα promoter from mung bean.

[0051] In some aspects, the heme protein comprises a plant-derived heme protein, a microbial-derived heme protein, or an animal-derived heme protein.

[0052] In some aspects, the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1 or SEQ NO:2.

[0053] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-7.

[0054] In some aspects, the nucleic acid further comprises a transcription terminator selected from the group consisting of: an extensin terminator from tobacco, an Ub10 terminator from Arabidopsis thaliana, an Hsp70 terminator from Arabidopsis thaliana, an Hsp18.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana, a G7 terminator from Arabidopsis thaliana, a 3g24240 terminator from Arabidopsis thaliana, a NOS terminator from Agrobacterium tumefaciens, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from cauliflower mosaic virus, 35s terminator from chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3'utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from potato, tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens, and Arc5 terminator from bean.

[0055] In some aspects, the nucleic acid further comprises a transcriptional or translational enhancer selected from the group consisting of 5'UTR TEV and Rb7Mar 3' matrix junction region.

[0056] In some aspects, the disclosure relates to an expression vector comprising a nucleic acid encoding a hemoglobin protein, wherein the nucleic acid is operably linked to a seed-specific promoter selected from the group consisting of: a β-conglycinin alpha subunit from the 7S storage (7s) promoter of soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from broad bean, a SBP promoter from broad bean, a legumin B4 promoter from broad bean, a napusin promoter from Brassica napus, a vicilin promoter from pea, an α-globulin promoter from cotton, a β-phaseolin promoter from common bean ... a α-globulin promoter from cotton, a β-phaseolin promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin (Phas) promoter from common bean, a β-phaseolin The promoters of the promoters of the invention include the γ-zein promoter from maize, the glutenin promoter from wheat, the VvβVPE promoter from grape, the peanut seed promoter (GSP) from peanut, the 7αP promoter from soybean, the AtLAC15 promoter from Arabidopsis thaliana, the SSPs promoter from chickpea, the lectin promoter from soybean, the oleosin promoter from Brassica napus, the AhLEC1A promoter from peanut, the Glu-1D-1 promoter from wheat, the sesame 2S albumin (2Salb) promoter from sesame, or the 8SGα promoter from mung bean.

[0057] In some aspects, the expression vector comprises a heme protein derived from a microorganism, a plant, or an animal.

[0058] In some aspects, the expression vector includes a nucleic acid encoding a heme protein having a sequence having at least 80% sequence identity to SEQ ID NO:1.

[0059] In some aspects, the expression vector comprises a nucleic acid encoding a heme protein having a sequence having at least 80% sequence identity to SEQ ID NO:2.

[0060] In some aspects, the expression vector includes a nucleic acid encoding a heme protein having a sequence having at least 80% sequence identity to SEQ ID NO: 1 operably linked to a beta-phaseolin (Phas) promoter.

[0061] In some aspects, the expression vector comprises a nucleic acid encoding a heme protein having a sequence having at least 80% sequence identity to SEQ ID NO: 2, the sequence being operably linked to a β-phaseolin (Phas) promoter. In some aspects, the expression vector further comprises a transcription terminator selected from the group consisting of: an extensin terminator from tobacco, an Ub10 terminator from Arabidopsis thaliana, an Hsp70 terminator from Arabidopsis thaliana, an Hsp18.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana, a G7 terminator from Arabidopsis thaliana, a 3g24240 terminator from Arabidopsis thaliana, a NOS terminator from Agrobacterium tumefaciens, an Ocs terminator from Agrobacterium tumefaciens, an 18.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana ... The terminator comprises the following: Mas terminator from Bacillus subtilis, 35s terminator from cauliflower mosaic virus, Rbc terminator from Chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3'utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from potato, tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens, and Arc5 terminator from Phaseolus vulgaris.

[0062] In some aspects, the expression vector includes a nucleic acid encoding a heme protein having a sequence having at least 80% sequence identity to SEQ ID NO: 1 operably linked to a beta-phaseolin (Phas) promoter and an Arc5 terminator.

[0063] In some aspects, the expression vector includes a nucleic acid encoding a heme protein having a sequence having at least 80% sequence identity to SEQ ID NO: 2, the sequence being operably linked to a beta-phaseolin (Phas) promoter and an Arc5 terminator.

[0064] In some aspects, the expression vector further comprises a transcriptional or translational enhancer selected from the group consisting of 5'UTR TEV and Rb7Mar 3' matrix linker region.

[0065] In some aspects, the expression vector includes a nucleic acid encoding a heme protein having a sequence having at least 80% sequence identity to SEQ ID NO: 1 operably linked to a β-phaseolin (Phas) promoter, an Arc5 terminator, and a Rb7Mar 3' matrix linker region.

[0066] In some aspects, the expression vector includes a nucleic acid encoding a heme protein having a sequence having at least 80% sequence identity to SEQ ID NO: 2, the sequence being operably linked to a β-phaseolin (Phas) promoter, an Arc5 terminator, and a Rb7Mar 3' matrix linker region.

[0067] In some aspects, the expression vector comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-7. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 ( Figure 1 ) shows the pIPTRA0: p35S + HbA-LL-HbB binary vector. This plasmid allows the expression of the hemoglobin (HbA-LL-HbB) gene driven by the constitutive promoter CaMV35S and contains the nopaline synthase terminator (TNOS). The hemoglobin gene consists of α-globulin and β-globulin connected by a 63 bp long linker (LL). The length of the vector backbone region is 11,238 bp. HbA-LL-HbB consists of a sequence optimized by soybean codons.

[0069] Figure 2 ( Figure 2 ) depicts the pIPTRA0:p7S+HbA-LL-HbB-Arc5T binary vector. This plasmid allows the expression of the hemoglobin (HbA-LL-HbB) gene driven by the 7S globulin (7s) promoter in combination with the ARC5 terminator and the Rb7 matrix array region (MAR). The hemoglobin gene consists of an alpha-globulin and a beta-globulin connected by a 63 bp long linker (LL). The length of the vector backbone region is 12,159 bp. HbA-LL-HbB consists of a soybean codon-optimized sequence.

[0070] Figure 3 ( Figure 3 ) shows a 35S+TEV+myoglobin cDNA+NOS linear construct, referred to as EC1. This linear construct allows expression of the myoglobin gene driven by the constitutive promoter CaMV35S promoter and contains the nopaline synthase terminator (TNOS). The length of the linear construct is 1,655 bp. The myoglobin gene consists of a sequence optimized by soybean codons.

[0071] Figure 4 ( Figure 4) shows the p7S+TEV+myoglobin cDNA+arc5+Rb7MAR linear construct, referred to as EC2. This linear construct allows expression of the myoglobin gene driven by the 7S globulin promoter in combination with the ARC5 terminator and the Rb7 matrix array region (MAR). The length of the linear construct is 3,220 bp. The myoglobin gene consists of a sequence optimized by soybean codons.

[0072] Figure 5 ( Figure 5 ) depicts a PPhas+myoglobin cDNA+arc5+Rb7MAR linear construct, referred to as EC3. This linear construct allows expression of the myoglobin gene driven by the phas promoter in combination with the ARC5 terminator and the Rb7 matrix array region (MAR). The Phas promoter contains a seed-specific enhancer (SSE). The length of the linear construct is 2,623 bp. The myoglobin gene consists of a sequence optimized by soybean codons.

[0073] Figure 6 ( Figure 6 ) depicts the T-DNA nucleotide sequence of pIPTRA0: p35S+HbA-LL-HbB (5′ to 3′) (4728 bp). Tvsp transcription terminator (italics). Bar gene cDNA, conferring glufosinate-ammonium resistance, complementary orientation ( Underlined ). Tobacco etch virus (TEV) translation enhancer, complementary orientation (bold). CaMV35S-derived dual promoter pr2x35S, complementary orientation (italic shaded). Tnos transcription terminator (underlined shaded). HbA-LL-HbB CDS, complementary orientation (shaded and bold). The hemoglobin gene consists of α-globin and β-globin connected by a 63 bp long linker (LL) (black background, white text).

[0074] Figure 7 ( Figure 7) depicts the T-DNA nucleotide sequence of pIPTRA0:p7S+HbA-LL-HbB-Arc5T (5′ to 3′) (5649 bp). Tvsp transcription terminator (italics). Bar gene cDNA, conferring glufosinate resistance, complementary orientation (underlined). Tobacco etch virus (TEV) translation enhancer, complementary orientation (bold). CaMV35S-derived dual promoter pr2x35S, complementary orientation (italic shaded). Tobacco Rb7 matrix linker region, complementary orientation (shaded underline). Arcelin-5 transcription terminator, complementary orientation (bold and shaded). HbA-LL-HbB CDS, complementary orientation (black background, white text, italics). The hemoglobin gene consists of α-globin and β-globin connected by a 63 bp long linker (LL) (lowercase letters and black background, white text). Tobacco etch virus (TEV) translation enhancer, complementary orientation. 7S globulin promoter, complementary orientation (black background with white text, underlined).

[0075] Figure 8 ( Figure 8 ) shows the nucleotide sequence of the EC1 linear construct (5' to 3') (1,655 bp). CaMV35S-derived dual promoter 2x35S (italics). Tobacco etch virus (TEV) translation enhancer (underlined). Myoglobin CDS (bold). TNOS transcription terminator (italic shaded).

[0076] Fig. 9 ( Fig. 9 ) shows the nucleotide sequence of the EC2 linear construct (5' to 3') (3,220 bp). 7S globulin (7s) promoter (italics). Tobacco etch virus (TEV) translation enhancer (underlined). Myoglobin CDS (bold). ARC5 terminator and Rb7 matrix array region (italic shaded).

[0077] Fig.10 ( Fig.10 ) shows the nucleotide sequence of the EC3 linear construct (5' to 3') (2,623 bp). Phas promoter (italics). Myoglobin CDS (underlined). ARC5 terminator and Rb7 matrix array region (bold).

[0078] Fig.11 ( Fig.11) depicts the regeneration of soybean transgenic lines (A, B). Images A and B show shoots regenerated on selection medium transformed with pIPTRA0:p7S+HbA-LL-HbB-Arc5T and EC3, respectively. Shoots from images A and B were transformed by agrobacterium and biolistic transformation method, respectively.

[0079] Fig.12 ( Fig.12 ) shows the results of PCR amplification from an event transformed with the pIPTRA0:p35S+HbA-LL-HbB binary vector. Specific primers were designed to detect the 2x35S promoter and the HbA CDS. The expected amplicon length is 412bp. MM: molecular marker (100bp). Samples 463 to 472 are a batch of potential transgenic plants generated in the study. C-1 and C-2 are negative controls prepared during DNA extraction. C+1 to C+2 are positive controls for the pIPTRA0:p35S+HbA-LL-HbB binary vector from two previously characterized plant samples. C+3 was amplified from the pIPTRA0:p35S+HbA-LL-HbB binary vector. C-3DNA represents another binary vector (negative control). Bco: PCR blank. WT: wild type soybean DNA

[0080] Fig.13 ( Fig.13 ) shows the results of PCR amplification from an event transformed with the pIPTRA0:p7S+HbA-LL-HbB-Arc5T binary vector. Specific primers were designed to detect the 7S promoter and HbA CDS. The expected amplicon length is 390bp. MM: molecular marker (100bp). Samples 563 to 562 are a batch of potential transgenic plants generated in the study. C-1 is a negative control prepared during DNA extraction. Bco is a PCR blank. WT: wild-type soybean DNA. C+I is a positive sample of the previously characterized pIPTRA0:p7S+HbA-LL-HbB-Arc5T. C-3DNA represents another binary vector (negative control). C+2 was amplified from the pIPTRA0:p7S+HbA-LL-HbB-Arc5T binary vector.

[0081] Fig.14 ( Fig.14) shows qPCR amplification results from events transformed with EC1, EC2, and EC3 vectors. Specific primers were designed to detect aadA1a CDS. Red line: marker Ct cutoff, positive must be <25 Ct value. WT control: wild type soybean DNA. Water control: water control. C+1 and C+2 are positive controls for aadA1 that have been previously characterized. Samples 1 to 4 of EC1, EC2, and EC3 are batches of potential transgenic plants generated in the study.

[0082] Fig.15 A-15C. Fig.15 AC depicts the WT ( Fig.15 A), pIPTRA0: p35S+HbA-LL-HbB( Fig.15 B) and pIPTRA0:p7S+HbA-LL-HbB-Arc5T( Fig.15 C) Protein extract coloration of soybean seeds from transgenic events. Fig.15 The protein extract of C is shown in pink ( Fig.15 Darker shading in the black and white image of C), which may be attributed to the expression and presence of heterologous hemoglobin genes. Arrows and circles were added to point to the darker shading.

[0083] Fig.16 A-16C( Fig.16 A-16C) shows the coloration of soybean half-cut seeds ( Fig.16 A) and coloration of protein extracts from soybean seeds ( Fig.16 B, 16C). Half-cut seeds of WT seeds are shown ( Fig.16 A) and half-cut seeds of transgenic EC3 event ( Fig.16 A). shows protein seed extraction of WT seeds ( Fig.16 B) and protein seed extraction of transgenic EC3 events ( Fig.16 C) Corresponding to EC3 transgenic line Fig.16 The pink color shown in A and 16C ( Fig.16 The darker shadows in the black and white images of A and 16C) may be attributed to the expression and presence of the heterologous porcine myoglobin gene.

[0084] Fig.17 A-17C( Fig.17A-17C) shows three replicates of protein blot (17A, 17B, 17C) analysis of soybean seeds. Soybean seed protein extracts were run on 12% SDS PAGE gels. The protein bands were then transferred to nitrocellulose membranes and the protein blots were developed with anti-porcine antibodies. White dots indicate control hemoglobin bands as well as putative hemoglobin bands of 16kDa (monomer), 32kDa (dimer), and 64kDa (tetramer). Lane 1: 100μg of WT extract. Lanes 2 to 6: WT extract + 10, 25, 50, 150, and 250ng of Hb standards, respectively. Lanes 7 to 14: 100μg of protein extracts of EC3 events 63, 64, 65, 66, 67, 68, 69, 70. Lane 15: Molecular weight standards.

[0085] Fig.18 ( Fig.18 ) depicts the hemoglobin expression level in seeds of soybean transgenic events. The total soluble protein (TSP%) represented by the Y axis was obtained by Western blot quantitative analysis. The expression of events transformed with pIPTRA0:p35S+HbA-LL-HbB binary vector (events 15 and 19) is shown in black bars. The expression of events transformed with pIPTRA0:p7S+HbA-LL-HbB-Arc5T binary vector (events 46 to 75) is shown in white bars. WT and empty vector events are not shown in the figure because the expression values ​​did not reach the limit of detection (LOD). The extracts were run on a 12% SDS PAGE gel, then transferred to a nitrocellulose membrane, and the Western blot was developed with an anti-porcine antibody. The hemoglobin percentage of TSP was quantified by comparing the intensity of the hemoglobin band from the seed extract with the intensity of the hemoglobin band from the hemoglobin standard. Each event consists of one biological replicate (pool of 3 seeds) and 3 technical replicates. The standard deviation of each event is presented as an error bar.

[0086] Fig.19 ( Fig.19 ) shows results from the identification and quantification of porcine hemoglobin from whole soybean seed protein extracts by liquid chromatography-mass spectrometry (LC-MS). Normalized porcine hemoglobin levels are shown in the graph for WT (wild type) and events 54 and 63 (both transformed with pIPTRA0:p7S+HbA-LL-HbB-Arc5T). Hemoglobin was not detected in the WT sample. No replicates were run.

[0087] Fig. 20 A-20B( Fig. 20 A-20B) shows the myoglobin expression level in soybean transgenic events. The total soluble protein (TSP%) indicated on the Y axis was obtained by ELISA quantitative analysis. Fig. 20A shows expression values ​​for WT and events transformed with empty vector (1542 event series), EC1 (1545 event series), and EC2 (1544 event series). Fig. 20 B shows the expression value of the event transformed with EC3 (1543 event series) and WT and the event transformed with empty vector, EC1 and EC2. Use α diagnostic ELISA kit (Cat. No. 600-640-PMY) to carry out quantitative myoglobin from seed extract. All samples are normalized relative to 50 μg / mL total soluble protein (TSP), and the myoglobin content is tested according to the manufacturer's scheme. The concentration of myoglobin is determined by reference to the standard curve. Each event is composed of a biological replicate (pool of 3 seeds) and 3 technical replicates. The standard deviation of each event is presented with error bars. DETAILED DESCRIPTION

[0088] The terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0089] It should be noted that, unless otherwise indicated, the term "a / an" entity refers to one or more entities in the entity; for example, "nucleic acid sequence" should be understood to mean one or more nucleic acid sequences. Thus, the terms "a / an" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0090] In addition, when used herein, "and / or" will be considered to explicitly disclose each of the two specified features or components with or without the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0091] It should be understood that when anywhere herein an aspect is described with the language "comprising," other similar aspects described with respect to "consisting of" and / or "consisting essentially of" are also provided.

[0092] As used herein, the term "about" means approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the limits above and below the numerical values ​​set forth. In general, the term "about" can modify numerical values ​​above and below the stated value by, for example, a variance of 10%, up or down (higher or lower).

[0093] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least", as well as all subsequent numbers or integers that can be logically included, as can be clearly seen from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20 or 21 nucleotides have the indicated properties. When at least appears before a series of numbers or ranges, it should be understood that "at least" can modify each number in the numbers in the series or range. "At least" is also not limited to integers (for example, "at least 5%" includes 5.0%, 5.1%, 5.18%, without considering the number of significant figures).

[0094] Throughout the present disclosure, various aspects of the present disclosure can be presented in a range format. Numerical ranges include numbers that limit the range. In the case of the range of narrated values, it should be understood that each intermediate integer value and each fraction between the upper limit and the lower limit of the range described and each subrange between such values ​​are also specifically disclosed. The upper and lower limits of any range can be independently included in the range or excluded from the range, and each range including any limit, not including two limits or including two limits is also included in the present disclosure. Therefore, the scope described herein should be understood as a shorthand for all values ​​in the range, including the endpoints described. For example, a range of 1 to 10 should be understood as including any number, a combination of numbers or a subrange from a group consisting of 1,2,3,4,5,6,7,8,9 and 10.

[0095] In the case of explicitly stating a value, it should be understood that the value of the same quantity or amount as the stated numerical value is also within the scope of the present disclosure. In the case of a disclosed combination, each sub-combination of the elements of the combination is also specifically disclosed and is within the scope of the present disclosure. On the contrary, in the case of separately disclosing different elements or groups of elements, their combination is also disclosed. In the case where any element of the present disclosure is disclosed as having multiple alternatives, the disclosed examples in which each alternative is excluded individually or in any combination with other alternatives are also hereby disclosed; more than one element of the present disclosure may have such exclusions, and all combinations of elements with such exclusions are hereby disclosed.

[0096] "Percentage identity" refers to the degree of identity between two sequences (e.g., amino acid sequence or nucleic acid sequence). Percentage identity can be determined by comparing two sequences and introducing a gap to maximize the identity between the sequences. Comparisons can be generated using programs known in the art. For purposes of this paper, the comparison of nucleotide sequences can be performed using the blastnn program set under default parameters, and the comparison of amino acid sequences can be performed using the blastp program set under default parameters (see the National Center for Biotechnology Information (NCBI) on the World Wide Web ncbi.nlm.nih.gov).

[0097] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be further understood that terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and in this disclosure, and will not be interpreted in an idealized or overly formal sense unless explicitly defined as such herein.

[0098] In describing the present invention, it should be understood that many techniques and steps are disclosed. Each of these techniques and steps has a separate benefit, and each can be used in combination with one or more techniques and steps, or in some cases, all other disclosed techniques can be used. Therefore, for clarity, this description will avoid repeating each possible combination of individual steps in an unnecessary manner. However, the specification and claims should be read with the understanding that such combinations are fully within the scope of the present invention and claims.

[0099] In some aspects, the present invention discusses the production of heme proteins in transgenic plants and the use of these heme proteins for meat replacement. In the following description, for the purpose of explanation, many specific details are set forth to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention can be practiced without these specific details.

[0100] As used herein, the term "derived from" refers to a component separated from or prepared using a specific molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from a specific molecule or organism. For example, a nucleic acid sequence (e.g., an expression vector) derived from a second nucleic acid sequence may include a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence.

[0101] "Nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably in this application. These terms refer only to the primary structure of the molecule. Therefore, these terms include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" are defined as molecules that are generally understood by those skilled in the art to include two or more covalently linked nucleotides. Such covalently linked nucleotides may also be referred to as nucleic acid molecules or oligomers. Polynucleotides may be prepared recombinantly, enzymatically or synthetically, for example by solid phase chemical synthesis, followed by purification preparation. When referring to the sequence of a polynucleotide or nucleic acid, the sequence or order of the core base portion of the covalently linked nucleotides or their modification is referred to.

[0102] As used herein, the term "heme protein" includes proteins that have the ability to bind a heme prosthetic group to its structure. As used herein, the term heme protein also refers to a key component of animal meat and / or animal protein, and can provide color and flavor to plant-based meat products. Myoglobin and hemoglobin are considered heme proteins, which are oxygen-binding proteins in animals. In addition, as used herein, the term heme protein refers to a protein containing heme, wherein the term containing means that the protein is connected to the protein by covalent or non-covalent bonds. As used herein, the term heme protein refers not only to the full-length protein, but also to fragments or variants thereof.

[0103] As used herein, the term "animal heme protein" or "animal-derived heme protein" refers to a heme protein expressed in an animal, but excludes human-derived heme proteins. According to some aspects of the present disclosure, animal heme proteins include heme proteins involved in oxygen transport, such as hemoglobin, myoglobin, neuroglobin, and cytoglobin; enzymes with a prosthetic heme group, such as cytochrome P450s, cytochrome c oxidase, ligninase, catalase, and peroxidase; and heme proteins involved in the electron transport chain, such as cytochrome a, cytochrome b, and cytochrome c.

[0104] As used herein, the term "plant-derived heme protein" means a heme protein whose genetic origin is naturally derived from a monocot or dicot plant, such as Nicotiana tabacum or Nicotiana sylvestris (tobacco); maize (corn), Arabidopsis thaliana, legumes such as soybean (soybean), chickpea (garbanzo / chickpea), pea (pea), bean (common bean), cowpea (cowpea), mung bean (mung bean), Lupinus albus (lupin) or alfalfa (alfalfa); Brassica napus (rapeseed oil); Triticum sps. (wheat, including wheat kernels and spelt); Gossypium hirsutum (Gossypium hirsutum (cotton); Oryza sativa (rice); Zizania sps. (wild rice); Helianthus annuus (sunflower); Sugar beet (sugarbeet); Pennisetum glaucum (pearl millet); Chenopodium sp. (quinoa); Sesamum sp. (sesame); Linumus itatissimum (flax); or barley (barley).

[0105] As used herein, the term "microbial-derived heme protein" means a heme protein whose genetic origin is naturally derived from bacteria, yeast, fungi, such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Synechocystis, Aquifex aeolicus, Methylacidiphilum infernorum, Thermophilus spp, A. eutrophus, Saccharomyces cerevisiae, Vitreoscilla sp, Pichia pastoris, Magnaporthe oryzae, Fusarium graminearum, Aspergillus oryzae, Trichoderma reesei, reesei, Myceliopthera thermophile, Kluyveramyces lactis, and Fusarium oxysporum.

[0106] As used herein, the term "recombinant protein" refers to a protein encoded by a gene (e.g., recombinant DNA) cloned in a system that supports gene expression and messenger RNA translation. Recombinant protein is an exogenous protein produced in an expression host. Modification of genes by recombinant DNA technology can result in the expression of mutant proteins.

[0107] As used herein, the term "recombinant heme protein" refers to a recombinant protein, wherein the recombinant protein is encoded by an exogenous cDNA encoding the heme protein. As used herein, the term "exogenous nucleic acid" means a cDNA encoding a recombinant heme protein; in addition, the term "exogenous nucleic acid" is used interchangeably with "recombinant nucleic acid" herein. The sequences and structures of many heme-containing proteins are known (Reedy et al., (2007), Nucleic Acids Res. 6: D307).

[0108] The term "plant" includes reference to whole plants, plant organs, plant tissues and plant cells and progeny thereof, and includes all monocotyledonous and dicotyledonous plants. The term plant as used herein also includes seeds, plant progeny, sexual or asexual propagation bodies, their descendants (such as cuttings or seeds), and tissues and organs before and after collection.

[0109] The term "transgenic plant" or "genetically engineered" means a plant that has been transformed with one or more exogenous nucleic acids (recombinant sequences). The term "transformation" refers to the process of introducing and expressing recombinant sequences in plant cells. In plant stable transformation, the exogenous DNA is fully integrated into the host genome, remains integrated, and continues to be expressed in the offspring of the plant. In plant transient transformation, the exogenous DNA is not integrated into the host genome and is not expressed in the offspring of the plant. Transformation can occur by Agrobacterium tumefaciens inoculation, viral infection, electroporation, heat shock, fat transfection, polyethylene glycol treatment, microinjection, silica beads, carbon non-tubes and particle bombardment methods.

[0110] In some respects, transgenic plant is soybean (soy) (soybean) plant. In some respects, genetically engineered plant is selected from the group consisting of: rice (rice), barley (Hordeum vulgare), wheat (wheat), corn (corn), rye (rye), oats (oat), beet (beet), sugar beet (sugar beet) (cultivar beet subspecies), parsnip (parsnip), beans, multi-vegetables, tubers and grass. In some respects, beans are beans or flower beans (kidney beans), peas (peas), adzuki beans (cowpeas), mung beans (mung beans), chickpeas (chickpeas), peanuts (groundnuts) or lentils (lentils). In some respects, leafy vegetables are alfalfa (clover), rocket (arugula) (arugula), mustard (mustard), lettuce (lettuce) (lettuce) or Brassica. In some aspects, the tuber is potato (potato), sweet potato (sweet potato) or cassava (cassava). In some aspects, the grass is triticale (wheat) or spelt (spelt).

[0111] Figure 1-10 Illustrative recombinant sequences of the present disclosure are provided in . These recombinant sequences are contained in plant transformation vectors. In some aspects, these vectors are introduced into Agrobacterium tumefaciens as circular plasmids. The t-DNA insert from the circular plasmid is introduced into plant cells by Agrobacterium tumefaciens-mediated transformation ( Figure 1-2 , 6-7 and SEQ ID NO: 3-4). In some aspects, these vectors are bombarded into plant cells as linear constructs ( Figure 3-5, 8-10 and SEQ ID NO: 5-7). In some aspects, the recombinant sequence includes a promoter, an enhancer sequence, a sequence encoding a hemoglobin protein, and a terminator ( Figure 1 , 3 , 6 and 8). In some aspects, the recombinant sequence includes a promoter, an enhancer sequence, a sequence encoding a hemoglobin protein, a terminator, and a matrix attachment region ( Figure 2 , 4 , 5, 7, 9 and 10). In some aspects, the heme protein is hemoglobin ( Figure 1-2 and 6-7). In other aspects, the heme protein is myoglobin ( Figure 3-5 and 8–10).

[0112] In some respects, the recombinant sequence comprises a sequence called a promoter, which refers to a nucleic acid sequence that promotes transcription initiation. The promoter may be a constitutive promoter. A constitutive promoter can initiate transcription in a plant cell under any circumstances, and the activity of the constitutive promoter is not affected by environmental conditions. Some promoters are tissue-specific because these promoters preferentially initiate transcription in certain organs. Other promoters are inducible, regulated by external stimuli such as different chemical, biological and abiotic environmental factors.

[0113] In some aspects, the promoter is a constitutive promoter derived from cauliflower mosaic virus (CaMV), such as the 35S promoter present as two units in tandem (2X 35S promoter) (SEQ ID NO: 8) ( Figure 1 , 3, 6 and 8). In some aspects, the promoter is a tissue-specific promoter. The tissue-specific promoter can be the β-conglycinin α′ subunit from the 7S storage protein (7s) promoter of soybean (Zakharov et al., (2004), J. Exp. Bot. 55: 1463) (SEQ ID NO: 11), the β-phaseolin (phas) ​​promoter from common bean (Zakharov et al., (2004), J. Exp. Bot. 55: 1463), the USP promoter from broad bean (Zakharov et al., (2004), J. Exp. Bot. 55: 1463), the SBP promoter from broad bean (Zakharov et al., (2004), J. Exp. Bot. 55: 1463), the legumin B4 promoter from broad bean (Zakharov et al., (2004), J. Exp. Bot. 55: 1463), the β-phaseolin (phas) ​​promoter from common ... The napus protein promoter from rapeseed (Vigeolas et al., (2007), Journal of Plant Biotechnology 5:431), the vicilin promoter from pea (Arun et al., (2014), Appl. Biochem. Biotechnol. 172:1763), the α-globulin promoter from cotton (Sunilkumar et al., (2002), Transgenic Research 11:347), the γ-zein promoter from maize (Marzábal et al., (1998), Plant Journal (Plant J.)》16:41), glutenin promoter from wheat (Lamacchia et al., (2001), Journal of Experimental Botany 52:243), VvβVPE promoter from grape (Gong et al., (2019), Planta, 250:657), peanut seed promoter (GSP) from peanut (Sunkara et al., (2014), Applied Biochemistry and Biotechnology 172:325), 7αP promoter from soybean (Fu et al., (2009); Northwest Sci. 37:105), AtLAC1 from Arabidopsis thaliana 5 promoter (El-Mezawy et al., (2009), Biotechnol. Lett. 31: 1961), SSPs promoter from chickpea (Verma and Bhatia, (2019), Functional and 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), AhLEC1A promoter from peanut (Tang et al., (2021) PLoS one, 16:e0242949), Glu-1D-1 promoter from wheat (Lamacchia et al., (2001) Journal of Experimental Botany 52:243), sesame 2S albumin (2Salb) promoter from sesame (Bhunia et al., (2014) Plant Mol. Biol. 86:351), 8SGα promoter from mung bean (Chen et al., (2014) Journal of Biotechnology (J. Biotechnol.) 174:49). In some aspects, the seed-specific promoter is 7S (. Figure 2 , 4 , 7 and 9) (SEQ ID NO: 11) and phas ( Figure 5 and 10 ). Constructs based on 7s or phas promoter showed higher expression of protein in seeds.

[0114] 7S and β-phaseolin are highly expressed seed storage proteins and their expression patterns have been characterized (Chandrasekharan et al., (2003), Plant J. 33:853; Hayashi et al., (2009), J. Hered. 100:802). The 7S globulin gene (β-conglycinin) is the major seed storage protein in soybean (soybean). This gene consists of three subunits: α, α′ and β, and accounts for 30-35% of the total seed protein (Thanh and Shibasaki, (1976), Biochim. Biophys. Acta. 439:326; Hayashi et al., (2009), J. Hered. 100:802). The 7S promoter was inserted into soybean to express human growth factor, and the transgenic line produced 2.3% tsp (total soluble protein) recombinant protein, which is 38 times that of the recombinant protein produced by the 35S promoter (Ding et al., (2006), Biotechnology Express 28: 869). Human bone morphogenetic protein was expressed under the control of the 7s promoter, resulting in a yield of up to 9.28% tsp (Queiroz et al., 2019, Plant Mol Biol 96: 429).

[0115] The phas gene encodes the major seed storage protein in bean. Studies have found that the phas gene is highly expressed in the cotyledon during embryogenesis (Li et al., (1999), Proceedings of the National Academy of Sciences of the United States of America (PNAS), 95:4772; Chandrasekharan et al., (2003), Plant Journal 33:853). This gene is strictly turned off during all vegetative stages of plant development (Li et al., (1999), Proceedings of the National Academy of Sciences of the United States of America, 95:4772). In Arabidopsis seeds and under the control of the phas promoter, the expression level of the recombinant protein reached up to 36% of the total soluble seed protein (Jaeger et al., (2002), Nature Biotechnology 20:1265).

[0116] In some aspects, the expression cassette comprises a 2x 35S promoter. In some aspects, the 2x 35S promoter comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 8.

[0117] In some aspects, the 2x 35S promoter 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:8.

[0118] In some aspects, the expression cassette includes a 7S promoter. In some aspects, the 7S promoter includes a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 11.

[0119] In some aspects, the 7S promoter 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:11.

[0120] In some aspects, the expression cassette includes a Phas promoter. In some aspects, the Phas promoter includes a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 14.

[0121] In some aspects, the Phas promoter 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:14.

[0122] In some aspects, the recombinant sequence includes sequences called terminators, which refer to nucleic acid sequences that define the ends of a gene.Useful terminators include, but are not limited to, the extensin terminator from tobacco (Rosenthal et al., (2018), Plant Mol. Biol. 96:429), the Ub10 terminator from Arabidopsis thaliana (Tian et al., 2002, Biodesign and Engineering (BIO-DES MANUF.)》2022:1), Hsp70 terminator from Arabidopsis thaliana (Tian et al., 2002, Biodesign and Manufacturing 2022:1), Hsp18.2 terminator from Arabidopsis thaliana (Tian et al., 2002, Biodesign and Manufacturing 2022:1), Act2 terminator from Arabidopsis thaliana (Tian et al., 2002, Biodesign and Manufacturing 2022:1), G7 terminator from Arabidopsis thaliana (Tian et al., 2002, Biodesign and Manufacturing 2022:1), 3g24240 terminator from Arabidopsis thaliana (Tian et al., 2002, Biodesign and Manufacturing 2022:1), NOS terminator from Agrobacterium tumefaciens (Tian et al., 2002, Biodesign and Manufacturing 2022:1) (SEQ ID NO: 10), Ocs terminator from Agrobacterium tumefaciens (Tian et al., 2002, Biodesign and Manufacturing, 2022: 1), Mas terminator from Agrobacterium tumefaciens (Tian et al., 2002, Biodesign and Manufacturing, 2022: 1), 35s terminator from cauliflower mosaic virus (Tian et al., 2002, Biodesign and Manufacturing, 2022: 1), Rbc terminator from Chrysanthemum (Tian et al., 2002, Biodesign and Manufacturing, 2022: 1), Ags terminator from Agrobacterium tumefaciens (Tian et al., 2002, Biodesign and Manufacturing 2022: 1), 3'utr-nos terminator from Agrobacterium tumefaciens (Tian et al., 2002, Biodesign and Manufacturing 2022: 1), 7s terminator from soybean (Tsubokura et al., (2012), Plant Molecular Biology 78: 301), E9 terminator from pea (Coruzzi et al., (1984), EMBO Reports Rep. 3:1671), ORF25 terminator from Agrobacterium tumefaciens (Barker et al., (1983), Plant Mol. Biol. 2:335), pinII terminator from potato (Keil et al., (1986), Nucleic Acids Res. 14:5641), tml terminator from Agrobacterium tumefaciens (Barker et al., (1983), Plant Mol. Biol. 2:335), Tr7 terminator from Agrobacterium tumefaciens (Dhaese et al., (1983), EMBO Proc. 2:419). In some aspects, the terminator is NOS (. Figure 1 ,3 , 6, 8) (SEQ ID NO: 10) or arc5 ( Figure 2 , 4 , 5, 7, 9, 10) (SEQ ID NO: 12). The Arc5 terminator from Phaseolus vulgaris provides sequences that terminate transcription and direct polyadenylation of mRNA (Goossens et al., (1999), Plant Physiol. 120: 1095), but has also been reported to enhance gene expression and contribute to seed-specific expression.

[0123] In some aspects, the expression cassette includes a NOS terminator sequence. In some aspects, the NOS terminator includes a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 10.

[0124] In some aspects, the NOS terminator 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:10.

[0125] In some aspects, the expression cassette includes an arc5 terminator sequence. In some aspects, the arc5 terminator includes a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 12.

[0126] In some aspects, the arc5 terminator 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:12.

[0127] In some aspects, the recombinant sequence includes a translation or transcription enhancer sequence. An example of a translation enhancer is 5′UTRTEV (Tobacco Etch Virus Translation Enhancer) (SEQ ID NO: 9). The 5′ leader of Tobacco Etch Virus (TEV) is one of the better studied potyvirus translation enhancers (which contains two cap-independent regulatory elements (CIRE) folded into a pseudoknot), which can independently enhance the translation of downstream transgenes (Carrington and Freed, (1993) Journal of Virology (J.Virol.), 64: 1590). In some aspects, the recombinant sequence includes a matrix attachment region (MAR) as an enhancer. Rb7 MAR (SEQ ID NO: 13) is a DNA element shown to increase transgenic expression in plants. It has been shown that when added to most transcription terminators, the addition of Rb7 MAR strongly enhances protein production (Diamos and Mason, (2018), Journal of Plant Biotechnology 16: 1971). In addition, MARs can further increase the stability of transgene expression levels and can confer protection against transgene silencing (Vain et al., (1999), Plant Journal 18:233). In some aspects, the arc5 terminator is fused to the Rb7 matrix attachment region (MAR), which increases the likelihood and magnitude of transgene expression.

[0128] In some aspects, the expression cassette includes an Rb7MAR enhancer. In some aspects, the Rb7MAR enhancer includes a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO: 13.

[0129] In some aspects, 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:13.

[0130] In some aspects, the expression cassette includes a TEV enhancer. In some aspects, the TEV enhancer includes a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity to SEQ ID NO:9.

[0131] In some aspects, the TEV 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:9.

[0132] In some aspects, specific combinations of regulatory elements (i.e., promoters, terminators, and enhancers) result in enhanced expression of the above-mentioned heme proteins in seeds, such as 5%, 8%, and 10% tsp. In some aspects, the expression cassette includes a seed-specific promoter such as 7S or phas, and a terminator such as arc5 fused to the Rb7MAR enhancer. In some aspects, the combinations identified herein are:

[0133] i.p7S+cDNAHP+arc5+Rb7MAR

[0134] ii.p7S+TEV+cDNAHP+arc5+Rb7MAR

[0135] iii.PPhas+TEV+cDNAHP+arc5+Rb7MAR

[0136] iv.PPhas+cDNAHP+arc5+Rb7MAR

[0137] cDNAHP identifies the cDNA of heme protein. In some aspects, heme protein is animal-derived heme protein. In some aspects, heme protein is derived from metazoan. In some aspects, heme protein is derived from red meat (e.g., beef, pork, goat and mutton), poultry (e.g., chicken and turkey) and seafood (e.g., fish, crustaceans and molluscs). In some aspects, animal-derived heme protein is myoglobin. In some aspects, animal-derived heme protein is hemoglobin. It is conventional for those skilled in the art to replace the orthologous sequence from other organisms, so the simple replacement of recombinant protein is also within the scope of the present disclosure.

[0138] In some aspects, the present disclosure provides a polynucleotide comprising a nucleic acid encoding a hemoglobin protein, wherein the nucleic acid is operably linked to a seed-specific promoter selected from the group consisting of: a β-conglycinin alpha subunit from a 7S storage (7s) promoter of soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from broad bean, a SBP promoter from broad bean, a legumin B4 promoter from broad bean, a napus protein promoter from Brassica napus, a vicilin promoter from pea, an α-globulin promoter from cotton, γ-zein promoter from corn, glutenin promoter from wheat, VvβVPE promoter from grape, peanut seed promoter (GSP) from peanut, 7αP promoter from soybean, AtLAC15 promoter from Arabidopsis, SSPs promoter from chickpea, lectin promoter from soybean, oleosin promoter from Brassica napus, AhLEC1A promoter from peanut, Glu-ID-1 promoter from wheat, sesame 2S albumin (2Salb) promoter from sesame and 8SGα promoter from mung bean.

[0139] In some aspects, the heme protein comprises a plant-derived heme protein, a microbial-derived heme protein, or an animal-derived heme protein.

[0140] In some aspects, the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1 or SEQ NO:2.

[0141] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-7.

[0142] In some aspects, the nucleic acid further comprises a transcription terminator selected from the group consisting of: an extensin terminator from tobacco, an Ub10 terminator from Arabidopsis thaliana, an Hsp70 terminator from Arabidopsis thaliana, an Hsp18.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana, a G7 terminator from Arabidopsis thaliana, a 3g24240 terminator from Arabidopsis thaliana, a NOS terminator from Agrobacterium tumefaciens, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from cauliflower mosaic virus, 35s terminator from chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3'utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from potato, tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens, and Arc5 terminator from bean.

[0143] In some aspects, the nucleic acid further comprises a transcriptional or translational enhancer selected from the group consisting of 5'UTR TEV and Rb7Mar 3' matrix junction region.

[0144] In some aspects, the polynucleotide 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:1.

[0145] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:1.

[0146] In some aspects, the polynucleotide 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:2.

[0147] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:2.

[0148] In some aspects, the polynucleotide 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:3.

[0149] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:3.

[0150] In some aspects, the polynucleotide 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:4.

[0151] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:4.

[0152] In some aspects, the polynucleotide 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:5.

[0153] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:5.

[0154] In some aspects, the polynucleotide 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:6.

[0155] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:6.

[0156] In some aspects, the polynucleotide 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:7.

[0157] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:7.

[0158] In some aspects, the polynucleotide 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:8.

[0159] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:8.

[0160] In some aspects, the polynucleotide 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:9.

[0161] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:9.

[0162] In some aspects, the polynucleotide 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:10.

[0163] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:10.

[0164] In some aspects, the polynucleotide 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:11.

[0165] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:11.

[0166] In some aspects, the polynucleotide 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:12.

[0167] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:12.

[0168] In some aspects, the polynucleotide 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:13.

[0169] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:13.

[0170] In some aspects, the polynucleotide 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:14.

[0171] In some aspects, the polynucleotide comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:14.

[0172] In some aspects, a plant is transformed with each of the expression cassettes ( Figure 1-10 In some aspects, the stably transformed plant comprises in its genome: a recombinant DNA construct wherein the heme protein is stably expressed and produces a pink color in seed cotyledons and seed protein extracts ( Figure 15-16 ). The presence of heme protein in transgenic organisms results in a visible color change (pink) of the protein extract when compared to WT (Carlsson et al., (2020), Scientific Reports 10:1).

[0173] In some aspects, the stably transformed plant comprises in its genome: a recombinant DNA construct wherein the heme protein is stably expressed, extracted by standard protein extraction protocols, and analyzed by Western blotting ( Fig.17 , 18 ), liquid chromatography-mass spectrometry (LC-MS) ( Fig.19 ) and / or ELISA assay ( Fig. 20 ) detection.

[0174] In some aspects, the stably transformed plant comprises in its genome: a recombinant DNA construct wherein the heme protein is stably expressed at about 5% tsp or more ( Fig. 20 B). In some aspects, the hemoglobin protein is stably expressed at about 8% tsp or more ( Fig. 20 B). In some aspects, the heme protein is stably expressed at about 10% tsp or more ( Fig. 20 B). In some aspects, the hemoglobin protein is stably expressed at about 25% or more ( Fig. 20 B).

[0175] In some aspects, the recombinant heme protein for conversion is hemoglobin and myoglobin. In some aspects, the hemoglobin described herein is separated from pig (wild boar (Sus scrofa)). In some aspects, the hemoglobin is recombinant HbA-LL-HbB, and the hemoglobin includes hemoglobin A subunit, long linker and hemoglobin B subunit. In some aspects, myoglobin described herein is separated from pig (domestic pig (Sus scrofa domesticus)). In some aspects, the expression cassette includes any sequence in the sequence disclosed in Table 1.

[0176] In some aspects, the expression cassette comprises SEQ ID NO: 11, SEQ ID NO: 12 and / or SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 12 and / or SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 14, SEQ ID NO: 9, SEQ ID NO: 12 and SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 14, SEQ ID NO: 12 and SEQ ID NO: 13.

[0177] In some aspects, the expression cassette comprises SEQ ID NO: 11, SEQ ID NO: 1, SEQ ID NO: 12 and / or SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 12 and / or SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 14, SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 12 and SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 14, SEQ ID NO: 1, SEQ ID NO: 12 and SEQ ID NO: 13.

[0178] In some aspects, the expression cassette comprises SEQ ID NO: 11, SEQ ID NO: 2, SEQ ID NO: 12 and / or SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 12 and / or SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 14, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID NO: 13. In some aspects, the expression cassette comprises SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID NO: 13.

[0179] In some aspects, a transgenic plant, plant tissue or plant cell is provided herein, comprising an expression cassette comprising an exogenous nucleic acid encoding a heme protein. In some aspects, the nucleic acid is operably linked to a seed-specific promoter and a transcription terminator. In some aspects, the heme protein is expressed in seeds at an amount of about 5% TSP. In some aspects, the heme protein is expressed in seeds at an amount of about 6% TSP. In some aspects, the heme protein is expressed in seeds at an amount of about 7% TSP. In some aspects, the heme protein is expressed in seeds at an amount of about 8% TSP. In some aspects, the heme protein is expressed in seeds at an amount of about 9% TSP. In some aspects, the heme protein is expressed in seeds at an amount of about 10% TSP. In some aspects, the heme protein is expressed in seeds at an amount of about 11% TSP. In some aspects, the heme protein is expressed in seeds at an amount of about 12% TSP. In some aspects, the heme protein is expressed in the seed at about 13% TSP. In some aspects, the heme protein is expressed in the seed at about 14% TSP. In some aspects, the heme protein is expressed in the seed at about 15% TSP. In some aspects, the heme protein is expressed in the seed at about 18% TSP. In some aspects, the heme protein is expressed in the seed at about 20% TSP. In some aspects, the heme protein is expressed in the seed at about 25% TSP. In some aspects, the heme protein is expressed in the seed at about 30% TSP. In some aspects, the heme protein is expressed in the seed at an amount between about 5% TSP and about 35% TSP. In some aspects, the heme protein is expressed in the seed at an amount between about 8% TSP and about 35% TSP. In some aspects, the heme protein is expressed in the seed in an amount between about 10% TSP and about 35% TSP. In some aspects, the heme protein is expressed in the seed in an amount between about 12% TSP and about 35% TSP. In some aspects, the heme protein is expressed in the seed in an amount between about 5% TSP and about 30% TSP. In some aspects, the heme protein is expressed in the seed in an amount between about 5% TSP and about 29% TSP. In some aspects, the heme protein is expressed in the seed in an amount between about 5% TSP and about 28% TSP. In some aspects, the heme protein is expressed in the seed in an amount between about 10% TSP and about 30% TSP. In some aspects, the heme protein is expressed in the seed in an amount between about 8% TSP and about 30% TSP. In some aspects, the heme protein is expressed in the seed in an amount between about 6% TSP and about 28% TSP.

[0180] In some aspects, the expression cassette 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:3.

[0181] In some aspects, the expression cassette 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:4.

[0182] In some aspects, the expression cassette 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:5.

[0183] In some aspects, the expression cassette 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:6.

[0184] In some aspects, the expression cassette 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 about 100% sequence identity to SEQ ID NO:7.

[0185] In some aspects, the expression cassette comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:3.

[0186] In some aspects, the expression cassette comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:4.

[0187] In some aspects, the expression cassette comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:5.

[0188] In some aspects, the expression cassette comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:6.

[0189] In some aspects, the expression cassette comprises a nucleic acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:7.

[0190] In some aspects, the stably transformed plant is soybean. Codon optimization is the process of improving the gene expression of the gene of interest and increasing the translation efficiency of the gene of interest by accommodating the codon bias of the host organism. In some aspects, the hemoglobin gene has been codon optimized for expression in soybean (SEQ ID NO: 1). In some aspects, the myoglobin gene has been codon optimized for expression in soybean (SEQ ID NO: 2).

[0191] In some aspects, the hemoglobin cDNA comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO:1.

[0192] In some aspects, the myoglobin cDNA comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO:2.

[0193] In some aspects, the hemoglobin cDNA comprises a nucleic acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% sequence identity to SEQ ID NO:1.

[0194] In some aspects, the myoglobin cDNA comprises a nucleic acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98% sequence identity to SEQ ID NO:2.

[0195] In some aspects, the recombinant sequence includes a gene encoding a selectable marker. In some aspects, the selectable marker is a BAR gene that produces phosphinothricin N-acetyltransferase protein and provides resistance to gluphosinate. In some aspects, the BAR gene is located in a plant transformation vector (circular plasmid) that is identical to the sequence of the heme protein ( Figure 1-2 In some aspects, the selectable marker is the aadA gene ( Figure 3-5 In some aspects, the aadA gene is in a separate linear construct and is co-bombarded with a linear construct that retains the sequence of the heme protein ( Figure 3-5 and 8–10).

[0196] In some aspects, disclosed herein is a method for stably expressing a heme protein in a plant, the method comprising a) transforming a plant with a plant transformation vector, b) regenerating a transgenic plant in vitro under selective pressure, and c) growing the transformed plant under conditions wherein the recombinant heme protein is expressed.

[0197] In some aspects, the expression level of the heme protein is referred to as "total soluble protein" ("TSP"). The expression level in TSP refers to the amount of the protein of interest relative to the total amount of protein that can be reasonably extracted from the plant using standard methods. Methods for extracting total protein from plant tissues such as seeds are known in the art (Cunha et al., (2011a), Transgenic Research 20:811, Cunha et al., (2011b), Transgenic Research 20:841, Ding et al., (2006), Biotechnology Express 28:869). The amount of the protein of interest can be measured using methods known in the art such as ELISA or Western blotting.

[0198] The heme proteins and transgenic plants described herein can be used to prepare food compositions. In some aspects, the recombinant heme proteins produced by transgenic plants can be used in their entirety, in their fractions and modifications, including dissolved, precipitated, partially or completely hydrolyzed, cross-linked, emulsified, texturized, cooked, extruded, reacted, structured versions, to prepare meat-like (meat analog) foods, including ground meat such as minced meat, meat strips, small pieces and steaks; reconstructed and shaped meat-like products, including burgers, fish fillets, meatballs, meat sticks, meat nuggets; reconstructed and stuffed / filled meat-like (meat analog) products, including sausages, ham-like products, spreadable foods; reconstructed and coated meat-like products, including nuggets, patties, meat strips, popcorn, rings, etc. Recombinant heme proteins can also be extracted from transgenic plants using standard methods known in the art.

[0199] In some aspects, the food composition is prepared using seeds of a transgenic plant expressing a recombinant heme protein. In some aspects, the food composition is prepared using a recombinant heme protein extracted and purified from the seeds.

[0200] The following experiments demonstrate different recombinant sequences containing heme proteins and methods for producing recombinant proteins in plants. Although the following examples describe expression in soybean, those skilled in the art will appreciate that the expression sequences and methods disclosed herein can be customized for expression in any monocot or dicot plant.

[0201] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, biotechnology, plant genetic engineering, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature.

[0202] All references cited above and all references cited herein are incorporated by reference in their entirety.

[0203] The present disclosure is to be considered illustrative of the invention and is not intended to limit the invention to the specific aspects illustrated by the following drawings or description.The invention will now be described with reference to the accompanying drawings showing specific aspects.

[0204] Examples

[0205] Example 1. Construction of plant transformation vector

[0206] A codon-optimized gene comprising the α and β subunits of the porcine hemoglobin gene, termed HbA-LL-HbB, was synthesized by Genscript. The HbA-LL-HbB gene was cloned into the internal pIPTRA0-2x35S-MCS vector using BamHI / HindIII restriction sites. The HbA-LL-HbB gene was cloned between the 35S promoter and the NOS terminator to generate the pIPTRA0:p35S+HbA-LL-HbB vector (SEQ ID NO: 3). Figure 1 A graphical representation of the pIPTRA0:p35S+HbA-LL-HbB vector is shown, and Figure 6 The nucleotide sequence of each of the regulatory elements involved is shown.

[0207] The 7S promoter fused to the TEV enhancer (p7S-TEV) and the arc5 terminator fused to the fish Rb7 matrix linker region (arc5T-Rb7MAR) were synthesized by Kingstar Technologies. The pIPTRA0:p35S+HbA-LL-HbB vector was modified to generate the pIPTRA0:p7S+HbA-LL-HbB-Arc5T vector (SEQ ID NO: 4). The 35S promoter was replaced with p7S-TEV using XbaI / BamHI restriction sites. The NOS terminator was replaced with the arc5T-Rb7MAR fusion using HindIII / SpeI restriction sites. The graphical representation and nucleotide sequence of the pIPTRA0:p7S+HbA-LL-HbB-Arc5T vector are shown in Figure 2 and 7 Shown in.

[0208] 35S+TEV+myoglobin cDNA+NOS (SEQ ID NO: 5), p7S+TEV+myoglobin cDNA+arc5+Rb7MAR (SEQ ID NO: 6) and PPhas+myoglobin cDNA+arc5+Rb7MAR (SEQ ID NO: 7) expression vectors are referred to as EC 1, EC2 and EC3, respectively. EC1, EC2 and EC3 were assembled by Golden Gate cloning in an internal pEXPLODER plasmid. The promoter, myoglobin and terminator were incorporated into the pEXPLODER plasmid. After successful assembly of EC1, EC2 and EC3 linear fragments, they were released from the circular plasmid by BsaI digestion and subsequently size-separated in a 0.5% (w / v) agarose gel. After gel purification, EC1, EC2 and EC3 were separated from the segment carrying the selectable marker by AscI digestion. The two resulting linear constructs (selectable marker + EC1, EC2 or EC3) were co-bombarded into soybean explants. A graphical representation of the three linearized fragments is shown in Figure 3 , 4 and 5. The nucleotide sequences of EC1, EC2, and EC3 are presented in Figure 8 , 9 and 10 are presented.

[0209] Example 2. Confirmation of transgenic events

[0210] For all constructs used in this regard, in vitro regeneration of putative transgenic lines was obtained ( Fig.11 ). DNA was extracted from leaf tissue of the regenerated explants for further genetic screening.

[0211] DNA from putative transformed lines with pIPTRA0:p35S+HbA-LL-HbB and pIPTRA0:p7S+HbA-LL-HbB-Arc5T was screened by PCR to confirm the presence of transgenic insertion in the host genome. Agarose gel images show that pIPTRA0:p35S+HbA-LL-HbB ( Fig.12 ) and IPTRA0:p7S+HbA-LL-HbB-Arc5T( Fig.13 ) PCR amplification results of putative transgenic lines. For lines transformed with pIPTRA0:p35S+HbA-LL-HbB and IPTRA0:p7S+HbA-LL-HbB-Arc5T, respectively, the presence of 412 bp and 390 bp bands confirmed the presence of transgenic inserts.

[0212] qPCR was performed on a segment of the aadA1a CDS to confirm the presence of the transgenic insert in lines putatively transformed with EC1, EC2, and EC3 ( Fig.14 ). The marker Ct cutoff value for positive lines must be <25.

[0213] Example 3. Total Soluble Protein Production in Soybean Transgenic Events Carrying the Porcine Hemoglobin Gene

[0214] Transgenic T0 plants transformed with pIPTRA0:p35S+HbA-LL-HbB and pIPTRA0:p7S+HbA-LL-HbB-Arc5T were grown and propagated to T1 seeds. T1 seeds were screened by PCR for the presence of the porcine hemoglobin gene; small segments of seeds were excised for PCR purposes.

[0215] A total of 3 PCR-positive seeds from each transgenic event were pooled and subjected to protein extraction. The pooled seeds were broken in a tissue lyser, treated with extraction buffer (50 mM Tris-Cl pH 6.8, NaCl 50 mM, Na2SO3 36 mM, PHIC 1:200), and centrifuged at 13000 rpm for 10 min at 4°C ( Fig.15). After protein extraction, the extracts were then run on a 10-well 12% SDS PAGE gel, loading 100 μg of protein per transgenic extract, molecular weight standards, 100 μg of protein extract of WT used as a negative control, and WT extract + 10, 25, 50, 150 and 250 ng of Hb standard (Sigma-Aldrich Hemoglobin Porcine Lyophilized Powder. Catalog No. H4131). The protein bands were then transferred to a nitrocellulose membrane and the western blot was developed with a 1 / 250 dilution of anti-porcine antibody (UsBiological Life sciences, Catalog No. 140639) Fig.17 Using Western blotting, the detected hemoglobin was quantified by comparing the intensity of the hemoglobin band from the seed extract with that from a hemoglobin standard, allowing calculation of the hemoglobin percentage of the TSP ( Fig.18 ; Table 1).

[0216] Table 1. Hemoglobin accumulation as a percentage of TSP content in independent transgenic soybean seed stocks. Table contains coefficients of variation (%). "nd": not determined, events with no coefficient of variation because two of three replicates were discarded.

[0217]

[0218]

[0219] Porcine hemoglobin from whole soybean seed protein extracts was also identified and quantified by liquid chromatography-mass spectrometry (LC-MS) ( Fig.18 ). Equal amounts of soybean seed protein from each transgenic event were digested with LysC / trypsin and then peptides were desalted on a C18 tip. LC-MS data were collected on a Bruker timsTOF-Pro2 and searched against a soybean database supplemented with the provided porcine hemoglobin sequence. The amount of hemoglobin was determined by normalizing its MS2 intensity relative to soybean cupin. Hemoglobin was not detected in the WT sample.

[0220] Transgenic events carrying porcine myoglobin gene: Transgenic T0 plants transformed with EC1, EC2 and EC3 linear constructs were grown and propagated to T2 seeds. T2 seeds were screened for the presence of porcine myoglobin gene by ddPCR; small segments of seeds were excised for PCR purposes.

[0221] A total of 3 PCR-positive seeds from each transgenic event were pooled and subjected to protein extraction. The pooled seeds were ground with Omni ceramic beads (1.4 mm) in extraction buffer (5% w / v SDS, 175 mM Tris-HCl, pH 8.0, 0.4% v / v β-mercaptoethanol); the extract was heated to 65°C for 25 minutes, centrifuged and the supernatant was transferred to a fresh tube. Myoglobin quantification from seed extracts was performed using the Alpha Diagnostic ELISA kit (Catalog No. 600-640-PMY). All samples were normalized to 50 μg / mL total soluble protein (TSP) and tested for myoglobin content according to the manufacturer's protocol. The concentration of myoglobin was determined by reference to a standard curve. Each event consisted of one biological replicate (pool of 3 seeds) and 3 technical replicates. Twenty μL (1 μg) was tested for each sample. The percentage of myoglobin in TSP was 40.4% (0.25 μg). Fig. 20 and are presented in Table 2.

[0222] Table 2. Myoglobin accumulation as a percentage (± sd) of TSP content in independent transgenic soybean seed stocks. TSP content above 5% is indicated in grey shading.

[0223]

[0224]

[0225]

[0226] sequence

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

Claims

1. A transgenic plant, plant tissue or plant cell comprising an exogenous nucleic acid encoding a hemoglobin protein, wherein the nucleic acid is operably linked to a seed-specific promoter and a transcription terminator, wherein the hemoglobin protein is expressed in the seed in an amount of at least about 5% of the total soluble protein (TSP).

2. The transgenic plant, plant tissue or plant cell of claim 1, wherein the nucleic acid is operably linked to a transcriptional or translational enhancer.

3. The transgenic plant, plant tissue or plant cell of claim 2, wherein the heme protein is expressed in the seed in an amount of at least about 8% TSP.

4. The transgenic plant, plant tissue or plant cell of claim 2, wherein the heme protein is expressed in the seed in an amount of at least about 10% TSP.

5. The transgenic plant, plant tissue or plant cell according to any one of claims 1 to 4, wherein the hemoglobin protein comprises a plant-derived hemoglobin protein, a microbial-derived hemoglobin protein or an animal-derived hemoglobin protein.

6. The transgenic plant, plant tissue or plant cell according to any one of claims 1 to 5, wherein the hemoprotein comprises a hemoprotein involved in oxygen transport, an enzyme having a prosthetic heme group or a hemoprotein involved in the electron transport chain.

7. The transgenic plant, plant tissue or plant cell of any one of claims 1 to 6, wherein the heme protein comprises hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b or cytochrome c.

8. The transgenic plant, plant tissue or plant cell according to any one of claims 1 to 7, wherein the heme protein is an animal-derived heme protein selected from the group consisting of hemoglobin and myoglobin.

9. The transgenic plant, plant tissue or plant cell of claim 1, wherein the seed-specific promoter comprises a β-conglycinin α subunit from the 7S storage (7s) promoter of soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from Vicia faba, a SBP promoter from Vicia faba, a legumin B4 promoter from Vicia faba, a napin (Napin) promoter from Brassica napus, a vicilin promoter from pea (Pisum sativum), an α-globulin promoter from cotton, a γ-zein promoter from corn, a glutenin promoter from wheat, a glutenin promoter from grape (Vitis spp), the VvβVPE promoter from peanut, the peanut seed promoter (GSP) from peanut, the 7αP promoter from soybean, the AtLAC15 promoter from Arabidopsis thaliana, the SSPs promoter from chickpea, the lectin promoter from soybean, the oleosin promoter from Brassica napus, the AhLEClA promoter from peanut, the Glu-1D-1 promoter from wheat, the sesame 2S albumin (2Salb) promoter from sesame, or the 8SGα promoter from mung bean.

10. The transgenic plant, plant tissue or plant cell of claim 9, wherein the seed-specific promoter is β-phaseolin (Phas).

11. The transgenic plant, plant tissue or plant cell according to claim 1, further comprising a terminator sequence, wherein the terminator sequence comprises an extensin terminator from tobacco, an Ub10 terminator from Arabidopsis thaliana, an Hsp70 terminator from Arabidopsis thaliana, an Hsp1 8.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana, a G7 terminator from Arabidopsis thaliana, a 3g24240 terminator from Arabidopsis thaliana, a NOS terminator from Agrobacterium tumefaciens, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from Agrobacterium tumefaciens, a Cauliflower Mosaic virus terminator, a Virus), 35s terminator from Rbc terminator from Chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3'utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from Solanum tuberosum, tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens, or Arc5 terminator from Phaseolus vulgaris.

12. The transgenic plant, plant tissue or plant cell according to claim 11, wherein the terminator is the Arc5 terminator from Phaseolus vulgaris.

13. The transgenic plant, plant tissue or plant cell of claim 1, further comprising a transcriptional or translational enhancer selected from the group consisting of: a 5' untranslated region (UTR) from Tobacco Etch Virus (TEV) and a Rb7Mar 3' matrix junction region.

14. The transgenic plant, plant tissue or plant cell according to claim 13, wherein the enhancer is the Rb7Mar 3' matrix junction region.

15. The transgenic plant, plant tissue or plant cell of claim 2, wherein the exogenous nucleic acid is operably linked to the β-conglycinin α subunit from the 7S storage protein (7s) promoter of soybean and the NOS terminator.

16. The transgenic plant, plant tissue or plant cell of claim 2, wherein the exogenous nucleic acid is operably linked to the β-conglycinin α subunit from the 7S storage protein (7S) promoter of soybean and the Arc5 terminator and Rb7MAR fused to arc5.

17. The transgenic plant, plant tissue or plant cell of claim 2, wherein the exogenous nucleic acid is operably linked to the β-conglycinin α subunit from the 7S storage protein (7S) promoter of soybean, the 5'UTR TEV enhancer, and the Arc5 terminator and Rb7MAR fused to arc5.

18. The transgenic plant, plant tissue or plant cell of claim 2, wherein the exogenous nucleic acid is operably linked to a β-phaseolin (Phas) promoter and a NOS terminator from common bean.

19. The transgenic plant, plant tissue or plant cell of claim 2, wherein the exogenous nucleic acid is operably linked to a β-phaseolin (Phas) promoter from common bean, an Arc5 terminator, followed by Rb7MAR.

20. The transgenic plant, plant tissue or plant cell of claim 2, wherein the exogenous nucleic acid is operably linked to a beta-phaseolin (Phas) promoter from common bean, a 5'UTR TEV enhancer and an Arc5 terminator followed by Rb7MAR.

21. The transgenic plant, plant tissue or plant cell of claim 1, wherein the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

1.

22. The transgenic plant, plant tissue or plant cell of claim 1, wherein the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

2.

23. The transgenic plant, plant tissue or plant cell of any one of claims 1 to 19, wherein the plant, plant tissue or plant cell is a plant.

24. The transgenic plant, plant tissue or plant cell of any one of claims 1 to 19, wherein the plant, plant tissue or plant cell is a plant tissue.

25. The transgenic plant, plant tissue or plant cell of any one of claims 1 to 19, wherein the plant, plant tissue or plant cell is a plant cell.

26. The transgenic plant, plant tissue or plant cell of any one of claims 1 to 25, wherein the transgenic plant, plant tissue or plant cell is derived from soybean (Glycine max), rice (Oryza sativa), barley (Hordeum vulgare), corn (Zea mays), rye (Secale cereale), oats (Avena sativa), sugar beet (Beta vulgaris), cultivated sugar beet subsp. vulgaris (Beta vulgaris subsp. vulgaris), parsnip (Pastina casativa), bean, pea, cowpea (Vigna angularis), mung bean (Vigna radiata), chickpea (Cicera rietinum), peanut (Arachis hypogaea), lentil (Lens culinaris), Medicago sativa, rocket (Eruca vesicaria), mustard (Brassica juncea), lettuce (Lactuca sativa), Brassica spp., potato, sweet potato (Ipomoea batatas, cassava (Manihot esculenta), wheat (Triticum aestivum) or spelt (Triticum spelta).

27. A method for obtaining a recombinant heme protein, the method comprising: i. Providing a transgenic plant capable of expressing at least about 5% TSP heme protein in seeds; ii. cultivating the transgenic plant; iii. collecting the transgenic plants; and iv. isolating and purifying animal heme protein from the collected plants.

28. The method of claim 27, wherein said collecting comprises collecting said seeds of said transgenic plants.

29. The method of claim 28, wherein the transgenic plant is selected from the group consisting of soybean, rice, barley, corn, rye, oats, sugar beet, Beta vulgaris, parsnips, beans, peas, cowpeas, mung beans, chickpeas, peanuts, lentils, clover, rocket, mustard, lettuce, Brassica, potato, sweet potato, cassava, wheat or spelt.

30. A transgenic seed comprising a recombinant heme protein of at least about 5% TSP.

31. A transgenic seed comprising a recombinant heme protein of at least about 8% TSP.

32. A transgenic seed comprising a recombinant heme protein of at least about 12% TSP.

33. A transgenic seed comprising a recombinant heme protein of at least about 15% TSP.

34. A transgenic seed comprising a recombinant heme protein of at least about 20% TSP.

35. A transgenic seed comprising a recombinant heme protein of at least about 25% TSP.

36. The transgenic seed of any one of claims 30 to 35, wherein the recombinant hemoglobin protein is an animal hemoglobin protein.

37. The transgenic seed of any one of claims 30 to 35, wherein the recombinant heme protein is myoglobin.

38. The transgenic seed of any one of claims 30 to 35, wherein the recombinant heme protein is hemoglobin.

39. The transgenic seed of any one of claims 30 to 38, wherein the transgenic seed is from a species selected from the group consisting of soybean, rice, barley, corn, rye, oats, sugar beet, Beta vulgaris, parsnips, beans, peas, cowpeas, mung beans, chickpeas, peanuts, lentils, clover, rocket, mustard, lettuce, Brassica, potato, sweet potato, cassava, wheat or spelt.

40. A food composition comprising the transgenic seed according to any one of claims 30 to 39.

41. A food composition comprising the hemoglobin protein of the transgenic plant, plant tissue or plant cell of claim 1.

42. A meat analog food composition comprising the transgenic seed according to any one of claims 30 to 39.

43. A meat analog food composition comprising the heme protein of the transgenic plant, plant tissue or plant cell of claim 1.

44. A polynucleotide comprising a nucleic acid encoding a hemoprotein, wherein the nucleic acid is operably linked to a seed-specific promoter selected from the group consisting of: a β-conglycinin alpha subunit from the 7S storage (7s) promoter of soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from broad bean, a SBP promoter from broad bean, a legumin B4 promoter from broad bean, a napus protein promoter from Brassica napus, a vicilin promoter from pea, an α-globulin promoter from cotton, a β-phaseolin promoter from corn, a β-phaseolin promoter from safflower, a β-phaseolin promoter from turmeric, a β-phaseolin promoter from sesame, a β-phaseolin promoter from safflower ... γ-zein promoter, glutenin promoter from wheat, VvβVPE promoter from grape, peanut seed promoter (GSP) from peanut, 7αP promoter from soybean, AtLAC15 promoter from Arabidopsis, SSPs promoter from chickpea, lectin promoter from soybean, oleosin promoter from Brassica napus, AhLEC1A promoter from peanut, Glu-1D-1 promoter from wheat, sesame 2S albumin (2Salb) promoter from sesame and 8SGα promoter from mung bean.

45. The polynucleotide of claim 44, wherein the hemoglobin protein comprises a plant-derived hemoglobin protein, a microbial-derived hemoglobin protein, or an animal-derived hemoglobin protein.

46. ​​The polynucleotide of claim 44, wherein the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

1.

47. The polynucleotide of claim 44, wherein the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

2.

48. The polynucleotide of claim 44, comprising a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3 to 7.

49. The polynucleotide of claim 44, wherein the seed-specific promoter is beta-phaseolin (Phas).

50. The polynucleotide of claim 44, further comprising a transcription terminator selected from the group consisting of: an extensin terminator from tobacco, an Ub10 terminator from Arabidopsis thaliana, an Hsp70 terminator from Arabidopsis thaliana, an Hsp18.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana, a G7 terminator from Arabidopsis thaliana, a 3g24240 terminator from Arabidopsis thaliana, a NOS terminator from Agrobacterium tumefaciens, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from Agrobacterium tumefaciens, 35s terminator from cauliflower mosaic virus, Rbc terminator from chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3'utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from potato, tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens and Arc5 terminator from bean.

51. The polynucleotide of claim 50, wherein the terminator is Arc5.

52. The polynucleotide of claim 44, further comprising a transcriptional or translational enhancer selected from the group consisting of 5'UTR TEV and Rb7Mar 3' matrix junction region.

53. The polynucleotide of claim 52, wherein the enhancer is the Rb7Mar 3' matrix junction region.

54. An expression vector comprising a nucleic acid encoding a hemoprotein, wherein the nucleic acid is operably linked to a seed-specific promoter selected from the group consisting of: a β-conglycinin alpha subunit from the 7S storage (7s) promoter of soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from broad bean, a SBP promoter from broad bean, a legumin B4 promoter from broad bean, a napus protein promoter from Brassica napus, a vicilin promoter from pea, an α-globulin promoter from cotton, a β-phaseolin promoter from corn, a β-phaseolin ... γ-zein promoter, glutenin promoter from wheat, VvβVPE promoter from grape, peanut seed promoter (GSP) from peanut, 7αP promoter from soybean, AtLAC15 promoter from Arabidopsis, SSPs promoter from chickpea, lectin promoter from soybean, oleosin promoter from Brassica napus, AhLEC1A promoter from peanut, Glu-1D-1 promoter from wheat, sesame 2S albumin (2Salb) promoter from sesame, or 8SGα promoter from mung bean.

55. The expression vector of claim 54, wherein the heme protein comprises a plant-derived heme protein, a microbial-derived heme protein, or an animal-derived heme protein.

56. The expression vector of claim 54, wherein the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

1.

57. The expression vector of claim 54, wherein the nucleic acid encoding the hemoglobin protein comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

2.

58. The expression vector of any one of claims 54 to 57, wherein the seed-specific promoter is beta-phaseolin (Phas).

59. according to the expression vector described in any one of claims 54 to 58, it further comprises the transcription terminator selected from the group consisting of: the extensin terminator from tobacco, the Ub10 terminator from Arabidopsis thaliana, the Hsp70 terminator from Arabidopsis thaliana, the Hsp18.2 terminator from Arabidopsis thaliana, the Act2 terminator from Arabidopsis thaliana, the G7 terminator from Arabidopsis thaliana, the 3g24240 terminator from Arabidopsis thaliana, the NOS terminator from Agrobacterium tumefaciens, the Ocs terminator from Agrobacterium tumefaciens. terminator from Agrobacterium tumefaciens, Mas terminator from Cauliflower mosaic virus, Rbc terminator from Chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3'utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from potato, tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens, and Arc5 terminator from Phaseolus vulgaris.

60. The expression vector of claim 59, wherein the terminator is Arc5.

61. The expression vector according to any one of claims 54 to 60, further comprising a transcriptional or translational enhancer selected from the group consisting of 5'UTR TEV and Rb7Mar 3' matrix junction region.

62. The expression vector of claim 61, wherein the enhancer is the Rb7Mar 3' matrix linker region.

63. The expression vector of any one of claims 54 to 62, wherein the expression vector is a plant expression vector or a plasmid.

64. The expression vector of claim 54, comprising a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3 to 7.

65. An expression vector comprising a nucleic acid encoding a recombinant protein, wherein the recombinant protein is expressed in a plant seed in an amount of at least about 5% total soluble protein (TSP), wherein the nucleic acid is operably linked to i) a phas promoter and ii) a terminator selected from the group consisting of: an extensin terminator from tobacco, an Ub10 terminator from Arabidopsis thaliana, an Hsp70 terminator from Arabidopsis thaliana, an Hsp18.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana, a G7 terminator from Arabidopsis thaliana, a 3g24240 ... NOS terminator from Agrobacterium tumefaciens, Ocs terminator from Agrobacterium tumefaciens, Mas terminator from Agrobacterium tumefaciens, 35s terminator from cauliflower mosaic virus, Rbc terminator from chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3'utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from potato, tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens and Arc5 terminator from bean.

66. The expression vector of claim 65, wherein the terminator is the Arc5 terminator from Phaseolus vulgaris.

67. The expression vector according to claim 65 or 66, further comprising a transcriptional or translational enhancer selected from the group consisting of 5'UTR TEV and Rb7Mar3' matrix linker region.

68. The expression vector of claim 67, wherein the enhancer is the Rb7Mar 3' matrix linker region.

69. The expression vector of claim 65, wherein the expression vector is a plant expression vector or a plasmid.

70. A polynucleotide comprising a nucleic acid encoding a recombinant protein, wherein the recombinant protein is expressed in a plant seed in an amount of at least about 5% total soluble protein (TSP), wherein the nucleic acid is operably linked to i) a phas promoter and ii) a terminator selected from the group consisting of: an extensin terminator from tobacco, an Ub10 terminator from Arabidopsis thaliana, an Hsp70 terminator from Arabidopsis thaliana, an Hsp18.2 terminator from Arabidopsis thaliana, an Act2 terminator from Arabidopsis thaliana, a G7 terminator from Arabidopsis thaliana, a 3g24240 terminator from Arabidopsis thaliana, a 5g24240 terminator from Arabidopsis thaliana, a 5g24241 terminator from Arabidopsis thaliana, a 5g24242 terminator from Arabidopsis thaliana, a 5g24243 terminator from Arabidopsis thaliana, a 5g24244 terminator from Arabidopsis thaliana, a 5g24242 terminator from Arabidopsis thaliana, a 5g24243 terminator from Arabidopsis thaliana NOS terminator from Agrobacterium tumefaciens, Ocs terminator from Agrobacterium tumefaciens, Mas terminator from Agrobacterium tumefaciens, 35s terminator from cauliflower mosaic virus, Rbc terminator from chrysanthemum, Ags terminator from Agrobacterium tumefaciens, 3'utr-nos terminator from Agrobacterium tumefaciens, 7s terminator from soybean, E9 terminator from pea, ORF25 terminator from Agrobacterium tumefaciens, pinII terminator from potato, tml terminator from Agrobacterium tumefaciens, Tr7 terminator from Agrobacterium tumefaciens and Arc5 terminator from bean.

71. The polynucleotide of claim 70, wherein the terminator is the Arc5 terminator from Phaseolus vulgaris.

72. The polynucleotide of claim 70 or 71, further comprising a transcriptional or translational enhancer selected from the group consisting of 5'UTR TEV and Rb7Mar 3' matrix junction region.

73. The polynucleotide of claim 72, wherein the enhancer is the Rb7Mar 3' matrix junction region.

74. A method for producing a recombinant protein at at least 5% TSP in plant seeds, the method comprising transforming the plant with an expression vector according to any one of claims 65 to 69 or a polynucleotide according to any one of claims 70 to 73.

Images