High expression of animal hemoproteins in plants

Abstract

The present disclosure provides methods for producing heme proteins in transgenic plants, plant tissues, or plant cells, as well as methods for describing the expression of these heme proteins in seeds. The present disclosure also provides transgenic plants that express heme proteins, myoglobin, and hemoglobin by introducing and integrating recombinant DNA constructs into the host genetic material of a plant of interest. Certain combinations of regulatory factors disclosed herein enable high levels of heme protein expression in seeds.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 367,299, filed on June 29, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] (Reference to Electronically Submitted Sequence Listing) The content of the sequence listing submitted electronically in XML format (name: 5061_002PC01Seqlisting_ST26.xml, size: 43,109 bytes, and creation date: June 16, 2023), filed together with this application, is incorporated herein by reference in its entirety.

[0003] This disclosure relates to the production of animal hemoglobin proteins in transgenic plants. This disclosure also relates to food compositions comprising recombinant hemoglobin proteins produced in genetically engineered plants. This disclosure also relates to improved expression cassettes for the production of animal hemoglobin proteins in transgenic plants and transgenic seeds, which contain selected regulatory elements and codon - optimized protein - coding sequences, and which result in substantially high (e.g., greater than 5%, greater than 8%, and greater than 10% of total soluble protein [TSP]) expression levels of recombinant proteins in plant seeds.

Background Art

[0004] Climate change and the projected increase in the world's population to 9.7 billion by 2050 require the practice of more sustainable lifestyles. Livestock production supplies most of the edible protein, but livestock cause approximately 18% of global greenhouse gas emissions (Stehfest et al. (2009), Clim. Change 95:83). Greenhouse gas emissions caused by livestock production are predicted to increase by 80% by 2050 (Tilman & Clark, (2014), Nature 515:518).

[0005] Meat production is plagued by other problems such as high resource intake, the presence of antibiotic residues in meat, zoonotic diseases, and ethical concerns regarding the utilization of animals. Public health problems such as type 2 diabetes, cardiovascular diseases, and cancer are also associated with meat consumption (Zhang et al. (2022), Curr. Opin. Food Sci. 43:43). Despite these problems, meat has a special status in human diet and demand continues to rise unprecedentedly. Over the past 20 years, the global demand for meat has increased by 58%. By 2050, studies predict that the total meat consumption will increase by 62 - 144% (Alexandratos & Bruinsma, (2012), Agricultural Development Economics Division; Valin et al. (2014), J. Agric. Econ. 45:51).

[0006] Proposed reduction efforts for livestock production and consumption include a shift to plant - derived protein foods. The demand for plant - derived proteins is increasing due to their health benefits, environmentally friendly production, animal welfare, and consumer curiosity based on taste (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 the consumer acceptance of alternative meat from plant proteins is the highest, followed by cultured meat (Onwezen et al. (2021), Appetite, 159:105058). However, the production of plant - derived alternative meat still faces several challenges such as the reconstruction of meat - like color, flavor, nutritional value, and structure (Zhang et al. (2022), Curr. Opin. Food Sci. 43:43).

[0007] Genetic engineering represents a convenient strategy for upgrading plant-derived recombinant protein products. These recombinant proteins in plants can help replace animal-derived proteins in human food and provide desired functional properties when used as components or as a whole along with the original plant parts. The expression of recombinant proteins in plants may help upgrade their color, flavor, nutritional value, and structure, either in their native form or when used as components. The present disclosure provides solutions for producing important animal heme proteins at high expression levels in transgenic plants and their seeds.

[0008] For further production of food, there have been several previous efforts to express plant-derived heme proteins in plants. For example, US Patent Application Publication No. US2019292555 discloses rice and Arabidopsis transgenic plants that express soybean leghemoglobin Lbc2 under the control of an alcohol-inducible promoter. This published patent application does not show any data related to the expression level of the recombinant leghemoglobin.

[0009] US Patent Application Publication US2019292217 describes transgenic Arabidopsis thaliana plants that overexpress an enzyme involved in the heme biosynthetic pathway (glutamyl-tRNA reductase (GluTR)-binding protein) and the expression of soybean leghemoglobin. This document does not provide any data regarding the expression level of leghemoglobin.

[0010] International Application Publication No. WO2022072846 discloses information about transgenic plants that express heme proteins with an altered fatty acid profile and upregulated heme biosynthesis, but no experiments are disclosed.

[0011] International Patent Application Publication No. WO9902687 discloses a method for increasing the iron content in transgenic rice plants by expressing rice or Arabidopsis thaliana hemoglobin, but the transgenic plants exhibit low hemoglobin expression levels.

[0012] A study has disclosed the production of human myoglobin in the leaves of Nicotiana benthamiana (Carlsson et al. (2020), Sci. Rep. 10:1). This document does not show data on heme loading into recombinant myoglobin, nor on the functionality or correct structural folding of this recombinant protein, nor on its incorporation into food.

[0013] However, attempts to produce hemoproteins in these plants do not result in high-level (e.g., greater than 5% TSP) recombinant hemoprotein expression in plant seeds. In molecular farming research, which has focused on gene expression via nuclear transformation, average expression levels of recombinant proteins of 0.5 - 2% TSP have been shown in stably transformed plants (Fischer & Emans, (2000), Transgenic Res. 9:279, Shanmugaraj et al. (2020), Plants, 9:842). These recombinant proteins produced in plants are mainly pharmaceutical proteins, proteins for diagnostic, research, and cosmetic industries. In chloroplasts, researchers have achieved higher yields of recombinant protein expression in the range of 3 - 46% TSP from plants (Dhingra & Daniell, (2006), Arabidopsis protocols, 245, Shanmugaraj et al. (2020), Plants, 9:842). The recombinant proteins expressed in chloroplasts are mainly pharmaceuticals, but also include herbicide tolerance genes. In seeds, recombinant proteins accumulate to lower average concentrations (0.05 - 1% TSP) (Jaeger et al. (2002), Nat. Biotechnol. 20:1265, Shanmugaraj et al. (2020), Plants, 9:842). However, regulators have been identified by independent research that produce significantly higher levels of protein in plant seeds (Jaeger et al. (2002), Nat. Biotechnol. 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 & 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), Nat. Biotechnol. 20:1265, Goossens et al. (1999), Plant Physiol. 120:1095). However, none of these independent studies have focused on complex proteins such as the heme proteins in the present disclosure and have only been verified in model plant species. The combination of regulatory factors described herein may result in stable seed protein production of more than 5%, 8%, or 10% TSP in commercially important seed crops such as legumes.

[0014] Therefore, the art has not yet provided a solution for producing heme proteins at high expression levels in the seeds of transgenic plants. SUMMARY OF THE INVENTION

[0015] In some embodiments, provided herein are transgenic plants, plant tissues, or plant cells comprising an exogenous nucleic acid encoding a heme protein. In some embodiments, the nucleic acid is operably linked to a seed - specific promoter and a transcription terminator. In some embodiments, the heme protein is expressed in the seed in an amount of at least about 5% of the total soluble protein (TSP).

[0016] In some embodiments, the nucleic acid is operably linked to a transcription enhancer or a translation enhancer.

[0017] In some embodiments, the heme protein is expressed in the seed in an amount of at least about 8% of the TSP.

[0018] In some embodiments, the heme protein is expressed in the seed in an amount of at least about 10% of the TSP.

[0019] In some embodiments, the heme protein includes a plant-derived heme protein, a microorganism-derived heme protein, an animal-derived heme protein, or a synthetic protein designed based on a natural heme protein.

[0020] In some embodiments, the heme protein includes a heme protein involved in oxygen transport, an enzyme having a heme prosthetic group, or a heme protein involved in the electron transport chain.

[0021] In some embodiments, the heme protein includes hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, or cytochrome c.

[0022] In some embodiments, the heme protein is an animal-derived heme protein selected from the group consisting of hemoglobin and myoglobin.

[0023] In some embodiments, the seed-specific promoter is a β-conglycinin α subunit of a 7S storage (7s) promoter from soybean, a β-phaseolin (Phas) promoter from kidney bean, a USP promoter from broad bean (Vicia faba), an SBP promoter from broad bean (Vicia faba), a legumin B4 promoter from broad bean (Vicia faba), a napin promoter from rapeseed (Brassica napus), a vicilin promoter from pea (Pisum sativum), an α-globulin promoter from cotton, a γ-zein promoter from maize, a glutenin promoter from wheat, a VvβVPE promoter from grapevine species (Vitis spp), a groundnut seed promoter (GSP) from peanut, a 7αP promoter from soybean, an AtLAC15 promoter from Arabidopsis thaliana, an SSP promoter from chickpea, a lectin promoter from soybean, an oleosin promoter from rapeseed, an AhLEC1A promoter from peanut, a Glu-1D-1 promoter from wheat, a 2S albumin (2Salb) promoter from sesame, or an 8SGα promoter from Japanese radish.

[0024] In some embodiments, the transgenic plant, plant tissue, or plant cell further comprises a terminator sequence.In some embodiments, the terminator sequence comprises an extensin terminator from tobacco, a 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 Arabidopsis thaliana, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from Agrobacterium tumefaciens, a 35s terminator from cauliflower mosaic virus, an Rbc terminator from Chrysanthemum, an Ags terminator from Agrobacterium tumefaciens, a 3’utr-nos terminator from Agrobacterium tumefaciens, a 7s terminator from soybean, an E9 terminator from Pisum sativum, an ORF25 terminator from Agrobacterium tumefaciens, a pinII terminator from Solanum tuberosum, a tml terminator from Agrobacterium tumefaciens, a Tr7 terminator from Agrobacterium tumefaciens, or an Arc5 terminator from Phaseolus vulgaris.

[0025] In some embodiments, the transgenic plant, plant tissue, or plant cell further comprises a transcription or translation enhancer selected from the group consisting of a 5' untranslated region (UTR) derived from tobacco etch virus (TEV), and an Rb7Mar 3' matrix attachment region as part of a transcription terminator.

[0026] In some embodiments, the exogenous nucleic acid is operably linked to the β-conglycinin α subunit of the 7S storage protein (7s) promoter derived from soybean, and the NOS terminator.

[0027] In some embodiments, the exogenous nucleic acid is operably linked to the β-conglycinin α subunit of the 7S storage protein (7S) promoter derived from soybean, as well as the Arc5 terminator and Rb7MAR fused to the Arc5 terminator.

[0028] In some embodiments, the exogenous nucleic acid is operably linked to the β-conglycinin α subunit of the 7S storage protein (7S) promoter derived from soybean, the 5'UTR TEV enhancer, as well as the Arc5 terminator and Rb7MAR fused to arc5.

[0029] In some embodiments, the exogenous nucleic acid is operably linked to the β-phaseolin (Phas) promoter derived from kidney bean, and the NOS terminator.

[0030] In some embodiments, the exogenous nucleic acid is operably linked to the β-phaseolin (Phas) promoter derived from kidney bean, and the Arc5 terminator fused to the Rb7MAR region.

[0031] In some embodiments, the exogenous nucleic acid is operably linked to the β-phaseolin (Phas) promoter derived from kidney bean, the 5'UTR TEV enhancer, and the Arc5 terminator fused to the Rb7MAR region.

[0032] In some embodiments, the nucleic acid encoding the heme protein comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 1.

[0033] In some embodiments, the nucleic acid encoding the heme protein comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 2.

[0034] In some embodiments, 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), table beet (Beta vulgaris subsp vulgaris), parsnip (Pastinaca sativa), common bean (Phaseolus vulgaris), pea (Pisum sativum), adzuki bean (Vigna angularis), mung bean (Vigna radiata), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentil (Lens culinaris), alfalfa (Medicago sativa), arugula (Eruca vesicaria), mustard (Brassica juncea), lettuce (Lactuca sativa), Brassica, potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), wheat (Triticum aestivum), or spelt wheat (Triticum spelta).

[0035] In some embodiments, provided herein is a method for obtaining a recombinant hemoprotein. In some embodiments, the method comprises: i) providing a transgenic plant capable of expressing at least about 5% of the total soluble protein (TSP) of the hemoprotein in seeds; ii) cultivating the transgenic plant; iii) harvesting the transgenic plant; and iv) isolating and purifying the animal hemoprotein from the harvested plant.

[0036] In some embodiments, harvesting comprises harvesting the seeds of the transgenic plant.

[0037] In some embodiments, provided herein are transgenic seeds comprising at least about 5% of the TSP of a recombinant hemoprotein.

[0038] In some embodiments, provided herein are transgenic seeds comprising at least about 8% of the TSP of a recombinant hemoprotein.

[0039] In some embodiments, provided herein are transgenic seeds comprising at least about 10% of the TSP of a recombinant hemoprotein.

[0040] In some embodiments, the transgenic seed is derived from a species selected from the group consisting of soybean (Glycine max), rice (Oryza sativa), barley (Hordeum vulgare), corn (Zea mays), rye (Secale cereale), oats (Avena sativa), sugar beet (Beta vulgaris), table beet (Beta vulgaris subsp vulgaris), parsnip (Pastinaca sativa), common bean (Phaseolus vulgaris), pea (Pisum sativum), adzuki bean (Vigna angularis), mung bean (Vigna radiata), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentil (Lens culinaris), alfalfa (Medicago sativa), arugula (Eruca vesicaria), mustard (Brassica juncea), lettuce (Lactuca sativa), Brassica genus, potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), wheat (Triticum aestivum), or spelt wheat (Triticum spelta).

[0041] In some embodiments, the recombinant hemoprotein is an animal hemoprotein.

[0042] In some embodiments, the recombinant hemoprotein is myoglobin.

[0043] In some embodiments, the recombinant hemoprotein is hemoglobin.

[0044] In some embodiments, a food composition comprising any of the transgenic seeds disclosed herein is provided herein.

[0045] In some embodiments, there are provided food compositions comprising a heme protein of any of the plants, plant tissues, or plant cells disclosed herein.

[0046] In some embodiments, there are provided meat analog food compositions comprising any of the transgenic seeds disclosed herein.

[0047] In some embodiments, there are provided meat analog food compositions comprising a heme protein of any of the plants, plant tissues, or plant cells disclosed herein.

[0048] In some embodiments, the disclosure also provides a polynucleotide comprising a nucleic acid encoding a heme protein, wherein the nucleic acid is operably linked to a β-conglycinin α subunit of a 7S storage (7s) promoter from soybean, a β-phaseolin (Phas) promoter from kidney bean, a USP promoter from broad bean (Vicia faba), an SBP promoter from broad bean (Vicia faba), a legumin B4 promoter from broad bean (Vicia faba), a napin promoter from rapeseed (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 VvβVPE promoter from grapevine species (Vitis spp), a glycinin seed promoter (GSP) from peanut, a 7αP promoter from soybean, an AtLAC15 promoter from Arabidopsis thaliana, an SSP promoter from chickpea, a lectin promoter from soybean, an oleosin promoter from rapeseed, an AhLEC1A promoter from peanut, a Glu-1D-1 promoter from wheat, a sesame 2S albumin (2Salb) promoter from sesame, or an 8SGα promoter from taro.

[0049] In some embodiments, the heme protein comprises a plant-derived heme protein, a microorganism-derived heme protein, or an animal-derived heme protein.

[0050] In some embodiments, the nucleic acid encoding the heme protein comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0051] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 3 to 7.

[0052] In some embodiments, the nucleic acid further comprises a transcription terminator selected from the group consisting of an extensin terminator from tobacco, a 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 Arabidopsis thaliana, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from Agrobacterium tumefaciens, a 35s terminator from cauliflower mosaic virus, an Rbc terminator from Chrysanthemum, an Ags terminator from Agrobacterium tumefaciens, a 3’utr-nos terminator from Agrobacterium tumefaciens, a 7s terminator from soybean, an E9 terminator from Pisum sativum, an ORF25 terminator from Agrobacterium tumefaciens, a pinII terminator from Solanum tuberosum, a tml terminator from Agrobacterium tumefaciens, a Tr7 terminator from Agrobacterium tumefaciens, or an Arc5 terminator from Phaseolus vulgaris.

[0053] In some embodiments, the nucleic acid further comprises a transcription or translation enhancer selected from the group consisting of a 5'UTR TEV and an Rb7Mar 3'matrix attachment region.

[0054] In some embodiments, the present disclosure is directed to an expression vector comprising a nucleic acid encoding a heme protein, wherein the nucleic acid is operably linked to a β-conglycinin α subunit of a 7S storage (7s) promoter from soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from broad bean (Vicia faba), an SBP promoter from broad bean (Vicia faba), a legumin B4 promoter from broad bean (Vicia faba), a napin promoter from rapeseed (Brassica napus), a vicilin promoter from pea (Pisum sativum), an α-globulin promoter from cotton, a γ-zein promoter from maize, a glutenin promoter from wheat, a VvβVPE promoter from a species of Vitis, a glycinin seed promoter (GSP) from peanut, a 7αP promoter from soybean, an AtLAC15 promoter from Arabidopsis thaliana, an SSP promoter from chickpea, a lectin promoter from soybean, an oleosin promoter from rapeseed, an AhLEC1A promoter from peanut, a Glu-1D-1 promoter from wheat, a 2S albumin (2Salb) promoter from sesame, or an 8SGα promoter from taro.

[0055] In some embodiments, the expression vector comprises a heme protein derived from a microorganism, a plant, or an animal.

[0056] In some embodiments, the expression vector comprises a nucleic acid encoding a heme protein having a sequence with at least 80% sequence identity to SEQ ID NO: 1.

[0057] In some embodiments, the expression vector comprises a nucleic acid encoding a heme protein having a sequence with at least 80% sequence identity to SEQ ID NO: 2.

[0058] In some embodiments, the expression vector comprises a nucleic acid encoding a heme protein having a sequence with at least 80% sequence identity to SEQ ID NO: 1, operably linked to a β-phaseolin (Phas) promoter.

[0059] In some embodiments, the expression vector comprises a nucleic acid encoding a heme protein having a sequence with at least 80% sequence identity to SEQ ID NO: 2, operably linked to a β-Phasolin (Phas) promoter.In some embodiments, the expression vector further comprises a transcription terminator selected from the group consisting of an extensin terminator from tobacco, a 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 Arabidopsis thaliana, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from Agrobacterium tumefaciens, a 35s terminator from cauliflower mosaic virus, an Rbc terminator from Chrysanthemum, an Ags terminator from Agrobacterium tumefaciens, a 3’utr-nos terminator from Agrobacterium tumefaciens, a 7s terminator from soybean, an E9 terminator from Pisum sativum, an ORF25 terminator from Agrobacterium tumefaciens, a pinII terminator from Solanum tuberosum, a tml terminator from Agrobacterium tumefaciens, a Tr7 terminator from Agrobacterium tumefaciens, or an Arc5 terminator from Phaseolus vulgaris.

[0060] In some embodiments, the expression vector comprises a nucleic acid encoding a heme protein, having a sequence with at least 80% sequence identity to SEQ ID NO: 1, operably linked to a β - phaseolin (Phas) promoter and an Arc5 terminator.

[0061] In some embodiments, the expression vector comprises a nucleic acid encoding a heme protein, having a sequence with at least 80% sequence identity to SEQ ID NO: 2, operably linked to a β - phaseolin (Phas) promoter and an Arc5 terminator.

[0062] In some embodiments, the expression vector further comprises a transcription or translation enhancer selected from the group consisting of 5’UTR TEV and the Rb7Mar 3’ matrix attachment region.

[0063] In some embodiments, the expression vector comprises a nucleic acid encoding a heme protein, having a sequence with at least 80% sequence identity to SEQ ID NO: 1, operably linked to a β - phaseolin (Phas) promoter, an Arc5 terminator, and an Rb7Mar 3’ matrix attachment region.

[0064] In some embodiments, the expression vector comprises a nucleic acid encoding a heme protein, having a sequence with at least 80% sequence identity to SEQ ID NO: 2, operably linked to a β - phaseolin (Phas) promoter, an Arc5 terminator, and an Rb7Mar 3’ matrix attachment region.

[0065] In some embodiments, 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

[0066]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

[0067] The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0068] The term "a" or "an" entity refers to one or more of that entity. For example, "nucleic acid sequence" is understood to represent one or more nucleic acid sequences, unless otherwise specified. Thus, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0069] Furthermore, as used herein, "and / or" is to be taken as each particular disclosure of two particular features or components, either having or not having the other. Thus, 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 encompass 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).

[0070] Of course, it should be understood that wherever an aspect is described herein in terms of "comprising", other similar aspects are also provided with respect to the terms "consisting of" and / or "consisting essentially of".

[0071] The term "about" is used herein to mean approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, the range is modified by extending the boundaries above and below the recited numerical values. Generally, the term "about" can modify the numerical values above and below the recited value by, for example, plus or minus 10 percent (higher or lower).

[0072] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" that may logically be included, as apparent from the context, and all subsequent numbers or integers. 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 having the property shown by 18, 19, 20, or 21 nucleotides. When present before a series of numbers, or a range, it is understood that "at least" can modify each of the series of numbers, or the range. "At least" is not limited to integers (e.g., without considering significant figures, "at least 5%" includes 5.0%, 5.1%, 5.18%).

[0073] Throughout this disclosure, various aspects of the disclosure are presented in a range format. Numerical ranges include the numerical values defining the range. When a range of values is recited, it should be understood that each intervening integer value between the upper and lower limits of that recited range, and each fraction thereof, are specifically disclosed along with each subrange between such values. The upper and lower limits of any range may independently be included in or excluded from the range, and each range where either, neither, or both are included is also encompassed by the disclosure. Accordingly, the ranges recited herein are understood to be abbreviated for all values within the range including the recited endpoints. For example, the range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0074] When values are explicitly enumerated, it should be understood that values that are approximately the same quantity or amount as the enumerated values are also within the scope of the present disclosure. When a combination is disclosed, each sub - combination of the elements of that combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, when different elements or groups of elements are disclosed individually, their combinations are also disclosed. If any element of the present disclosure is disclosed as having a plurality of alternatives, embodiments of the present disclosure in which each alternative is excluded, either alone or in any combination with other alternatives, are also disclosed herein, and a plurality of elements of the present disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0075] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences, or nucleic acid sequences). Percent identity can be determined by aligning the two sequences and introducing gaps to maximize the identity between the sequences. The alignment can be generated using programs known in the art. For the purposes of this specification, the alignment of nucleotide sequences can be performed with the Blastn program set with default parameters, and the alignment of amino acid sequences can be performed with the Blastp program set with default parameters (see the National Center for Biotechnology Information (NCBI), ncbi.nlm.nih.gov, on the World Wide Web).

[0076] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and this disclosure, and it will be further understood that they are not to be interpreted in an idealized or overly formal sense unless explicitly defined as such herein.

[0077] In describing the present invention, it will be understood that several techniques and steps are disclosed. Each of these has individual benefits and each can also be used in combination with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for clarity, this specification refrains from repeating all possible combinations of the individual steps in an unnecessary manner. Nevertheless, this specification and the claims should be read with the understanding that such combinations are fully within the scope of the present invention and the claims.

[0078] In some aspects, the production of heme proteins in transgenic plants and the use of these heme proteins for alternative meat are considered herein. In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without these specific details.

[0079] As used herein, the term "derived from" refers to a component that is isolated from or made using a particular molecule or organism, or is isolated from or made using information (e.g., an amino acid or nucleic acid sequence) from a particular molecule or organism. For example, a nucleic acid sequence (e.g., an expression vector) derived from a second nucleic acid sequence may contain a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence.

[0080] "Nucleic acid", "polynucleotide", and "oligonucleotide" are used interchangeably in this application. These terms refer only to the primary structure of a molecule. Thus, 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 a molecule containing two or more covalently linked nucleotides, as commonly understood by those skilled in the art. Such covalently linked nucleotides may also be referred to as a nucleic acid molecule, or an oligomer. Polynucleotides can be made recombinantly, enzymatically, or synthetically, for example, by solid-phase chemical synthesis followed by purification. When referring to the sequence of a polynucleotide or nucleic acid, reference is made to the sequence, or order, of the nucleobase portions of the covalently linked nucleotides, or modifications thereof.

[0081] As used herein, the term "hemoprotein" includes proteins having the ability to bind a family of heme-deficient molecules to its structure. As used herein, the term "hemoprotein" also refers to an animal protein and / or an important component of the meat of an animal protein, and can provide color and taste to plant-derived meat products. Myoglobin and hemoglobin are considered hemoproteins and are oxygen-binding proteins of animals. Also, as used herein, the term "hemoprotein" refers to a heme-containing protein, and the term "containing" means that the protein is bound through a covalent or non-covalent bond to the protein. As used herein, the term "hemoprotein" refers not only to the full-length protein, but also to fragments or variants thereof.

[0082] As used herein, the term "animal heme protein" or "animal-derived heme protein" refers to heme proteins expressed in animals, excluding 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 having a heme prosthetic group family 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.

[0083] As used herein, the term "plant-derived hemoprotein" means a hemoprotein whose gene source is from a monocotyledonous or dicotyledonous plant such as Nicotiana tabacum or Nicotiana sylvestris (tobacco), Zea mays (corn), Arabidopsis thaliana, leguminous plants such as Glycine max, Cicer arietinum (garbanzo or chickpea), Pisum sativum, Phaseolus vulgaris (kidney bean), Vigna radiata, Lupinus albus (lupin), or Medicago sativa (alfalfa), Brassica napus (canola), Triticum sps. (including wheat, wheat fruit, and spelt), Gossypium hirsutum, Oryza sativa (rice), Zizania sps. (wild rice), Helianthus annuus (sunflower), Beta vulgaris, Pennisetum glaucum (pearl millet), Chenopodium sp. (quinoa), Sesamum sp. (sesame), Linum usitatissimum (flax), or Hordeum vulgare (barley).

[0084] As used herein, the term "microorganism-derived hemoprotein" means a hemoprotein whose gene source is derived from bacteria, yeasts, fungi such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Synechocystis sp., Aquifex aeolicus, Methylacidiphilum infernorum, Thermophilus spp., A. eutrophus, Saccharomyces cerevisiae, Vitreoscilla sp., Pichia pastoris, Magnaporthe oryzae, Fusarium graminearum, Aspergillus oryzae, Trichoderma reesei, Myceliopthera thermophile, Kluyveramyces lactis, and Fusarium oxysporum.

[0085] As used herein, the term "recombinant protein" refers to a protein encoded by a cloned gene (e.g., recombinant DNA) in a system that supports gene expression and messenger RNA translation. A recombinant protein is a foreign protein produced in an expression host. Modification of a gene by recombinant DNA technology can result in the expression of a mutant protein.

[0086] As used herein, the term "recombinant hemoprotein" refers to a recombinant protein, which is encoded by a foreign cDNA encoding a hemoprotein. As used herein, the term "exogenous nucleic acid" means a cDNA encoding a recombinant hemoprotein, and the term "exogenous nucleic acid" is used interchangeably with "recombinant nucleic acid" herein. The sequences and structures of many heme-containing polypeptides are known (Reedy, et al. (2007), Nucleic Acids Res. 6:D307).

[0087] The term "plant" includes reference to the whole plant, plant organs, plant tissues, and plant cells, and their progeny, and includes all monocots and dicots. The term whole plant as used herein also includes seeds, plant progeny, whether sexual or not, cuttings, or other propagules such as seeds, and tissues and organs, both before and after harvest.

[0088] The term "transgenic plant" or "genetically engineered" means a plant transformed with one or more exogenous nucleic acids (recombinant sequences). The term "transformation" refers to the process by which a recombinant sequence is introduced into and expressed in a plant cell. In stable plant transformation, the foreign DNA is completely integrated into the host genome and remains integrated and continues to be expressed in the progeny of the plant. In transient plant transformation, the foreign DNA is not integrated into the host genome and is not expressed in the progeny of the plant. Transformation can occur via Agrobacterium inoculation, virus infection, electroporation, heat shock, lipofection, polyethylene glycol treatment, microinjection, silica beads, carbon nanotubes, and particle bombardment.

[0089] In some embodiments, the transgenic plant is a soybean (Glycine max) plant. In some embodiments, the genetically engineered plant is selected from the group consisting of rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), corn (Zea mays), rye (Secale cereale), oats (Avena sativa), sugar beet (Beta vulgaris), sugar beet (Beta vulgaris subsp. vulgaris), parsnip (Pastinaca sativa), legumes, leafy vegetables, tubers, and forage grasses. In some embodiments, the legumes are common bean or kidney bean (Phaseolus vulgaris), pea (Pisum sativum), adzuki bean (Vigna angularis), mung bean (Vigna radiata), chickpea (Cicer arietinum), peanut (Arachis hypogaea), or lentil (Lens culinaris). In some embodiments, the leafy vegetables are alfalfa (Medicago sativa), arugula (Eruca vesicaria), mustard (Brassica juncea), lettuce (Lactuca sativa), or rape. In some embodiments, the tubers are potato (Solanum tuberosum), sweet potato (Ipomoea batatas), or cassava (Manihot esculenta). In some embodiments, the forage grasses are wheat (Triticum aestivum), or spelt wheat (Triticum spelta).

[0090] Exemplary recombinant arrays of the present disclosure are provided in FIGS. 1-10. These recombinant arrays are contained within plant transformation vectors. In some embodiments, these vectors are introduced into Agrobacterium tumefaciens as circular plasmids. The t-DNA insert from the circular plasmid is introduced into plant cells via Agrobacterium-mediated transformation (FIGS. 1-2, 6-7, and SEQ ID NOs: 3-4). In some embodiments, these vectors are delivered to plant cells as linear constructs (FIGS. 3-5, 8-10, and SEQ ID NOs: 5-7). In some embodiments, the recombinant array includes a promoter, an enhancer sequence, a sequence encoding a heme protein, and a terminator (FIGS. 1, 3, 6, and 8). In some embodiments, the recombinant array includes a promoter, an enhancer sequence, a sequence encoding a heme protein, a terminator, and a matrix attachment region (FIGS. 2, 4, 5, 7, 9, and 10). In some embodiments, the heme protein is hemoglobin (FIGS. 1-2 and FIGS. 6-7). In other embodiments, the heme protein is myoglobin (FIGS. 3-5 and FIGS. 8-10).

[0091] In some embodiments, the recombinant array includes a sequence named promoter that refers to a nucleic acid sequence that promotes the initiation of transcription. The promoter may be a constitutive promoter. A constitutive promoter can initiate transcription in plant cells under any circumstances, and its activity is not affected by environmental conditions. Some promoters are tissue-specific in that they preferentially initiate transcription in specific organs. Other promoters are inducible and are regulated by external stimuli such as different chemical, biological, and abiotic environmental factors.

[0092] In some embodiments, the promoter is a constitutive promoter, such as the 35S promoter, which exists as a tandem (2×35S promoter) (SEQ ID NO: 8) (Figures 1, 3, 6, and 8) double unit derived from the cauliflower mosaic virus (CaMV). In some embodiments, the promoter is a tissue-specific promoter. Tissue-specific promoters include the β-conglycinin α subunit of the 7S storage (7s) promoter from 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 (Vicia faba) (Zakharov et al. (2004), J. Exp. Bot. 55:1463), the SBP promoter from broad bean (Vicia faba) (Zakharov et al. (2004), J. Exp. Bot. 55:1463), the legumin B4 promoter from broad bean (Vicia faba) (Zakharov et al. (2004), J. Exp. Bot. 55:1463), the napin promoter from rapeseed (Brassica napus) (Vigeolas et al. (2007), Plant Biotechnol. J. 5: 431), the vicilin promoter from pea (Pisum sativum) (Arun et al. (2014), Appl. Biochem. Biotechnol. 172:1763), the α-globulin promoter from cotton (Sunilkumar et al. (2002), Transgenic Res. 11:347), the γ-zein promoter from maize (Marzabal et al. (1998), Plant J. 16:41), the glutenin promoter from wheat (Lamacchia et al. (2001), J. Exp. Bot. 52:243), the VvβVPE promoter from species of the genus Vitis (Vitis spp) (Gong et al.(2019), Planta, 250:657), the Glycine max seed promoter (GSP) (Sunkara et al. (2014), Appl. Biochem. Biotechnol. 172: 325), the 7αP promoter from Glycine max (Fu et al. (2009); Northwest Sci. 37:105), the AtLAC15 promoter from Arabidopsis thaliana (El-Mezawy et al. (2009), Biotechnol. Lett. 31:1961), the SSP promoter from Vigna radiata (Verma & Bhatia, (2019), Funct. Integr. Genomics, 19:373), the lectin promoter from Glycine max (Ma et al. (2008), J. Plant Growth Regul. 27:68), the oleosin promoter from Brassica napus (Keddie et al. (1994) Plant Mol. Biol. 24:327), the AhLEC1A promoter from Glycine max (Tang et al. (2021) PloS one, 16:e0242949), the Glu-1D-1 promoter from Triticum aestivum (Lamacchia et al. (2001), J. Exp. Bot. 52:243), the sesame 2S albumin (2Salb) promoter from Sesamum indicum (Bhunia et al. (2014), Plant Mol. Biol. 86:351), or the 8SGα promoter from Pyrus pyrifolia (Chen et al. (2014), J. Biotechnol. 174:49) may also be used. In some embodiments, the seed-specific promoter is 7S (Figures 2, 4, 7, and 9) (SEQ ID NO: 11), and phas (Figures 5, and 10). Constructs based on either the 7s promoter or the phas promoter show higher expression of the protein in seeds.

[0093] 7S, and β-phaseolin proteins 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 a major seed storage protein in soybean (Glycine max). This gene consists of three subunits, α, α’, and β, and contains 30–35% of the total seed protein (Thanh and Shibasaki, (1976), Biochim. Biophys. Acta. 439:326; Hayashi et al. (2009), J. Hered. 100:802). Inserting the 7S promoter into soybean to express human growth factor, the transgenic lines produced 2.3% TSP (total soluble protein) for the recombinant protein, which was 38-fold higher than that of the 35S promoter (Ding et al. (2006), Biotechnol. Lett. 28:869). Human bone morphogenetic protein was expressed under the control of the 7S promoter, and a maximum yield of 9.28% TSP was obtained (Queiroz et al., 2019, Plant Mol. Biol. 96:429).

[0094] The phas gene in common bean (Phaseolus vulgaris) encodes the major seed storage protein. According to research, the phas gene has been found to be highly expressed in cotyledons during embryogenesis (Li et al. (1999), PNAS, 95:4772; Chandrasekharan et al. (2003), Plant J. 33:853). This gene is strictly turned off during all vegetative stages of plant development (Li et al. (1999), PNAS, 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), Nat. Biotechnol. 20:1265).

[0095] In some embodiments, the expression cassette comprises a 2×35S promoter. In some embodiments, the 2×35S 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 with SEQ ID NO: 8.

[0096] In some embodiments, the 2×35S 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 with SEQ ID NO: 8.

[0097] In some embodiments, the expression cassette comprises a 7S promoter. In some embodiments, the 7S 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 with SEQ ID NO: 11.

[0098] In some embodiments, 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 with SEQ ID NO: 11.

[0099] In some embodiments, the expression cassette comprises a Phas promoter. In some embodiments, the Phas 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 with SEQ ID NO: 14.

[0100] In some embodiments, 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 with SEQ ID NO: 14.

[0101] In some embodiments, the recombinant array includes a sequence named a terminator that refers to a nucleic acid sequence that defines the end 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, BIO-DES MANUF. 2022:1), the Hsp70 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022:1), the Hsp18.2 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022:1), the Act2 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022:1), the G7 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022:1), the 3g24240 terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022:1), the NOS terminator from Arabidopsis thaliana (Tian et al. 2002, BIO-DES MANUF. 2022:1) (SEQ ID NO: 10), the Ocs terminator from Agrobacterium tumefaciens (Tian et al. 2002, BIO-DES MANUF. 2022:1), the Mas terminator from Agrobacterium tumefaciens (Tian et al. 2002, BIO-DES MANUF.2022: 1), the 35s terminator derived from cauliflower mosaic virus (Tian et al. 2002, BIO-DES MANUF. 2022:1), the Rbc terminator derived from Chrysanthemum (Tian et al. 2002, BIO-DES MANUF. 2022:1), the Ags terminator derived from Agrobacterium tumefaciens (Tian et al. 2002, BIO-DES MANUF. 2022:1), the 3’utr-nos terminator derived from Agrobacterium tumefaciens (Tian et al. 2002, BIO-DES MANUF. 2022:1), the 7s terminator derived from soybean (Tsubokura et al. (2012), Plant Mol. Biol. 78:301), the E9 terminator derived from Pisum sativum (Coruzzi et al. (1984), EMBO Rep. 3:1671), the ORF25 terminator derived from Agrobacterium tumefaciens (Barker et al. (1983), Plant Mol. Biol. 2:335), the pinII terminator derived from Solanum tuberosum (Keil et al. (1986), Nucleic Acids Res. 14:5641), the tml terminator derived from Agrobacterium tumefaciens (Barker et al. (1983), Plant Mol. Biol. 2:335), the Tr7 terminator derived from Agrobacterium tumefaciens (Dhaese et al. (1983), EMBO Rep.(2:419). In some embodiments, the terminator is NOS (FIGS. 1, 3, 6, 8) (SEQ ID NO: 10), or arc5 (FIGS. 2, 4, 5, 7, 9, 10) (SEQ ID NO: 12). The Arc5 terminator from Phaseolus vulgaris provides a sequence that terminates transcription and directs polyadenylation of the mRNA (Goossens et al. (1999), Plant Physiol. 120:1095), and has also been reported to enhance gene expression and contribute to seed-specific expression.

[0102] In some embodiments, the expression cassette comprises a NOS terminator sequence. In some embodiments, the NOS terminator 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 with SEQ ID NO: 10.

[0103] In some embodiments, 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 with SEQ ID NO: 10.

[0104] In some embodiments, the expression cassette comprises an arc5 terminator sequence. In some embodiments, the arc5 terminator 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 with SEQ ID NO: 12.

[0105] In some embodiments, 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.

[0106] In some embodiments, the recombinant sequence comprises a translation or transcription enhancer sequence. An example of a translation enhancer is the 5’UTR TEV (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 and contains two cap-independent regulatory elements (CIREs) that fold into a pseudoknot, which can independently enhance the translation of downstream transgenes (Carrington & Freed, (1993) J. Virol., 64:1590). In some embodiments, the recombinant sequence comprises a matrix attachment region (MAR) as an enhancer. The Rb7 MAR (SEQ ID NO: 13) is a DNA element that has been shown to increase transgene expression in plants. Addition of the Rb7 MAR has been shown to strongly enhance protein production when added to most transcription terminators (Diamos & Mason, (2018), Plant Biotechnol. J. 16:1971). Further, the MAR can further improve the stability of transgene expression levels and confer protection against transgene silencing (Vain et al. (1999), Plant J. 18:233). In some embodiments, the arc5 terminator is fused to the Rb7 matrix attachment region (MAR) that increases the potential and magnitude of transgene expression.

[0107] In some embodiments, the expression cassette comprises the Rb7MAR enhancer. In some embodiments, the Rb7MAR enhancer 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 with SEQ ID NO: 13.

[0108] In some embodiments, 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 with SEQ ID NO: 13.

[0109] In some embodiments, the expression cassette comprises the TEV enhancer. In some embodiments, the TEV enhancer 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 with SEQ ID NO: 9.

[0110] In some embodiments, 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 with SEQ ID NO: 9.

[0111] In some embodiments, a particular combination of regulatory elements (i.e., promoters, terminators, and enhancers) results in enhanced expression of the heme protein in the seeds described above, for example, at TSP above 5%, 8%, and 10%. In some embodiments, 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 embodiments, the combinations specified herein are as follows: i.p7S + cDNAHP + arc5 + Rb7MAR ii.p7S + TEV + cDNAHP + arc5 + Rb7MAR iii.PPhas + TEV + cDNAHP + arc5 + Rb7MAR iv.PPhas + cDNAHP + arc5 + Rb7MAR

[0112] cDNAHP specifies the cDNA of the heme protein. In some embodiments, the heme protein is an animal-derived heme protein. In some embodiments, the heme protein is derived from metazoans. In some embodiments, the heme protein is derived from red meat (e.g., beef, pork, goat, and lamb), poultry (e.g., chicken and turkey), and seafood (e.g., fish, crustaceans, and mollusks). In some embodiments, the animal-derived heme protein is myoglobin. In some embodiments, the animal-derived heme protein is hemoglobin. Since it is routine for those skilled in the art to substitute orthologous sequences from other organisms, mere substitutions of the recombinant protein are also within the scope of the present disclosure.

[0113] In some embodiments, the disclosure also provides a polynucleotide comprising a nucleic acid encoding a heme protein, wherein the nucleic acid is operably linked to a β-conglycinin α subunit of a 7S storage (7s) promoter from soybean, a β-phaseolin (Phas) promoter from common bean, a USP promoter from broad bean (Vicia faba), an SBP promoter from broad bean (Vicia faba), a legumin B4 promoter from broad bean (Vicia faba), a napin promoter from rapeseed (Brassica napus), a vicilin promoter from pea (Pisum sativum), an α-globulin promoter from cotton, a γ-zein promoter from maize, a glutenin promoter from wheat, a VvβVPE promoter from species of the genus Vitis (Vitis spp), a glycinin seed promoter (GSP) from peanut, a 7αP promoter from soybean, an AtLAC15 promoter from Arabidopsis thaliana, an SSP promoter from chickpea, a lectin promoter from soybean, an oleosin promoter from rapeseed, an AhLEC1A promoter from peanut, a Glu-1D-1 promoter from wheat, a sesame 2S albumin (2Salb) promoter from sesame, or an 8SGα promoter from taro.

[0114] In some embodiments, the heme protein comprises a plant-derived heme protein, a microorganism-derived heme protein, or an animal-derived heme protein.

[0115] In some embodiments, the nucleic acid encoding the heme protein comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

[0116] In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 3-7.

[0117] In some embodiments, the nucleic acid further comprises a transcription terminator selected from the group consisting of an extensin terminator from tobacco, a 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 Arabidopsis thaliana, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from Agrobacterium tumefaciens, a 35s terminator from cauliflower mosaic virus, an Rbc terminator from Chrysanthemum, an Ags terminator from Agrobacterium tumefaciens, a 3’utr-nos terminator from Agrobacterium tumefaciens, a 7s terminator from soybean, an E9 terminator from Pisum sativum, an ORF25 terminator from Agrobacterium tumefaciens, a pinII terminator from Solanum tuberosum, a tml terminator from Agrobacterium tumefaciens, a Tr7 terminator from Agrobacterium tumefaciens, or an Arc5 terminator from Phaseolus vulgaris.

[0118] In some embodiments, the nucleic acid further comprises a transcription or translation enhancer selected from the group consisting of a 5'UTR TEV and an Rb7Mar 3'matrix attachment region.

[0119] In some embodiments, 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.

[0120] In some embodiments, 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.

[0121] In some embodiments, 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.

[0122] In some embodiments, 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.

[0123] In some embodiments, 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.

[0124] In some embodiments, 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.

[0125] In some embodiments, 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.

[0126] In some embodiments, 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.

[0127] In some embodiments, 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.

[0128] In some embodiments, 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 with SEQ ID NO: 5.

[0129] In some embodiments, 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 with SEQ ID NO: 6.

[0130] In some embodiments, 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 with SEQ ID NO: 6.

[0131] In some embodiments, 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 with SEQ ID NO: 7.

[0132] In some embodiments, 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 with SEQ ID NO: 7.

[0133] In some embodiments, 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.

[0134] In some embodiments, 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.

[0135] In some embodiments, 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.

[0136] In some embodiments, 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.

[0137] In some embodiments, 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.

[0138] In some embodiments, 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 with SEQ ID NO: 10.

[0139] In some embodiments, 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 with SEQ ID NO: 11.

[0140] In some embodiments, 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 with SEQ ID NO: 11.

[0141] In some embodiments, 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 with SEQ ID NO: 12.

[0142] In some embodiments, 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 with SEQ ID NO: 12.

[0143] In some embodiments, 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.

[0144] In some embodiments, 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.

[0145] In some embodiments, 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.

[0146] In some embodiments, 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.

[0147] In some embodiments, plants are transformed in each of the expression cassettes (FIGS. 1-10). In some embodiments, stably transformed plants contain the recombinant DNA construct in their genome, the heme protein is stably expressed, and produce a pink color in seed cotyledons and seed protein extracts (FIGS. 15-16). The presence of the heme protein in the transgenic organism resulted in a visible color change in the protein extract (pink color) when compared to the wild type (Carlsson et al. (2020), Sci. Rep. 10:1).

[0148]

[0148] In some embodiments, stably transformed plants contain the recombinant DNA construct in their genomes, the heme protein is stably expressed, extracted via standard protein extraction protocols, and detected via Western blot (Figures 17, 18), liquid chromatography mass spectrometry (LC-MS) (Figure 19), and / or ELISA assay (Figure 20).

[0149] In some embodiments, stably transformed plants contain the recombinant DNA construct in their genomes, and the heme protein is stably expressed in an amount of at least about 5% of the TSP (Figure 20B). In some embodiments, the heme protein is stably expressed in an amount of at least about 8% of the TSP (Figure 20B). In some embodiments, the heme protein is stably expressed in an amount of at least about 10% of the TSP (Figure 20B). In some embodiments, the heme protein is stably expressed in an amount of at least about 25% of the TSP (Figure 20B).

[0150] In some embodiments, the recombinant heme proteins used for transformation are hemoglobin and myoglobin. In some embodiments, the hemoglobin described herein is isolated from swine (Sus scrofa). In some embodiments, the hemoglobin is recombinant HbA-LL-HbB and includes a hemoglobin A subunit, a long linker, and a hemoglobin B subunit. In some embodiments, the myoglobin described herein is isolated from swine (Sus scrofa domesticus). In some embodiments, the expression cassette includes any of the sequences disclosed in Table 1.

[0151] In some embodiments, the expression cassette includes SEQ ID NO: 11, SEQ ID NO: 12, and / or SEQ ID NO: 13. In some embodiments, the expression cassette includes SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 12, and / or SEQ ID NO: 13. In some embodiments, the expression cassette includes SEQ ID NO: 14, SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 13. In some embodiments, the expression cassette includes SEQ ID NO: 14, SEQ ID NO: 12, and SEQ ID NO: 13.

[0152] In some embodiments, the expression cassette comprises SEQ ID NO: 11, SEQ ID NO: 1, SEQ ID NO: 12, and / or SEQ ID NO: 13. In some embodiments, 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 embodiments, 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 embodiments, the expression cassette comprises SEQ ID NO: 14, SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 13.

[0153] In some embodiments, the expression cassette comprises SEQ ID NO: 11, SEQ ID NO: 2, SEQ ID NO: 12, and / or SEQ ID NO: 13. In some embodiments, 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 embodiments, 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 embodiments, the expression cassette comprises SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 12, and SEQ ID NO: 13.

[0154] In some embodiments, provided herein are transgenic plants, plant tissues, or plant cells comprising an expression cassette comprising an exogenous nucleic acid encoding a heme protein. In some embodiments, the nucleic acid is operably linked to a seed-specific promoter and a transcription terminator. In some embodiments, the heme protein is expressed in seeds in an amount of about 5% of total soluble protein (TSP). In some embodiments, the heme protein is expressed in seeds in an amount of about 6% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 7% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 8% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 9% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 10% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 11% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 12% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 13% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 14% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 15% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 18% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 20% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 25% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of about 30% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of from about 5% of TSP to about 35% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of from about 8% of TSP to about 35% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of from about 10% of TSP to about 35% of TSP. In some embodiments, the heme protein is expressed in seeds in an amount of from about 12% of TSP to about 35% of TSP.In some embodiments, the heme protein is expressed in the seed in an amount of about 5% TSP to about 30% TSP. In some embodiments, the heme protein is expressed in the seed in an amount of about 5% TSP to about 29% TSP. In some embodiments, the heme protein is expressed in the seed in an amount of about 5% TSP to about 28% TSP. In some embodiments, the heme protein is expressed in the seed in an amount of about 10% TSP to about 30% TSP. In some embodiments, the heme protein is expressed in the seed in an amount of about 8% TSP to about 30% TSP. In some embodiments, the heme protein is expressed in the seed in an amount of about 6% TSP to about 28% TSP.

[0155] In some embodiments, 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.

[0156] In some embodiments, 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.

[0157] In some embodiments, 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.

[0158] In some embodiments, 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.

[0159] In some embodiments, 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.

[0160] In some embodiments, 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.

[0161] In some embodiments, 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.

[0162] In some embodiments, 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.

[0163] In some embodiments, 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.

[0164] In some embodiments, the expression cassette comprises a nucleic acid sequence having at least 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.

[0165] In some embodiments, the stably transformed plant is soybean. Codon optimization is a process used to improve gene expression and increase the translation efficiency of a gene of interest by corresponding to the codon bias of the host organism. In some embodiments, the hemoglobin gene is codon optimized for expression in soybean (SEQ ID NO: 1). In some embodiments, the myoglobin gene is codon optimized for expression in soybean (SEQ ID NO: 2).

[0166] In some embodiments, 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.

[0167] In some embodiments, 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.

[0168] In some embodiments, 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.

[0169] In some embodiments, the myoglobin 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: 2.

[0170] In some embodiments, the recombinant array contains a gene encoding a selectable marker. In some embodiments, the selectable marker is the BAR gene that produces a phosphinothricin N-acetyltransferase protein and provides resistance to glufosinate. In some embodiments, the BAR gene is located within the same plant transformation vector (circular plasmid) as the sequence of the heme protein (Figs. 1-2, and 6-7). In some embodiments, the selectable marker is the aadA gene that produces an aminoglycoside-3'-adenyltransferase protein and provides resistance to aminoglycoside spectinomycin and streptomycin (Figs. 3-5, and 8-10). In some embodiments, the aadA gene is located within a separate linear construct and is co-bombarded with a linear construct that retains the sequence of the heme protein (Figs. 3-5, and 8-10).

[0171] In some embodiments, a method for stably expressing a heme protein in a plant is disclosed herein, the method comprising: a) transforming the plant with a plant transformation vector; b) regenerating transgenic plants in vitro under a selection pressure; and c) cultivating the transformed plants under conditions in which the recombinant heme protein is expressed.

[0172] In some embodiments, 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 Res. 20:811, Cunha et al. (2011b), Transgenic Res. 20:841, Ding et al. (2006), Biotechnol. Lett. 28:869). The amount of the protein of interest can be measured using methods known in the art such as ELISA or Western blot.

[0173] Food compositions may be prepared using the heme proteins and transgenic plants described herein. In some embodiments, the recombinant heme protein produced by the transgenic plant may be used in its entirety to prepare meat-like (meat analog) foods, including ground meat such as minced meat, meat strips, cubes, and steaks, reconstituted and shaped meat-like products including burgers, fillets, balls, sticks, slabs, reconstituted and stuffed meat-like products including sausages, ham-like products, spreadables, reconstituted and coated meat-like products including nuggets, patties, strips, poppers, rings, etc., and their fragments and modifications, including solubilized, precipitated, partially or fully hydrolyzed, cross-linked, emulsified, texturized, cooked, extruded, reacted, structured versions. The recombinant heme protein may also be extracted from the transgenic plant using standard methods known in the art.

[0174] In some embodiments, the food composition is prepared using seeds of a transgenic plant that expresses the recombinant heme protein. In some embodiments, the food composition is prepared using the recombinant heme protein extracted and purified from the seeds.

[0175] The following experiments demonstrate different recombinant sequences containing heme proteins and methods for producing recombinant proteins in plants. The following examples describe expression in soybean, but it will be understood by those skilled in the art that the expression sequences and methods disclosed herein can be adapted for expression in any monocotyledonous or dicotyledonous plant.

[0176] The practice of the present disclosure, unless otherwise indicated, employs conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, biotechnology, plant genetic engineering, and immunology, within the skill of the art. Such techniques are fully explained in the literature.

[0177] All references cited above and all references cited in this specification are hereby incorporated by reference in their entirety into this specification.

[0178] This disclosure should be regarded as examples of the present invention and is not intended to limit the present invention to the specific embodiments illustrated by the following figures or description. Here, the present invention will be described by referring to the accompanying drawings representing specific embodiments.

Examples

[0179] Example 1. Construction of a plant transformation vector A codon-optimized gene containing the α-subunit and β-subunit of the porcine hemoglobin gene, designated HbA-LL-HbB, was synthesized by Genscript. The HbA-LL-HbB gene was cloned into the in-house pIPTRA0-2x35S-MCS vector using the 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 shows a graphic representation of the pIPTRA0:p35S+HbA-LL-HbB vector, and Figure 6 shows the nucleotide sequence of each of the regulatory factors involved.

[0180] The 7S promoter fused to the TEV enhancer (p7S-TEV), and the arc5 terminator fused to the Rb7 matrix attachment region (arc5T-Rb7MAR) were synthesized by Genscript. 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 the XbaI / BamHI restriction sites. The NOS terminator was replaced by fusion with arc5T-Rb7MAR using the HindIII / SpeI restriction sites. The graphic representation and nucleotide sequence of the pIPTRA0:p7S+HbA-LL-HbB-Arc5T vector are shown in Figure 2 and 7, respectively.

[0181] The 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 EC1, EC2, and EC3, respectively. EC1, EC2, and EC3 were assembled via Golden Gate cloning in the in-house pEXPLODER plasmid. The promoter, myoglobin, and terminator were incorporated into the pEXPLODER plasmid. After successful assembly of the linear fragments of EC1, EC2, and EC3, they were released from the circular plasmid via 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 section carrying the selectable marker via AscI digestion. The two resulting linear constructs (selectable marker + EC1, EC2, or EC3) were co-bombarded into the soybean extract. The graphic representations of the three linearized fragments are shown in Figures 3, 4, and 5. The nucleotide sequences of EC1, EC2, and EC3 are shown in Figures 8, 9, and 10, respectively.

[0182] Example 2. Confirmation of Transgenic Events In vitro regeneration of putative transgenic lines was obtained for all constructs used in this manner (Figure 11). For further gene screening, DNA was extracted from the regenerated transplanted leaf tissue.

[0183] DNA from putative transformed lines using pIPTRA0:p35S+HbA-LL-HbB and pIPTRA0:p7S+HbA-LL-HbB-Arc5T was PCR-screened for the presence of transgenic inserts in the host genome. The agarose gel photographs show the results of PCR amplification for putative transformed lines for pIPTRA0:p35S+HbA-LL-HbB (Figure 12) and IPTRA0:p7S+HbA-LL-HbB-Arc5T (Figure 13). The presence of 412 bp and 390 bp bands confirms the presence of transgenic inserts for the lines transformed with pIPTRA0:p35S+HbA-LL-HbB and IPTRA0:p7S+HbA-LL-HbB-Arc5T, respectively.

[0184] qPCR of the aadA1a CDS section was performed to confirm the presence of transgenic inserts in the lines presumably transformed with EC1, EC2, and EC3 (Figure 14). The marker Ct cutoff for positive lines must be less than 25.

[0185] Example 3. Total Soluble Protein Production in Soybean Transgenic Events Carrying the Porcine Hemoglobin Gene Transgenic T0 plants transformed with pIPTRA0:p35S+HbA-LL-HbB and pIPTRA0:p7S+HbA-LL-HbB-Arc5T were cultured and grown into T1 seeds. The T1 seeds were screened for the presence of the porcine hemoglobin gene via PCR, and small sections of the seeds were excised for PCR purposes.

[0186] For each transgenic event, a total of three PCR-positive seeds were pooled and subjected to protein extraction. The pooled seeds were ground in a tissue lyser and treated with an extraction buffer (50 mM Tris-Cl pH 6.8, 50 mM NaCl, 36 mM Na2SO3, PHIC 1:200), and centrifuged at 13,000 rpm for 10 minutes at 4°C (Figure 15). After protein extraction, the extracts were run on 10-well and 12% SDS PAGE gels, loading 100 μg of protein from each transgenic extract, a molecular weight standard, 100 μg of protein extract from wild type used as a negative control, and wild type extract + 10, 25, 50, 150, and 250 ng of Hb standard (Sigma-Aldrich freeze-dried porcine hemoglobin powder, catalog number H4131). Subsequently, the protein bands were transferred to a nitrocellulose membrane, and Western blotting was developed using an anti-pig antibody (UsBiological Life sciences, catalog number 140639) diluted 1 / 250 (Figure 17). Using Western blotting, the detected hemoglobin was quantified by comparing the intensity of the hemoglobin band from the seed extract with the intensity from the hemoglobin standard, enabling the calculation of the percentage of hemoglobin in TSP (Figure 18, Table 1).

[0187] Table 1. Accumulation of hemoglobin as a percentage of TSP content in independent transgenic soybean seed stocks. The coefficient of variation (%) is included in the table. "n.d": undetermined; events without a coefficient of variation because two of the three replicates were discarded.

Table 1-1

Table 1-2

[0188] The identification and quantification of porcine hemoglobin from whole soybean seed protein extracts were also performed via liquid chromatography-mass spectrometry (LC-MS) (Figure 18). Equal amounts of soybean seed protein per transgenic event were digested with LysC / trypsin, and then the peptides were desalted on a C18 tip. LC-MS data were acquired on a Bruker timsTOF-Pro2 and searched against a soybean database supplemented with the provided porcine hemoglobin sequences. The amount of hemoglobin was determined by normalizing its MS2 intensity to soybean coipins. Hemoglobin was not detected in wild-type samples.

[0189] Transgenic events carrying the porcine myoglobin gene: Transgenic T0 plants transformed with the EC1, EC2, and EC3 linear constructs were cultured and grown to T2 seeds. T2 seeds were screened for the presence of the porcine myoglobin gene via ddPCR, and small sections of the seeds were excised for PCR purposes.

[0190] A total of three PCR-positive seeds per transgenic event were pooled and protein extraction was performed. The pooled seeds were ground in extraction buffer (5% w / v SDS, 175 mM Tris-HCl, pH 8.0, 0.4% v / v β-mercaptoethanol) containing Omni ceramic beads (1.4 mm), and 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 an Alpha Diagnostics ELISA kit (Catalog number 600-640-PMY). All samples were normalized to 50 μg / mL of total soluble protein (TSP) and tested for myoglobin content according to the manufacturer's protocol. The concentration of myoglobin was determined by referring to a standard curve. Each event consisted of one biological replicate (pool of three seeds) with three technical replicates. 20 μL (1 μg) was tested for each sample. The percentage of myoglobin in TSP is shown in Figure 20 and Table 2.

[0191] Table 2. Myoglobin accumulation as a percentage of TSP content (±s.d.) in independent transgenic soybean seed stocks. TSP content exceeding 5% is shaded in gray.

Table 2-1

Table 2-2

[0001]

[0192] Array

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Claims

**Claim 1** A transgenic plant, plant tissue, or plant cell comprising an exogenous nucleic acid encoding a heme protein, wherein said nucleic acid is operably linked to a seed-specific promoter and a transcription terminator, and wherein said heme protein is expressed in seeds in an amount of at least about 5% of the total soluble protein (TSP). **Claim 2** The transgenic plant, plant tissue, or plant cell according to claim 1, wherein said nucleic acid is operably linked to a transcription or translation enhancer. **Claim 3** The transgenic plant, plant tissue, or plant cell according to claim 2, wherein said heme protein is expressed in said seeds in an amount of at least about 8% of the TSP. **Claim 4** The transgenic plant, plant tissue, or plant cell according to claim 2, wherein said heme protein is expressed in said seeds in an amount of at least about 10% of the TSP. **Claim 5** The transgenic plant, plant tissue, or plant cell according to any one of claims 1 to 4, wherein said heme protein comprises a plant-derived heme protein, a microorganism-derived heme protein, or an animal-derived heme protein. **Claim 6** The transgenic plant, plant tissue, or plant cell according to any one of claims 1 to 5, wherein said heme protein comprises a heme protein involved in oxygen transport, an enzyme having a heme prosthetic group, or a heme protein involved in said electron transport chain. **Claim 7** The transgenic plant, plant tissue, or plant cell according to any one of claims 1 to 6, wherein said heme protein comprises hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, or cytochrome c. **Claim 8** The transgenic plant, plant tissue, or plant cell according to any one of claims 1 to 7, wherein said heme protein is an animal-derived heme protein selected from the group consisting of hemoglobin and myoglobin. **Claim 9** The transgenic plant, plant tissue, or plant cell according to claim 1, wherein the seed-specific promoter comprises a β-conglycinin α subunit of a 7S storage (7s) promoter derived from soybean, a β-phaseolin (Phas) promoter derived from kidney bean, a USP promoter derived from broad bean (Vicia faba), an SBP promoter derived from broad bean (Vicia faba), a legumin B4 promoter derived from broad bean (Vicia faba), a napin promoter derived from rapeseed (Brassica napus), a vicilin promoter derived from pea (Pisum sativum), an α-globulin promoter derived from cotton, a γ-zein promoter derived from maize, a glutenin promoter derived from wheat, a VvβVPE promoter derived from a species of the genus Vitis (Vitis spp), a groundnut seed promoter (GSP) derived from peanut, a 7αP promoter derived from soybean, an AtLAC15 promoter derived from Arabidopsis thaliana, an SSP promoter derived from chickpea, a lectin promoter derived from soybean, an oleosin promoter derived from rapeseed, an AhLEC1A promoter derived from peanut, a Glu-1D-1 promoter derived from wheat, a sesame 2S albumin (2Salb) promoter derived from sesame, or an 8SGα promoter derived from taro.

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

11. Further comprising a terminator sequence, said terminator sequence being an extensin terminator derived from tobacco, a Ub10 terminator derived from Arabidopsis thaliana, an Hsp70 terminator derived from Arabidopsis thaliana, an Hsp18.2 terminator derived from Arabidopsis thaliana, an Act2 terminator derived from Arabidopsis thaliana, a G7 terminator derived from Arabidopsis thaliana, a 3g24240 terminator derived from Arabidopsis thaliana, a NOS terminator derived from Arabidopsis thaliana, an Ocs terminator derived from Agrobacterium tumefaciens, a Mas terminator derived from Agrobacterium tumefaciens, a 35s terminator derived from cauliflower mosaic virus, an Rbc terminator derived from Chrysanthemum, an Ags terminator derived from Agrobacterium tumefaciens, a 3'utr-nos terminator derived from Agrobacterium tumefaciens, a 7s terminator derived from soybean, an E9 terminator derived from Pisum sativum, an ORF25 terminator derived from Agrobacterium tumefaciens, a pinII terminator derived from Solanum tuberosum, a tml terminator derived from Agrobacterium tumefaciens, a Tr7 terminator derived from Agrobacterium tumefaciens, or a PhaseolusThe transgenic plant, plant tissue, or plant cell according to claim 1, comprising an Arc5 terminator derived from Ralstonia solanacearum pv. vulgaris).

12. The transgenic plant, plant tissue, or plant cell according to claim 11, wherein the terminator is an Arc5 terminator derived from kidney bean (Phaseolus vulgaris).

13. The transgenic plant, plant tissue, or plant cell according to claim 1, further comprising a transcription or translation enhancer, wherein the translation or translation enhancer is selected from the group consisting of a 5' untranslated region (UTR) derived from tobacco etch virus (TEV) and an Rb7Mar 3' matrix attachment region.

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

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

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

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

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

19. The transgenic plant, plant tissue, or plant cell according to claim 2, wherein the exogenous nucleic acid is operably linked to a β-phaseolin (Phas) promoter derived from kidney bean, an Arc5 terminator, and then an Rb7MAR.

20. The transgenic plant, plant tissue, or plant cell according to claim 2, wherein the exogenous nucleic acid is operably linked to a β-phaseolin (Phas) promoter derived from kidney bean, a 5'UTR TEV enhancer, and an Arc5 terminator, and then an Rb7MAR.

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

1.

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

2.

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

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

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

26. The transgenic plant, plant tissue, or plant cell according to any one of claims 1 to 25, wherein the transgenic plant, plant tissue, or plant cell is derived from Glycine max, Oryza sativa, Hordeum vulgare, Zea mays, Secale cereale, Avena sativa, Beta vulgaris, Beta vulgaris subsp vulgaris, Pastinaca sativa, Phaseolus vulgaris, Pisum sativum, Vigna angularis, Vigna radiata, Cicer arietinum, Arachis hypogaea, Lens culinaris, Medicago sativa, Eruca vesicaria, Brassica juncea, Lactuca sativa, Brassica, Solanum tuberosum, Ipomoea batatas, Manihot esculenta, Triticum aestivum, or Triticum spelta.

27. A method for obtaining a recombinant hemoprotein, the method comprising: i. providing a transgenic plant capable of expressing at least about 5% TSP of a hemoprotein in seeds; ii. cultivating the transgenic plant; iii. harvesting the transgenic plant. iv. isolating and purifying the animal hemoglobin from the harvested plant, and a method comprising the steps.

28. The method according to claim 27, wherein the harvesting step comprises harvesting the seeds of the transgenic plant.

29. The transgenic plant is selected from the group consisting of soybean (Glycine max), rice (Oryza sativa), barley (Hordeum vulgare), corn (Zea mays), rye (Secale cereale), oats (Avena sativa), sugar beet (Beta vulgaris), beet (Beta vulgaris subsp vulgaris), parsnip (Pastinaca sativa), common bean (Phaseolus vulgaris), pea (Pisum sativum), adzuki bean (Vigna angularis), mung bean (Vigna radiata), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentil (Lens culinaris), alfalfa (Medicago sativa), arugula (Eruca vesicaria), mustard (Brassica juncea), lettuce (Lactuca sativa), rape (Brassica), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), wheat (Triticum aestivum), or spelt wheat (Triticum spelta). The method according to claim 28.

30. Transgenic seeds comprising at least about 5% TSP of recombinant hemoglobin.

31. Transgenic seeds comprising at least about 8% TSP of recombinant hemoglobin.

32. Transgenic seeds comprising at least about 12% TSP of recombinant hemoglobin.

33. Transgenic seeds comprising at least about 15% TSP of recombinant hemoglobin.

34. Transgenic seeds comprising at least about 20% TSP of recombinant hemoglobin.

35. Transgenic seeds comprising at least about 25% TSP of a recombinant heme protein.

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

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

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

39. The transgenic seed is selected from the group consisting of soybean (Glycine max), rice (Oryza sativa), barley (Hordeum vulgare), corn (Zea mays), rye (Secale cereale), oats (Avena sativa), sugar beet (Beta vulgaris), sugar beet (Beta vulgaris subsp vulgaris), parsnip (Pastinaca sativa), common bean (Phaseolus vulgaris), pea (Pisum sativum), adzuki bean (Vigna angularis), mung bean (Vigna radiata), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentil (Lens culinaris), alfalfa (Medicago sativa), arugula (Eruca vesicaria), mustard (Brassica juncea), lettuce (Lactuca sativa), brassica (Brassica), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), wheat (Triticum aestivum), or spelt wheat (Triticum spelta). The method according to any one of claims 30 to 38.

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

41. A food composition comprising the heme protein of the transgenic plant, plant tissue, or plant cell according to 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 hemoprotein of the transgenic plant, plant tissue, or plant cell according to 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 the β-conglycinin α subunit of the 7S storage (7s) promoter derived from soybean, the β-phaseolin (Phas) promoter derived from kidney bean, the USP promoter derived from broad bean (Vicia faba), the SBP promoter derived from broad bean (Vicia faba), the legumin B4 promoter derived from broad bean (Vicia faba), the napin promoter derived from rapeseed (Brassica napus), the vicilin promoter derived from pea (Pisum sativum), the α-globulin promoter derived from cotton, the γ-zein promoter derived from corn, the glutenin promoter derived from wheat, the VvβVPE promoter derived from a species of the genus Vitis (Vitis spp), the groundnut seed promoter (GSP) derived from peanut, the 7αP promoter derived from soybean, the AtLAC15 promoter derived from Arabidopsis thaliana, the SSP promoter derived from chickpea, the lectin promoter derived from soybean, the oleosin promoter derived from rapeseed, the AhLEC1A promoter derived from peanut, the Glu-1D-1 promoter derived from wheat, the sesame 2S albumin (2Salb) promoter derived from sesame, or the 8SGα promoter derived from taro.

45. The polynucleotide according to claim 44, wherein the hemoprotein comprises a plant-derived hemoprotein, a microorganism-derived hemoprotein, or an animal-derived hemoprotein.

46. The polynucleotide according to claim 44, wherein the nucleic acid encoding the hemoprotein comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

1.

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

2.

48. The polynucleotide according to claim 44, comprising a nucleic acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 3 to 7. **Claim 49** The polynucleotide according to claim 44, wherein the seed-specific promoter is β-phaseolin (Phas). **Claim 50** A transcription terminator selected from the group consisting of a tobacco-derived extensin terminator, an Arabidopsis thaliana-derived Ub10 terminator, an Arabidopsis thaliana-derived Hsp70 terminator, an Arabidopsis thaliana-derived Hsp18.2 terminator, an Arabidopsis thaliana-derived Act2 terminator, an Arabidopsis thaliana-derived G7 terminator, an Arabidopsis thaliana-derived 3g24240 terminator, an Arabidopsis thaliana-derived NOS terminator, an Agrobacterium tumefaciens-derived Ocs terminator, an Agrobacterium tumefaciens-derived Mas terminator, a cauliflower mosaic virus-derived 35s terminator, a Chrysanthemum-derived Rbc terminator, an Agrobacterium tumefaciens-derived Ags terminator, an Agrobacterium tumefaciens-derived 3'utr-nos terminator, a soybean-derived 7s terminator, a Pisum sativum-derived E9 terminator, an Agrobacterium tumefaciens-derived ORF25 terminator, a Solanum tuberosum-derived pinII terminator, an Agrobacterium tumefaciens-derived tml terminator, an Agrobacterium tumefaciens-derived Tr7 terminator, or a Phaseolus vulgaris-derived Arc5 terminator, the polynucleotide according to claim 44, further comprising.

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

52. The polynucleotide according to claim 44, further comprising a transcription or translation enhancer selected from the group consisting of 5'UTR TEV and the Rb7Mar 3'matrix attachment region.

53. The polynucleotide according to claim 52, wherein the enhancer is the Rb7Mar 3'matrix attachment region.

54. An expression vector comprising a nucleic acid encoding a heme protein, wherein the nucleic acid is a polynucleotide comprising a nucleic acid encoding a heme protein, and the nucleic acid is operably linked to a seed-specific promoter selected from the group consisting of the β-conglycinin α subunit of the 7S storage (7s) promoter from soybean, the β-phaseolin (Phas) promoter from kidney bean, the USP promoter from broad bean (Vicia faba), the SBP promoter from broad bean (Vicia faba), the legumin B4 promoter from broad bean (Vicia faba), the napin promoter from rapeseed (Brassica napus), the vicilin promoter from pea (Pisum sativum), the α-globulin promoter from cotton, the γ-zein promoter from corn, the glutenin promoter from wheat, the VvβVPE promoter from a species of the genus Vitis (Vitis spp), the glycinin seed promoter (GSP) from peanut, the 7αP promoter from soybean, the AtLAC15 promoter from Arabidopsis thaliana, the SSP promoter from chickpea, the lectin promoter from soybean, the oleosin promoter from rapeseed, 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 taro.

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

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

1.

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

2.

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

59. A transcription terminator selected from the group consisting of a tobacco-derived extensin terminator, an Arabidopsis thaliana-derived Ub10 terminator, an Arabidopsis thaliana-derived Hsp70 terminator, an Arabidopsis thaliana-derived Hsp18.2 terminator, an Arabidopsis thaliana-derived Act2 terminator, an Arabidopsis thaliana-derived G7 terminator, an Arabidopsis thaliana-derived 3g24240 terminator, an Arabidopsis thaliana-derived NOS terminator, an Agrobacterium tumefaciens-derived Ocs terminator, an Agrobacterium tumefaciens-derived Mas terminator, a cauliflower mosaic virus-derived 35s terminator, a Chrysanthemum-derived Rbc terminator, an Agrobacterium tumefaciens-derived Ags terminator, an Agrobacterium tumefaciens-derived 3′utr-nos terminator, a soybean-derived 7s terminator, a Pisum sativum-derived E9 terminator, an Agrobacterium tumefaciens-derived ORF25 terminator, a Solanum tuberosum-derived pinII terminator, an Agrobacterium tumefaciens-derived tml terminator, an Agrobacterium tumefaciens-derived Tr7 terminator, or a Phaseolus vulgaris-derived Arc5 terminator, and the expression vector according to any one of claims 54 to 58.

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

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

62. The expression vector according to claim 61, wherein the enhancer is the Rb7Mar 3'matrix attachment region.

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

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

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

66. The expression vector according to claim 65, wherein the terminator is the Arc5 terminator derived from common bean (Phaseolus vulgaris).

67. The expression vector according to claim 65 or 66, further comprising a transcription or translation enhancer selected from the group consisting of 5'UTR TEV and the Rb7Mar 3'matrix attachment region.

68. The expression vector according to claim 67, wherein the enhancer is the Rb7Mar 3'matrix attachment region.

69. The expression vector according to claim 65, which 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% of the total soluble protein (TSP), and the nucleic acid comprises: i) a phas promoter, and ii) an extensin terminator from tobacco, a 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 Arabidopsis thaliana, an Ocs terminator from Agrobacterium tumefaciens, a Mas terminator from Agrobacterium tumefaciens, a 35s terminator from cauliflower mosaic virus, an Rbc terminator from Chrysanthemum, an Ags terminator from Agrobacterium tumefaciens, a 3'utr-nos terminator from Agrobacterium tumefaciens, a 7s terminator from soybean, an E9 terminator from Pisum sativum, an ORF25 terminator from Agrobacterium tumefaciens, a pinII terminator from Solanum tuberosum, a tml terminator from Agrobacterium tumefaciens, an AgrobacteriumA polynucleotide operably linked to a terminator selected from the group consisting of the Tr7 terminator derived from tumefaciens) or the Arc5 terminator derived from common bean (Phaseolus vulgaris).

71. The polynucleotide according to claim 70, wherein the terminator is the Arc5 terminator derived from common bean (Phaseolus vulgaris).

72. The polynucleotide according to claim 70 or 71, further comprising a transcription or translation enhancer selected from the group consisting of 5'UTR TEV and the Rb7Mar 3'matrix attachment region.

73. The polynucleotide according to claim 72, wherein the enhancer is the Rb7Mar 3'matrix attachment region.

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

Images