WO2022072846A2

WO2022072846A2 - Transgenic plants with altered fatty acid profiles and upregulated heme biosynthesis

Info

Publication number: WO2022072846A2
Application number: PCT/US2021/053199
Authority: WO
Inventors: Ranjani VARADAN; Martin Andrew HOYT; Allen HENDERSON; Rachel FRASER; Sergey SOLOMATIN; Xin Li; Celeste HOLZ-SCHIETINGER; Patrick O'reilly Brown
Original assignee: Impossible Foods Inc.
Priority date: 2020-10-02
Filing date: 2021-10-01
Publication date: 2022-04-07
Also published as: WO2022072846A3

Abstract

This disclosure generally relates to transgenic plants that recombinantly express nucleic acid sequences encoding polypeptides capable of upregulating heme biosynthesis, wherein the plants have an altered fatty acid profile and methods of producing heme-loaded heme polypeptides.

Description

TRANSGENIC PLANTS WITH ALTERED FATTY ACID PROFILES AND UPREGULATED HEME BIOSYNTHESIS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos. 63/087,146, filed on October 2, 2020, and 63/180,849, filed on April 28, 2021, each of which are incorporated by reference in their entirety.

TECHNICAL FIELD

This disclosure relates to methods and materials for altering the fatty acid profile and upregulating heme biosynthesis in plants, and more particularly, to methods for recombinantly producing heme-loaded heme polypeptides in transgenic plants that have altered fatty acid profiles, and plant cells, or seeds having upregulated heme biosynthesis and altered fatty acid profiles.

BACKGROUND

Plants make large amounts of tetrapyrrole molecules including chlorophyll, sirohemes and heme B. The tetrapyrrole biosynthetic pathway provides key co-factors and pigments for processes such as growth and essential redox reactions. Plants control tetrapyrrole synthesis by a number of routes, including a diurnal switch, a redox response system and negative feedback loops to prevent the formation of reactive oxidative species such as photoreactive intermediates, and to accurately distribute metabolic intermediates amongst end products of the pathway. (Mochizuki et al., Trends Plant Sci 15 (9): 488- 498, 2010).

SUMMARY

This document is based on methods and materials for making transgenic plants, transgenic plant cells, and transgenic seeds in which heme biosynthesis is specifically upregulated in order to increase the heme loading of recombinant heme polypeptides produced in the transgenic plants, cells, and seeds. As described herein, using transgenic plants with altered fatty acid profiles, e.g., a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, or a high saturated fatty acid content, can improve stability and/or improve the flavor profile of the recombinant heme polypeptides produced in the transgenic plants, cells, and seeds.

One molecule of the heme co-factor is typically synthesized for each polypeptide that is made. Therefore, in order to increase the specific production of heme B for incorporation into heme-containing proteins, it is important to separate the production of heme from the control mechanisms of the tetrapyrrole biosynthesis pathway, and specifically upregulate heme biosynthesis and not the other tetrapyrroles. "Up-regulation" in the context of heme biosynthesis refers to increased biosynthesis of heme without a corresponding increase in the biosynthesis of other tetrapyrroles.

In one aspect, provided herein is a transgenic plant and seeds of the transgenic plant. The transgenic plant and seeds include at least one recombinant nucleic acid, wherein the recombinant nucleic acid includes: (i) a first promoter operably linked to a nucleic acid encoding a heme-containing polypeptide and; (ii) a second promoter operably linked to a nucleic acid encoding a polypeptide that upregulates heme biosynthesis, wherein heme-loading of the heme-containing polypeptide is increased in the transgenic plant relative to that of a corresponding control plant that does not comprise the second promoter operably linked to the nucleic acid encoding the polypeptide that upregulates heme biosynthesis, wherein the transgenic plant has a fatty acid profile selected from the group consisting of a high monounsaturated and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, and a high saturated fatty acid content.

In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters are inducible. In some embodiments, the first and second promoters are seed specific promoters. In some embodiments, the seed specific promoter is selected from the group consisting of a soy beta-conglycinin seed specific promoter, a G1 -Glycinin seed specific promoter, a Kunitz trypsin inhibitor (KTI) promoter, and an oleosin promoter.

In some embodiments, the recombinant nucleic acid further comprises a first targeting sequence operably linked to the nucleic acid encoding the heme-containing polypeptide, and a second targeting sequence operably linked to the nucleic acid encoding the polypeptide that upregulates heme biosynthesis. In some embodiments, the first and second targeting sequences target the polypeptides to the same intracellular location within the transgenic plant. In some embodiments, the first and second targeting sequences are the same.

In some embodiments, the targeting sequence encodes a vacuole targeting signal peptide. In some embodiments, the vacuole targeting signal peptide is a soy conglycinin vacuole targeting signal peptide, a soy glycinin vacuole targeting signal peptide, or a plant seed storage protein vacuole targeting signal peptide. In some embodiments, the targeting sequence encodes a plastid targeting signal peptide. In some embodiments, the plastid targeting signal peptide is a RuBisCO signal peptide. In some embodiments, the targeting sequence is a soy beta-conglycinin targeting sequence. In some embodiments, the targeting sequence is a G1 -Glycinin targeting sequence.

In some embodiments, the polypeptide that upregulates heme biosynthesis is a ferrochelatase, a glutamyl-tRNA reductase (GluTR) binding protein, a truncated glutamate tRNA reductase protein (GTR), an aminolevulinic acid synthase, or a combination of two or more of the polypeptides. In some embodiments, the polypeptide that upregulates heme biosynthesis is endogenous to the transgenic plant. In some embodiments, the polypeptide that upregulates heme biosynthesis is heterologous to the transgenic plant.

In some embodiments, the ferrochelatase is a barley ferrochelatase, a tobacco ferrochelatase, a soy ferrochelatase, or a microbial ferrochelatase. In some embodiments, the microbial ferrochelatase is a Bradyrhizobium ferrochelatase or an Aspergillus ferrochelatase.

In some embodiments, the aminolevulinic acid synthase is a bacterial aminolevulinic acid synthase.

In some embodiments, the heme-containing polypeptide is a globin polypeptide in Pfam 00042. In some embodiments, the globin polypeptide is a plant globin polypeptide. In some embodiments, the globin polypeptide is a leghemoglobin, a non-symbiotic hemoglobin, an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a myoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a neuroglobin, a chlorocruorin, a truncated hemoglobin, a truncated 2/2 globin, a hemoglobin 3, a cytochrome, or a peroxidase. In some embodiments, the globin polypeptide is expressed in the cytosol of the seeds of the transgenic plant cells. In some embodiments, the globin polypeptide is expressed in the vacuole of the seeds of transgenic plant cells.

In some embodiments, the transgenic plant is a soy plant. In some embodiments, the transgenic plant is a rice plant. In some embodiments, the transgenic plant is selected from the group consisting of: a barley, a wheat, a com, a rye, an oat, a beet, a sugar beet, a parsnip, a bean, a leafy vegetable, a tuber, and a grass. In some embodiments, the bean is an adzuki, a mung, a pea, a peanut, a lentil, or a garbanzo. In some embodiments, the leafy vegetable is an alfalfa, an arugula, a mustard, or a. Brassica. In some embodiments, the grass is triticale or spelt. In some embodiments, the tuber is a potato, a sweet potato, or a cassava.

In some embodiments, the plant has a low polyunsaturated fatty acid content. In some embodiments, the bean or the seed has a low linolenic acid content. In some embodiments, the bean or the seed has a high monounsaturated fatty acid content. In some embodiments, the bean or the seed has a high oleic acid content. In some embodiments, the bean or the seed has a low unsaturated fatty acid content. In some embodiments, the bean or the seed has a high saturated fatty acid content. In some embodiments, the plant has a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content. In some embodiments, the plant has a high medium chain fatty acid content. In some embodiments, the plant has a high saturated fatty acid content.

In another aspect, provided herein is a method of making a textured soy protein having reduced off-flavors. The method can include obtaining a soy protein from a soybean plant yielding a seed oil with a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content and processing the soy protein to obtain the textured soy protein.

In another aspect, provided herein is a method of making a textured soy protein having reduced off-flavors. The method can include obtaining a soy protein from a soybean plant yielding a seed oil with a low linolenic acid content and processing the soy protein to obtain the textured soy protein.

In any of the methods of making textured soy protein, the methods further can include a step selected from the group consisting of: extracting the soy protein with supercritical CO2, extracting the soy protein with supercritical CO2 and a subsequent extraction with an organic solvent, fractioning the soy protein with ammonium-sulfate, and treating the soy protein with a cyclodextrin.

In some embodiments, a plant-based food composition comprising a hemecontaining polypeptide from the seeds is disclosed. In some embodiments, the plantbased food composition is a meat substitute. In some embodiments, the meat substitute is a beef substitute. In some embodiments, the meat substitute is a chicken substitute.

In another aspect, provided herein is a plant-based food composition comprising soybean protein, wherein the soybean protein is isolated from a soybean plant that yields a seed oil with a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content.

In another aspect, provided herein is a plant-based food composition comprising soybean protein, wherein the soybean protein is isolated from a soybean plant that yields a seed oil with a low linolenic acid content.

In another aspect, provided herein is a plant-based food composition comprising soybean protein, wherein the soybean protein is isolated from a soybean plant having decreased expression and/or activity of one or more lipases, lipoxygenases, and/or hydroperoxide lyases.

In another aspect, provided herein is a plant-based food composition comprising soybean protein, wherein the soybean protein is isolated from a soybean plant having decreased expression and/or activity of one or more lipases, lipoxygenases, and/or hydroperoxide lyases that yields a seed oil with a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content.

In some embodiments, the soybean is textured soybean protein. In some embodiments, the plant-based food composition further includes a heme-containing polypeptide. In some embodiments, the plant-based food composition further includes a heme-containing polypeptide from the seeds of disclosed plants. In some embodiments, the plant-based food composition is a meat substitute. In some embodiments, the meat substitute is a beef substitute. In some embodiments, the meat substitute is a chicken substitute.

In another aspect, provided herein is a genetically-modified plant comprising at least one genetic modification, wherein the genetic modification comprises an inversion of an endogenous nucleic acid, wherein the inversion results in i) a nucleic acid encoding a heme-containing polypeptide being operably linked to a nucleic acid encoding a seedspecific signal peptide and a seed-specific promoter of a seed-storage protein, and (ii) a nucleic acid encoding the seed-storage protein being operably linked to a promoter of the heme-containing polypeptide, wherein the heme-containing polypeptide is expressed in seeds of the plant and the seed-storage protein is expressed in root nodules of the plant.

In some embodiments, the heme-containing polypeptide is a leghemoglobin. In some embodiments, the nucleic acid encoding the heme-containing polypeptide has about 80% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid encoding the seed-storage storage protein is a beta-conglycinin subunit. In some embodiments, the beta-conglycinin subunit is the beta-conglycinin alpha prime subunit 2. In some embodiments, the heme-containing polypeptide is expressed in the protein storage vacuole of the seeds of the genetically modified plant. In some embodiments, the heme-containing polypeptide is expressed in the cytosol of the seeds of the genetically modified plant cells. In some embodiments, the heme-containing polypeptide is expressed in the vacuole of the seeds of genetically modified plant cells.

In some embodiments, the genetically modified plant is a soy plant. In some embodiments, the genetically modified plant is a rice plant. In some embodiments, the genetically modified plant is selected from the group consisting of: a barley, a wheat, a corn, a rye, an oat, a beet, a sugar beet, a parsnip, a bean, a leafy vegetable, a tuber, and a grass. In some embodiments, the bean is an adzuki, a mung, a pea, a peanut, a lentil, or a garbanzo. In some embodiments, the leafy vegetable is an alfalfa, an arugula, a mustard, or a Brassica. In some embodiments, the grass is triticale or spelt. In some embodiments, the tuber is a potato, a sweet potato, or a cassava.

In some embodiments, the seeds of the transgenic plant are disclosed.

In another aspect, provided herein a method of making a genetically modified plant including a) obtaining a plant cell comprising endogenously in the genome in the 5’ to 3’ direction: a first nucleic acid encoding a seed-storage polypeptide; a second nucleic acid, which includes, in the 5’ to 3’ direction, a nucleic acid encoding a seed-specific signal peptide and a seed-specific promoter, wherein the seed-specific promoter is operably liked to the nucleic acid encoding the seed-specific signal peptide and the first nucleic acid encoding the seed-storage polypeptide, and a nodule-specific promoter sequence; and a third nucleic acid encoding a heme-containing polypeptide operably linked to the nodule-specific promoter sequence, b) cleaving, using a first site-specific nuclease, between the first nucleic acid encoding the seed-storage polypeptide and the second nucleic acid encoding the seed-specific signal peptide, and cleaving, using a second site-specific nuclease, between the nodule-specific promoter sequence and the third nucleic acid encoding the heme-containing polypeptide, c) selecting a genetically modified plant cell including an inversion of the second nucleic acid in its genome, wherein the genome of the genetically modified plant cell includes in the 5’ to 3’ direction the first nucleic acid encoding the seed-storage polypeptide, the second nucleic acid, which includes in the 5’ to 3’ direction the nodule-specific promoter sequence, the seed-specific promoter sequence, and the seed-specific signal peptide, and the third nucleic acid encoding the heme-containing polypeptide operably linked to the seedspecific promoter sequence and the seed-specific signal peptide, and d) producing the genetically modified plant from the genetically modified plant cell. In some embodiments, the method further comprising screening the genetically modified plant for expression of the first nucleic acid encoding the seed-storage protein in nodules and/or for expression of the third nucleic acid encoding the heme-containing polypeptide in the seeds.

Unless otherwise defined, all 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. The word “comprising” in the claims may be replaced by “consisting essentially of’ or with “consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

Figure 1 is a schematic of tetrapyrrole synthesis.

Figure 2 contains the nucleic acid sequence encoding the Leghemoglobin (Lbc2) from Glycine max (SEQ ID NO:1), the nucleic acid sequence encoding a truncated glutamyl tRNA reductase from Glycine max (corresponding to amino acids 1, and 91 to 542 (end) of Uniprot Q9ZPK4_SOYBN-Glutamyl tRNA reductase) (SEQ ID NO: 2), the nucleic acid sequence encoding the chloroplast ribulose- 1-5 -bisphosphate carboxylase/oxygenase small unit plastid targeting sequence (SEQ ID NO: 3), a nucleic acid sequence encoding a vacuole targeting signal sequence (Conglycinin signal peptide) (SEQ ID NO: 4), a nucleic acid sequence encoding a soluble ferrochelatase from Glycine max (SEQ ID NO: 5, the nucleic acid encodes a polypeptide having residues 105 to 500 and 522 to 531 of SEQ ID NO:9), a nucleic acid sequence encoding a full-length ferrochelatase from Glycine max (SEQ ID NO: 6, encodes residues 105 to 531 of the amino acid sequence set forth in SEQ ID NO: 9), a nucleic acid sequence encoding a glutamyl tRNA reductase binding protein from Glycine max (SEQ ID NO: 7), a nucleic acid sequence encoding a 5-aminolevulinic acid synthase from Bradyrhizobium japonicum (SEQ ID NO: 8), and an amino acid sequence of a ferrochelatase from Glycine max (Uniprot I1K551, SEQ ID NO: 9).

DETAILED DESCRIPTION

In general, this document provides methods and materials for making and using transgenic plants to increase the specific biosynthesis of heme for incorporation into heme-containing proteins. In some embodiments, the heme-containing protein can be produced in the seeds of the transgenic plant to facilitate isolation of the protein. Depending on the desired use of the heme-containing protein, transgenic plants with altered fatty acid profiles, e.g., high monounsaturated fatty acid (e.g., oleic acid, C18: l) content and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, or a high saturated fatty acid content, can be used to improve stability, color, and/or improve the flavor profile of the recombinant polypeptides produced in the transgenic plants, cells, and seeds. As used herein, “polyunsaturated fatty acid content” refers to the total amount of linoleic acid (C18:2) and a-linolenic acid (C18:3). In some embodiments, the total polyunsaturated fatty acid content is low due to a reduction in the a-linolenic acid content. In some embodiments, the total polyunsaturated fatty acid content is low due to a reduction in the linoleic acid content. In some embodiments, the total polyunsaturated fatty acid content is low due to a reduction in both the linoleic acid and a-linolenic acid content. “Medium chain fatty acid content” is used herein to refer to the total amount of caproic acid (C6:0), caprylic acid (C8:0), capric acid (C10:0) and lauric acid (C12:0). “Saturated fatty acid content” is used herein to refer to long chain fatty acids such as myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), and arachidic acid (C20:0). As described below, transgenic plants that are used to produce the heme-containing polypeptide can be selected based on their fatty acid profile (e.g., in their seed oil) or can be modified to alter the fatty acid profile (e.g., in their seed oil) to the desired profile (e.g., high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, or a high saturated fatty acid content).

In some embodiments, high monounsaturated fatty acid content can refer to a monounsaturated fatty acid content of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some embodiments, high oleic acid content can refer to an oleic acid content of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some embodiments, low polyunsaturated fatty acid content can refer to a polyunsaturated fatty acid content up to 55%, up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 9%, up to 8%, up to 7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1%. In some embodiments, low linolenic acid content can refer to a linolenic acid content of up to 10%, up to 9%, up to 8%, up to 7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1%. In some embodiments, low linoleic acid content can refer to a linoleic acid content of up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 9%, up to 8%, up to 7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1%. In some embodiments, low unsaturated fatty acid content can refer to an unsaturated fatty acid content of up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, or up to 10%. In some embodiments, high medium chain fatty acid content can refer to a medium chain fatty acid content of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%. In some embodiments, high saturated fatty acid content can refer to a saturated fatty acid content of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.

In some embodiments, a high monounsaturated and a low polyunsaturated fatty acid content can refer to a monounsaturated and a polyunsaturated fatty acid content of at least 25% monounsaturated and up to 55% polyunsaturated fatty acid content, at least 30% monounsaturated and up to 50% polyunsaturated fatty acid content, at least 40% monounsaturated and up to 40% polyunsaturated fatty acid content, at least 50% monounsaturated and up to 30% polyunsaturated fatty acid content, at least 60% monounsaturated and up to 20% polyunsaturated fatty acid content, at least 70% monounsaturated and up to 10% polyunsaturated fatty acid content, or at least 80% monounsaturated and up to 5% polyunsaturated fatty acid content.

In some embodiments, a plant, bean, seed, or oil as described herein can have a lower amount (e.g., reduced by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) of a polyunsaturated fatty acid content, linolenic acid content, linoleic acid content, unsaturated fatty acid content, or a combination thereof as compared to a corresponding control plant, bean, seed, or oil. In some embodiments, a plant, bean, seed, or oil as described herein can have a higher amount (e.g., increased by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) of a monounsaturated fatty acid, oleic acid content, medium chain fatty acid content, saturated fatty acid content, or a combination thereof as compared to a corresponding control plant, bean, seed, or oil. A standard soybean oil, for example, can contain 15.7% saturated fatty acids, 22.8% total monounsaturated fatty acids (including 22.6% oleic acid), and 57.7% total polyunsaturated fatty acids (including 7% linolenic acid and 51% linoleic acid) (U.S. Department of Agriculture FoodData Central). A standard sunflower oil, for example, can contain 10.3% saturated fatty acids, 19.5% total monounsaturated fatty acids (including 19.5% oleic acid), and 65.7% total polyunsaturated fatty acids (including 0% linolenic acid and 65.7% linoleic acid) (U.S. Department of Agriculture FoodData Central).

In addition, in some embodiments, plants having a desired fatty acid profile (e.g., high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, or a high saturated fatty acid content) can be used as a source for textured vegetable protein (e.g., textured soy protein) to improve the flavor profile and/or functional characteristics and/or properties of the protein (e.g., one or more of reduced off-flavors, altered gel-strength, altered viscosity, improved oxidative stability, and improved whiteness). As described herein, functional proteins can have one or more of the following properties: non-denatured; capable of forming a gel upon heating (e.g., a suspension of about 25 to about 250 mg/mL (e.g., about 25 to about 50 mg/mL, about 25 to about 100 mg/mL, about 25 to about 150 mg/mL, about 25 to about 200 mg/mL, about 50 to about 250 mg/mL, about 100 to about 250 mg/mL, about 150 to about 250 mg/mL, or about 200 to about 250 mg/mL) at a pH of about 7.0) thermally transitions to a gel upon heating to about 65°C); thermally denatures during incubation between about 50°C and about 85°C, with greater than about 80% of the protein denaturing after about 20 minutes at about 85°C, as measured either by differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF); in a solution or suspension of purified protein at or above about 50 mg/mL (5% w/v), protein forms a freestanding gel (with, e.g., a 100 Pa storage modulus) when heated at or above about 85°C for about 20 minutes; can denature and gel between about pH 5.5 and about pH 10.0; can denature and gel in solutions with ionic strength (I) below about 0.5M, when I is calculated based on the concentration of non-protein solutes; at a protein concentration of about 10 mg/mL, particle size distribution D10, D50 and D90 are less than about 0.1 pm, 1.0 pm and 5 pm, respectively; has enzymatic activity; and/or has an emulsion activity index (EAI) of greater than or equal to about 50 m²/g protein across a pH range of about 4.0 to about 8.0.

In some embodiments, the transgenic plants, cells, and seeds described herein include at least one recombinant nucleic acid that includes a) a promoter operably linked to a nucleic acid encoding a heme-containing polypeptide and b) a promoter operably linked to a nucleic acid that encodes a polypeptide that specifically upregulates heme biosynthesis. "Polypeptide" as used herein refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics, regardless of post- translational modification, e.g., phosphorylation or glycosylation. The subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds. Full- length polypeptides, truncated polypeptides, point mutants, insertion mutants, inversion mutants, splice variants, chimeric proteins, and fragments thereof are encompassed by this definition.

To specifically upregulate heme biosynthesis, one or more of the following polypeptides can be expressed in the transgenic plant: a ferrochelatase, a glutamyl-tRNA reductase (GluTR) binding protein, a truncated glutamate tRNA reductase protein (GTR), or an aminolevulinic acid synthase. For example, a transgenic plant may express one, two, three, or four of such polypeptides. For example, a transgenic plant may express a ferrochelatase, a ferrochelatase and a GluTR binding protein, a ferrochelatase and a truncated GTR, a ferrochelatase and an aminolevulinic acid synthase, a GluTR binding protein, a GluTR binding protein and a truncated GTR, a GluTR binding protein and an aminolevulinic acid synthase, a truncated GTR, a truncated GTR and an aminolevulinic acid synthase, a ferrochelatase, a GluTR binding protein and a truncated GTR, a ferrochelatase, a GluTR binding protein and an aminolevulinic acid synthase, a GluTR binding protein, a truncated GTR, and an aminolevulinic acid synthase, or a ferrochelatase, a GluTR binding protein, a truncated GTR, and an aminolevulinic acid synthase. See, for example, U.S. Publication No. 2019/0292217 and International Publication No. WO 2018/102721.

It will be appreciated that the polypeptide that upregulates heme biosynthesis can be a variant (e.g., comprise a mutation such as an amino acid substitution, e.g., a nonconservative or conservative amino acid substitution, an amino acid deletion, an amino acid insertion, or non-native sequence) relative to a wild-type heme biosynthesis polypeptide. For example, a domain such as a transmembrane domain can be removed from a polypeptide that upregulates heme biosynthesis to increase solubility of the polypeptide or a signal peptide can be deleted. For example, a transmembrane domain near the C-terminus of a ferrochelatase (e.g., residues 501-521 of the Glycine max ferrochelatase set forth in SEQ ID NO: 9) can be deleted and/or the signal peptide of the ferrochelatase (residues 1 to 104 of SEQ ID NO: 9) can be deleted. For example, a ferrochelatase polypeptide can include residues 105 to 531 of the amino acid sequence set forth in Uniprot I1K551 (see SEQ ID NO: 9, Figure 2) or can include residues 105 to 500 and 522 to 531 the amino acid sequence set forth in Uniprot I1K551 (see SEQ ID NO: 9, Figure 2). A truncated glutamate tRNA reductase protein can have one or more N- terminal residues (e.g., 5, 10, 15, 20, 25, 30, 35, or 40 residues) removed as described below to, for example, remove feedback inhibition by heme. In some instances, a variant polypeptide can include at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 mutations. In some instances, a variant polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more mutations. In some instances, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50% of the sequence of a polypeptide of the disclosure can be mutated. In some instances, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50% of the sequence of a polypeptide can be mutated.

Ferrochelatase is the enzyme at the branch point of the biosynthetic pathway that pulls the biosynthetic flux towards heme rather than chlorophyll. See, Figure 1. Overexpression of a ferrochelatase in combination with a heme protein can increase the heme-loading of the heme polypeptide. In some embodiments, a ferrochelatase lacking a C-terminal transmembrane domain can be used. Non-limiting examples of suitable ferrochelatases include a barley ferrochelatase, a tobacco ferrochelatase, a soy ferrochelatase, a mung bean ferrochelatase (e.g., GenBank Accession No.

XP 014509945, C-terminal domain from residues 500-520 can be deleted) or a microbial ferrochelatase (e.g., a Bradyrhizobium ferrochelatase such as Bradyrhyzobium japonicum ferrochelatase (GenBank Accession No. AJA60352.1) or an Aspergillus ferrochelatase such as Aspergillus niger ferrochelatase). As used herein, the term microbial refers to a bacterial, viral, or fungal polypeptide.

In some embodiments, a GluTR binding protein mediates heme biosynthetic flux through a spatially separated system that leads only to heme and avoids some of the feedback loops. See, for example, Czarnecki et al., J Exp Bot, 63 (4): 1675-1687, 2012. Non-limiting examples of suitable GluTR binding proteins include the soy, Arabidopsis thaiHana, barley, Medicago trunculata, adzuki bean, and kidney bean GluTR binding protein. Overexpressing the GluTR binding protein in combination with a heme polypeptide can lead to increased heme-loading of the heme polypeptide.

Glutamyl tRNA reductase converts glutamate molecules that are ligated to tRNA^Glu into glutamate 1 -semialdehyde, an intermediate in the synthesis of 5- aminolevulinate, chlorophyll and heme. GluTR activity is inhibited by binding of heme to its N-terminus (see, e.g., Vothknecht et al., Phytochemistry 47: 513-519, 1998). Removal of the first 30 amino acids of a GluTR (e.g., the soy or barley GluTR) removes the feedback inhibition by heme. Therefore, overexpressing a truncated GluTR protein (e.g., a truncated GluTR protein from Glycine max that is 453 amino acids in length, encoded by SEQ ID NO:2) in combination with a heme polypeptide can lead to increased heme-loading of the heme polypeptide. It will be appreciated that for different glutamyl tRNA reductase polypeptides, the optimal truncation may be greater or less than 30 amino acids.

5-Aminolevulinic acid (ALA), a non-protein amino acid, is the first committed intermediate in the common tetrapyrrole pathway for synthesis of heme, chlorophyll, and cytochrome. In nature, there are two known alternate routes by which this committed intermediate is generated. One route is the C4 pathway (Shemin pathway), which involves the condensation of succinyl-CoA and glycine to ALA by ALA synthase (ALAS). The C4 pathway is restricted to mammals, fungi and purple nonsulfur bacteria. The second route is the C5 pathway, which involves three enzymatic reactions resulting in the biosynthesis of ALA from glutamate. The C5 pathway is active in most bacteria, all archaea and plants. See, e.g., Zhang, et al., Sci Rep., 5:8584 (2015).

Expression of bacterial aminolevulinic acid synthase (ALAS) can increase the flux through the tetrapyrrole biosynthesis pathway as plants have no control mechanism for the C4 route of ALA production. The increased flux can be captured by the overexpressed heme polypeptide, leading to increased heme-loading of the heme polypeptide. The bacterial ALAS can be a Rhodobacter ALAS or rhizobia ALAS such as Bradyrhizobium japonicum ALAS (see SEQ ID NO: 8).

The term “heme-containing protein” can be used interchangeably with “hemecontaining polypeptide” or “heme protein” or “heme polypeptide” or “heme-loaded heme-containing polypeptide” and includes any polypeptide covalently or noncovalently bound to a heme. The terms “heme cofactor” and “heme” are used interchangeably and refer to the iron-containing (Fe²⁺ or Fe³⁺) compound of the porphyrin class which forms a nonprotein part of the heme-containing protein. In some embodiments, the hemecontaining polypeptide is a globin such as one in Pfam 00042 and can include a globin fold, which comprises a series of seven to nine alpha helices. Globin type proteins can be of any class (e.g., class I, class II, or class III), and in some embodiments, can transport or store oxygen. For example, a heme-containing protein can be a non-symbiotic type of hemoglobin or a leghemoglobin.

A heme-containing polypeptide can be a monomer, i.e., a single polypeptide chain, or can be a dimer, a trimer, tetramer, and/or higher order oligomer. The life-time of the oxygenated Fe²⁺ state of a heme-containing protein can be similar to that of myoglobin or can exceed it by 10%, 20%, 30%, 50%, 100% or more under conditions in which the heme-protein-containing consumable is manufactured, stored, handled or prepared for consumption. The life-time of the unoxygenated Fe²⁺ state of a hemecontaining protein can be similar to that of myoglobin or can exceed it by 10%, 20%, 30%, 50%, 100% or more under conditions in which the heme-protein-containing consumable is manufactured, stored, handled or prepared for consumption

Non-limiting examples of heme-containing polypeptides can include leghemoglobin, an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a myoglobin (e.g., bovine myoglobin), an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a neuroglobin, a chlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated 2/2 globin, a hemoglobin 3 (e.g., Glb3), a cytochrome, or a peroxidase.

Heme-containing proteins that can produced in the plants, plant cells, and seeds described herein can be from mammals (e.g., farms animals such as cows, goats, sheep, pigs, ox, or rabbits), birds, plants, algae, fungi (e.g., yeast or filamentous fungi), ciliates, or bacteria. For example, a heme-containing protein can be from a mammal such as a farm animal (e.g., a cow, goat, sheep, pig, fish, ox, or rabbit) or a bird such as a turkey or chicken. Heme-containing proteins can be from a plant such as Nicotiana tabacum or Nicotiana sylvestris (tobacco); Zea mays (corn), Arabidopsis thaliana, a legume such as Glycine max (soybean), Cicer arietinum (garbanzo or chick pea), Pisum sativum (pea) varieties such as garden peas or sugar snap peas, Phaseolus vulgaris varieties of common beans such as green beans, black beans, navy beans, northern beans, or pinto beans, Vigna unguiculata varieties (cow peas), Vigna radiata (mung beans), Lupinus albus (lupin), or Medicago sativa (alfalfa); Brassica napus (canola); Triticum sps. (wheat, including wheat berries, and spelt); Gossypium hirsutum (cotton); Oryza sativa (rice); Zizania sps. (wild rice); Helianthus annuus (sunflower); Beta vulgaris (sugarbeet); Pennisetum glaucum (pearl millet); Chenopodium sp. (quinoa); Sesamum sp. (sesame); Linum usitatissimum (flax); or Hordeum vulgare (barley). Heme-containing proteins can be isolated from fungi such as Saccharomyces cerevisiae. Pichia pasloris. Magnaporthe oryzae, Fusarium graminearum. Aspergillus oryzae, Trichoderma reesei. Myceliopthera thermophile, Kluyveramyces lactis, o Fusarium oxysporum. Heme-containing proteins can be isolated from bacteria such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Synechocistis sp., Aquifex aeolicus, Methylacidiphilum infernorum, or thermophilic bacteria such as Thermophilus spp. The sequences and structure of numerous heme-containing proteins are known. See for example, Reedy, et al., Nucleic Acids Research, 2008, Vol. 36, Database issue D307- D313 and the Heme Protein Database available on the world wide web at http://hemeprotein.info/heme.php. In some embodiments, a leghemoglobin can be a soy, pea, or cowpea leghemoglobin. In some embodiments, the heme-containing polypeptide is a flavohemoglobin, a protein composed of a heme binding domain and a ferredoxin reductase-like FAD- and NAD- binding domain. It is also known as flavohemoprotein, nitric oxide dioxygenase, nitric oxide oxygenase and flavodoxin reductase. Flavohemoglobin genes from E. coli, A. eutrophus, Saccharomyces cerevisiae and Vitreoscilla sp. are abbreviated as HMP, FHP, YHB1 (or YHG), and VHP respectively. See, WO 2006121757.

It will be appreciated that a heme-containing polypeptide can be a variant (e.g., comprise a mutation such as an amino acid substitution, e.g., a non-conservative or conservative amino acid substitution, an amino acid deletion, an amino acid insertion, or non-native sequence) relative to a wild-type heme-containing polypeptide.

The term “sequence identity” refers to a measure of the amount of shared nucleotides of two nucleic acid sequences. The sequence identity between two nucleic acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson’s web site (e.g., www.fir.com/blast/) or the U.S. government’s National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two amino acid sequences using the BLASTN algorithm. To compare two nucleic acid sequences, the options of B12seq are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two nucleic acid sequences: C:\B12seq -i c:\seql.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the full-length nucleic acid sequence followed by multiplying the resulting value by 100. It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., 30 bacteria or fungus) is obtained, using appropriate codon bias tables for that species.

Recombinant Nucleic Acid Constructs

As described herein, in some embodiments, the transgenic plants, transgenic plant cells, or transgenic seeds contain at least one recombinant nucleic acid that includes a) a promoter operably linked to a nucleic acid encoding a heme-containing polypeptide and b) a promoter operably linked to a nucleic acid that encodes a polypeptide that specifically upregulates heme biosynthesis. In some embodiments, the promoter operably linked to a nucleic acid encoding the heme-containing polypeptide and the promoter operably linked to a nucleic acid encoding a polypeptide that specifically upregulates heme biosynthesis are on separate nucleic acid constructs. In some embodiments, the heme-containing polypeptide can be produced in methyl otrophic yeast such as Pichia (e.g., Pichia pastoris). See, for example, U.S. Publication No. 2018/0127764.

The recombinant nucleic acid is exogenous to the plant, plant cell, or seed. As used herein, the term “exogenous” with respect to a nucleic acid indicates that the nucleic acid is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. A heterologous polypeptide as used herein refers to a polypeptide that is not a naturally occurring polypeptide in a plant cell, e.g., a transgenic soybean plant transformed with and expressing the coding sequence for a leghemoglobin from an alfalfa plant. In the plants, cells, and seeds described herein, the hemecontaining polypeptide being expressed in the plant can be heterologous to the plant. In the plants, cells, and seeds described herein, the polypeptide that upregulates heme biosynthesis that is being expressed in the plant can be heterologous to the plant.

An exogenous nucleic acid also can be a sequence that is native to a plant (i.e., it is endogenous to the plant) and that has been reintroduced into cells of that plant such as a nucleic acid encoding a soybean ferrochelatase being re-introduced into a soybean plant. In the plants, cells, and seeds described herein, the heme-containing polypeptide being expressed in the plant can be endogenous to the plant. In the plants, cells, and seeds described herein, the polypeptide that upregulates heme biosynthesis that is being expressed in the plant can be endogenous to the plant. An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. It will be appreciated that an exogenous nucleic acid may have been introduced into a progenitor and not into the cell under consideration. For example, a transgenic plant containing an exogenous nucleic acid can be the progeny of a cross between a stably transformed plant and a non-transgenic plant. Such progeny are considered to contain the exogenous nucleic acid. "Isolated nucleic acid" as used herein includes a naturally-occurring nucleic acid, provided one or both of the sequences immediately flanking that nucleic acid in its naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a nucleic acid that exists as a purified molecule or a nucleic acid molecule that is incorporated into a vector or a virus. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries, genomic libraries, or gel slices containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.

"Nucleic acid" and "polynucleotide" are used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA or RNA containing nucleic acid analogs. A nucleic acid can be double-stranded or singlestranded (i.e., a sense strand or an antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes and nucleic acid primers. A polynucleotide may contain unconventional or modified nucleotides.

In the recombinant nucleic acid constructs described herein, the promoters can be the same or different, and can vary in strength or tissue-specificity. In some embodiments, the recombinant nucleic acid construct can be configured such that the nucleic acid encoding the heme-containing polypeptide and the nucleic acid encoding the polypeptide that upregulates heme biosynthesis are contiguous and a single promoter is used to drive transcription of both nucleic acid sequences. The term “promoter” means a DNA sequence recognized by enzymes/proteins required to initiate the transcription of a specific nucleic acid sequence. A promoter typically refers, e.g., to a sequence of nucleic acid to which an RNA polymerase and/or any associated factors binds and at which transcription is initiated. In some embodiments, the recombinant nucleic includes at least one promoter that is positioned 5’ to the nucleic acid sequence encoding a heme polypeptide or a polypeptide that upregulates heme biosynthesis. As used herein, “operably linked” refers to a segment of DNA being linked to another segment of DNA when placed into a functional relationship with the other segment. In some embodiments, the promoter can be a seed-specific promoter. For example, a seed specific promoter can be from the soy beta-conglycinin gene (see, for example, Chen, et al., Dev Genet., 10(2): 112-22 (1989)) or beta-conglycinin alpha prime subunit 2, a G1 -Glycinin seed specific promoter (Ding, et al. , Biotechnol Lett., 28(12):869-75 (2006)), a KTI promoter (see, for example, Perez-Grau and Goldberg, Plant Cell., 1(11): 1095-1109 (1989)), or an oleosin promoter such as P24 (see, for example, Keddie, et al., Plant Mol BioL, 24(2):327-40 (1994)). Other non-limiting examples include promoters from the following seed-genes: zygote and embryo LEC1; suspensor G564; maize MAC1 (see, Sheridan, Genetics 142: 1009-1020 (1996)); maize Cat3, (see, GenBank No. L05934, Abler, Plant Mol. Biol. 22: 10131-1038, (1993)); Arabidopsis viviparous-1, (see, GenBank No. U93215); Arabidopsis atmycl, (see, Urao, Plant Mol. Biol. 32:571-57 (1996), Conceicao, Plant 5:493-505 (1994)); Brassica napus napin gene family, including napA, (see, GenBank No. J02798, Josefsson ///. 26: 12196- 1301 (1987), and Sjodahl, Planta 197:264-271 (1995)). See also U.S. Publication No. 2016/0340411, U.S. Patent No. 8,115,058, and International Publication Nos. WO 2015/038796 and WO 2006/121757 for additional seed-specific promoters. Producing a heme-containing polypeptide such as leghemoglobin in seeds can be used to facilitate isolation of the protein (e.g., in soluble form).

In some embodiments, the promoter can be a constitutive promoter such as the cauliflower mosaic virus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the 1' or 2' promoters derived from T-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34S promoter, actin promoters such as the rice actin promoter, or a ubiquitin promoter such as the maize ubiquitin-1 promoter. See also U.S. Patent No. 8,115,058 for additional constitutive promoters.

An inducible promoter can include, for example, a core or basal promoter sequence and one or more elements such as transcriptional activator binding sites or other regulatory element to allow control of transcription. A core promoter refers to the minimal sequence necessary for assembly of a transcription complex required for transcription initiation. Basal promoters frequently include a "TATA box" element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation. Basal promoters also may include a "CCAAT box" element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.

For example, an inducible promoter can be a modified cauliflower mosaic virus (CaMV) 35S promoter that is responsive to tetracycline. See, Gatz, et al., Plant J. 2, 397- 404 (1992), and Weinmann, et al., Plant J., 5, 559-569 (1994). For example, an inducible promoter can be dexamethasone-inducible, or dexamethasone-inducible and tetracycline-inactivatable. See, Aoyama and Chua, Plant J., 11, 605-612 (1997), Craft, et al., Plant J., 41, 899-918 (2005); Samalova, et al., Plant J., 41, 919-935 (2005); Bbhner, et al., Plant J., 19, 87-95 (1999); and Bbhner, S. and Gatz, Mol. Gen. Genet. 264, 860-870 (2001). For example, an inducible promoter can be responsive to copper. See, Mett, et al., Proc. Natl Acad. Set. USA, 90, 4567-4571 (1993). For example, an inducible promoter can be responsive to an insecticide (e.g., tebufenozide or methoxyfenozide). See, Koo, et al., Plant J., 37, 439-448 (2004); Martinez, et al., Plant J., 5, 559-569 (1999); and Padidam, et al., Transgenic Res., 12, 101-109 (2003). For example, an inducible promoter can be estrogen responsive (e.g., 17 beta estradiol). See, Bruce, et al., (2000) Plant Cell, 12, 65-80 (2000); and Zuo, et aL, Plant J., 24, 265-273 (2000).

For example, an inducible promoter can be responsive to salicylic acid, ethylene, or jasmonic acid. See, for example, Liu, et al., Plant Biotech. J., 11, 43-52 (2013) and Liu, et aL, BMC Biotechnol. , 11, 108 (2011). Salicylic acid (SA) interacts with either the Arabidopsis PR1 promoter or SA-responsive elements (SARE), which drives the expression of the nucleic acid of interest. Ethylene (ET) interacts with ethylene responsive element (ERE), which drives the expression of the nucleic acid of interest. Methyl jasmonate interacts with jasmonic acid responsive element (JAR) which drives the expression of the nucleic acid of interest. In some embodiments, the inducer (e.g., ethylene gas) can be added to the malting chamber to induce expression of the nucleic acid.

For example, an inducible promoter can be ethanol responsive (e.g., ethanol or acetaldehyde). See, Caddick, et al., Nat. Biotechnol., 16, 177-180 (1998); Rosian, et al., Plant J., 28, 225-235 (2001); and Salter, et al., Plant J., 16, 127-132 (1998). In the presence of ethanol or acetaldehyde, the Aspergillus nidulans ALCR transcription factor (alcR) drives expression from the palcA promoter by binding to upstream sequences (alcA) from the A. nidulans alcA locus. The palcA promoter is positioned upstream of a target DNA for expression.

In some embodiments, ethanol-inducible expression can be based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. See, Werner, et al., Proc Natl Acad Sci USA, 108(34): 14061-14066 (2011).

In some embodiments, the promoter can be a germination specific promoter. Such a promoter results in expression of the target product during germination and/or early seedling growth in one or more of the radical, hypocotyl, cotyledons, epicotyl, root tip, shoot tip, meristematic cells, seed coat, endosperm, true leaves, internodal tissue, and nodal tissue. See, for example, promoters from genes encoding the glyoxysomal enzymes isocitrate lyase (ICL) and malate synthase (MS) from several plant species (Zhang et al., Plant Physiol. 104: 857-864, 1994); Reynolds and Smith, Plant Mol. Biol. 27: 487-497, 1995); Comai et al., Plant Physiol. 98: 53-61, 1992). Promoters also can be from other genes whose mRNAs appear to accumulate specifically during the germination process, for example class I P-l,3-glucanase B from tobacco (Vogeli-Lange et al, Plant J., 5: 273-278, 1994); canola cDNAs CA25, CA8, AX92 (Harada et al., Mol. Gen. Genet., 212: 466-473, 1988); Dietrich et al., J. Plant Nutr., 8: 1061-1073, 1992), lipid transfer protein (Sossountzove et al, Plant Cell, 3: 923-933, 1991); or rice serine carboxypeptidases (Washio et al., Plant Phys., 105: 1275-1280, 1994); and repetitive proline rich cell wall protein genes (Datta et al., Plant Mol. Biol. 14: 285-286, 1990). See U.S. Publication No. 2016/0024512. The a-amylase promoter also can be used as a germination specific promoter. See, Eskelin, et al., Plant Biotechnology Journal, 7: 657-672 (2009).

In some embodiments, the promoter can express in green tissues of plant. Nonlimiting examples of such promoters include the promoter of a maize aldolase gene FDA (U.S. Publication No. 2004/0216189) or the promoter of an aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi et al. (2000) Plant Cell Physiol. 41(1): 42- 48).

Other promotors can include aleurone-specific promoters, endosperm-specific promoters, embryo-specific promoters, leaf-and-stem-specific promoters, panicle-specific promoters, root-specific promoters, and pollen-specific promoters (see, for example U.S. Patent No. 8,115,058).

In some embodiments, the nucleic acid construct further includes a targeting sequence that can be used to direct the heme polypeptide and/or heme biosynthesis polypeptide to one of several different intracellular compartments, including, for example, the endoplasmic reticulum (ER), mitochondria, plastids (such as chloroplasts) such as the RuBisCo plastid targeting sequence, the vacuole, the Golgi apparatus, protein storage vesicles (PSV) and, in general, membranes, to structures such as the roots, or cells in, for example, the hypocotyl. For example, the heme polypeptide and/or heme biosynthesis polypeptide can be directed to the same intracellular location or to different intracellular locations. Some signal peptide sequences are conserved, such as the Asn- Pro-Ile-Arg (SEQ ID NO: 10) amino acid motif found in the N-terminal propeptide signal that targets proteins to the vacuole (Marty, Plant Cell, 11 : 587-599, 1999). Other signal peptides do not have a consensus sequence per se, but are largely composed of hydrophobic amino acids, such as those signal peptides targeting proteins to the ER (Vitale and Denecke, Plant Cell, 11 : 615-628, 1999). Still others do not appear to contain either a consensus sequence or an identified common secondary sequence, for instance the chloroplast stromal targeting signal peptides (Keegstra and Cline, Plant Cell, 11 : 557- 570, 1999). Chloroplast targeting peptides commonly have a high content of hydroxylated amino acid residues (Ser, Thr, and Pro), lack acidic amino acid residues (Asp and Glu), and tend to form a-helical structures in hydrophobic environments (see, e.g., Shen, et al., Scientific Reports, 7, 46231, 2017). In some embodiments, a chloroplast targeting sequence can be a pea, rice, tobacco, Arabidopsis, or soy rubisco small subunit (rbcS) transit peptide (Van den Broeck, et al., Nature, 313, 358-363, 1985). In some embodiments, a portion of the N-terminus of the rbcS protein can be included in the targeting sequence. For example, a portion of the N-terminal unfolded region (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 amino acids) of the rbcS protein can be included in the chloroplast target sequence (see, e.g., Shen, et al., 2017, supra), for a total length of 50- 80 amino acids (e.g., 60, 65, 70, 75 amino acids). Furthermore, some targeting peptides are bipartite, directing proteins first to an organelle and then to a membrane within the organelle (e.g. within the thylakoid lumen of the chloroplast; see Keegstra and Cline, 1999, supra). In addition to the diversity in sequence and secondary structure, placement of the signal peptide is also varied. Proteins destined for the vacuole, for example, can have targeting signal peptides found at the N-terminus, at the C-terminus and at a surface location in mature, folded proteins.

In some embodiments, a nucleic acid construct includes a root targeting sequence such as domain A of the CaMV 35S promoter (e.g., containing a tandem repeat of the sequence TGACG separated by 7 base pairs). See, for example, Benfey, et al., The EMBO Journal, 8(8):2195-2202, 1989.

In some embodiments, a nucleic acid sequence encoding a soy conglycinin vacuole targeting signal peptide, a soy glycinin vacuole targeting signal peptide, or a plant seed storage protein vacuole targeting signal peptide is used as a targeting sequence.

In some embodiments, heme-containing polypeptides can be produced in methylotrophic yeast such as Pichia (e.g., Pichia pastoris) or in transgenic plants, transgenic cells, or transgenic seeds using inducible promoters and/or the positive feedback loop disclosed in U.S. Publication No. 2018/0127764.

Producing Transgenic Plant Cells and Plants

Transgenic plant cells and plants comprising at least one recombinant nucleic acid construct described herein can be produced using a variety of techniques. For example, Agrobaclerium-m dx&i transformation, viral vector-mediated transformation, electroporation, or particle gun transformation can be used for introducing nucleic acids into monocotyledonous or dicotyledonous plants. See, for example, U.S. Patent Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. In some embodiments, gene editing techniques using site-specific nucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENS), or the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system can be used to produce plants or cells having altered characteristics including one or more of fatty acid profile, flavor profile, or sugar content. See, for example, Aroroa and Narula, Front. Plant Sc. 8: 1932 (2017). If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art. In some embodiments, seed-specific expression of a protein, e.g., leghemoglobin, endogenously expressed elsewhere in the plant, e.g., in a root or nodule, can be obtained in a plant or plant cells using a gene-editing technique that includes site-specific cleavage of the genome to produce a cleaved fragment of DNA, inversion of the cleaved fragment, and repair of the sites of DNA cleavage to rejoin the fragment within the genome.

In some embodiments, the inverted DNA fragment is about 4 Mb in length. In some embodiments, the inverted DNA fragment is about lOObp to about 8Mbp in length. For example, the inverted DNA fragment can be about lOObp to about 1Mb, about lOObp to about 2Mb, about lOObp to about 3Mb, about lOObp to about 4Mb, about lOObp to about 5Mb, about lOObp to about 6Mb, about lOObp to about 7Mb, about lOObp to about 8Mb, about 7Mb to about 8Mb, about 6Mb to about 8Mb, about 5Mb to about 8Mb, about 4Mb to about 8Mb, about 3Mb to about 8Mb, about 2Mb to about 8Mb, about 1Mb to about 8Mb, or about lOObp to about 8Mb in length. In some embodiments, the inverted DNA fragment is about 2Mb to about 4Mb, about 3Mb to about 5Mb, about 4Mb to about 6Mb, about 3Mb to about 4Mb, about 3.5Mb to about 4.5Mb, or about 4Mb to about 5Mb in length.

For example, a site-specific nuclease can be used to cleave between the signal peptide sequence and the coding region sequence of the nucleic acid encoding a seedstorage polypeptide, such that the seed-specific promoter is linked to the signal peptide sequence, and a second site-specific nuclease can be used to cleave between the promoter of a nucleic acid encoding a heme-containing polypeptide and the nucleic acid encoding the heme-containing polypeptide. Site-specific cleavage can produce a fragment of DNA that can be naturally inverted before repair with, for example, host repair enzymes, such that the nucleic acid encoding the heme-containing polypeptide is operably linked to the seed-specific promoter and signal peptide sequence of the nucleic acid encoding the seedstorage polypeptide. Seed-storage polypeptides can include, but are not limited to, beta- conglycinin alpha prime subunit 2. Heme-containing polypeptides can include, but are not limited to, leghemoglobin C2 (lbc2). The inverted fragment of endogenous DNA created by site-specific cleavage can include promoters, signal peptides, open reading frames, activators, terminators, genes, encoded polypeptides, or regulatory sequences. The polynucleotides and constructs described herein can be used to transform a number of monocotyledonous and dicotyledonous plants including, for example, Arabidopsis lhaHana. Oryza sativa (rice), Glycine max or Glycine soja (soybean), a beet, a sugar beet, parsnip, a sunflower, a bean such as an adzuki, a mung, a pea, a peanut, a lentil, or a garbanzo, a leafy vegetable such as an alfalfa, an arugula, a mustard, or a Brassica, or a grass such as a barley, an oat, a wheat, a corn, a rye, triticale, or spelt. Suitable soybean plants can produce seeds of any color, including black, brown, red, yellow, or green, and can be variegated or bicolored. In some embodiments, the color of a seed or a bean can be influenced by a target protein (e.g., a heme-containing protein) and/or environmental conditions (e.g., temperature and/or pH).

In some embodiments, a soy plant can be used that produces an oil with a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content. See, for example, U.S. Patent No. 5,981,781, which describes soy plants that produce an oil with an oleic acid content of greater than 75% and a polyunsaturated fatty acid content of less than 10%. See also U.S. Patent No. 9,918,485, which describes soy plants that produce an oil with an oleic acid content of at least 60%. See also, the Plenish® high oleic soybeans from Pioneer. Typical soybean plants produce oils with high levels of polyunsaturated fatty acids and the oil is more prone to oxidation than oils with higher levels of monounsaturated and saturated fatty acids.

In some embodiments, a soy plant can be used that produces an oil with a high saturated fatty acid content. See, for example, U.S. Patent No. 6,365,802, which describes soy plants that produce an oil with 40% or more stearic acid (e.g., 50% stearic acid). U.S. Publication No. 2004/0049813 describes soy plants that produce an oil with a combined palmitic acid and stearic acid content of greater than 21%, an oleic acid content of greater than 60% and a polyunsaturated fatty acid content of less than 7%. Higher saturated fatty acid content typically is associated with animal fat (typically 22-37% C16:0 and 8-30% C18:0) and/or palm oil (typically 39-48% C16:0 and 3.5-6% C18:0), and may impart a different flavor profile to the heme or other proteins isolated from the plant.

In some embodiments, a soy plant can be used that has a higher protein content. For example, International Publication No. WO 2020/106488 describes a soy plant with decreased expression or activity of one or more HECT E3 ligases (HEL) that has an increased protein content. Specifically, knocking out HELI, or both HELI and HEL2 increased seed protein content significantly compared to wild type plants, and knocking out both HELI and HEL2 showed higher seed protein content than the HELI knockout. In addition, the knockout of HELI showed increased oleic and stearic acid contents and reduced linolenic, palmitic and stachyose contents. Knockout of both HELI and HEL2 increased oleic content and reduced linoleic, linolenic, palmitic, stearic, stachyose, and total soluble carbohydrate contents.

In some embodiments, at least one characteristic selected from: (A) increased oleic acid content, (B) decreased linoleic acid content, (C) decreased linolenic acid content, (D) decreased stearic acid content, and (E) decreased palmitic acid content, can be obtained in seeds of plants by altering expression or activity of one or more ELECT E3 ligase (HEE) polypeptides (e.g., decreasing expression or activity). Modifications can include deletions, insertions, or substitution into one or more ELECT E3 ligase genes. See, for example International Publication No. WO 2020/106488.

In some embodiments, the fatty acid profile of a plant can be modified by breeding or genetic engineering. See, for example, Nguyen, et al., Curr Genomics, 17(3):241-260 (2016), Clemente and Cahoon, Plant Physiol., 151(3): 1030-1040 (2009), and U.S. Patent No. 6,323,392. For example, to increase oleic acid content and reduce linoleic acid content, a plant can have decreased activity of delta- 12 desaturase, which converts oleic acid to linoleic acid. For example, to increase saturated fatty acids, the plants can be modified such that they contain increased oleoyl- or stearoyl-ACP thioesterase activity and decreased fatty acid desaturase activities, including delta-9, delta-12, and delta-15 desaturase activities. Plants also can be modified such that they contain increased 3 -ketoacyl -ACP synthase II (KAS II). Increased thioesterase activity may not be necessary if delta-9 desaturase activity is completely inhibited. Plants also can exhibit increased palmitoyl-ACP thioesterase activity.

In some embodiments, the expression and/or activity of one or more enzymes that act on lipids or fatty acids in a plant can be modified by breeding or genetic engineering. For example, lipases hydrolyze lipids into free fatty acids, which can generate off-flavors upon oxidation. Lipoxygenases, such as 9-lipoxygenases or 13 -lipoxygenases, catalyze the dioxygenation of polyunsaturated fatty acids, such as linoleic acid, alpha-linolenic acid, or arachidonic acid, into hydroperoxides. Hydroperoxide lyases can catalyze the cleavage of C-C bonds in hydroperoxides of fatty acids, which can result in the formation of aldehydes. Such aldehydes, for example, hexenal, hexanal, nonenal, nonanal, or nonadienal, can contribute off-flavors or aromas or undergo isomerization, dehydrogenation, reduction to alcohols, or oxidation to esters. For example, to decrease off-flavors or aromas and/or to improve oxidative stability, a plant can be modified to have decreased expression and/or activity of one or more lipases, lipoxygenases, and/or hydroperoxide lyases. In some embodiments, plants can have a desired fatty acid profile (e.g., a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, or a high saturated fatty acid content) and decreased expression and/or activity of one or more lipases, lipoxygenases, and/or hydroperoxide lyases. In some embodiments, a plant, bean, or seed as described herein can have a lower expression and/or activity (e.g., reduced by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) of a lipase, lipoxygenase, hydroperoxide lyase, or a combination thereof as compared to a corresponding control plant, bean, or seed. Without being bound by any particular theory, it is believed that in some embodiments, a low lipase, lipoxygenase, and/or hydroperoxide lyase activity can yield a low flavor plant (and/or protein product produced therefrom). See, for example International Publication No. WO 2017/218883.

In some embodiments, hybrids of plants with modified FAD-2 or FAD-3 enzymes can result in altered fatty acid profiles. For example, a hybrid between a female Brassica napus parent with a homozygous modified FAD-2 (delta- 12 fatty acid desaturase 2) in either A- or C-genome and a male Brassica napus plant with a homozygous modified FAD-2 in both the A- and C-genomes can result in progeny that produce elevated oleic acid (C18: l) of at least 80% by weight and reduced linolenic acid (C18:3) content of no more than 3% by weight of total fatty acid content in their seeds. See, for example, U.S. Patent No. 6,323,392. In another example, oleic acid content of seeds is increased to between 70% and 90% and linolenic acid content is decreased to less than about 3% by weight of the total fatty acids in the seed of a plant (e.g., a soybean, canola, or sunflower plant) with four or more modifications to the genes encoding a FAD2 and a FAD3 polypeptide, such as FAD2-1 A, FAD2-1B, FAD3a, and FAD3b compared to the seed of a corresponding control plant without the introduced genetic modifications. See, for example International Publication No. WO 2019/173125.

For example, to increase production of medium-chain fatty acids, which are typically found in coconut oil, a thioesterase (e.g., the Umbellularia californica Nutt. FATB1) that releases medium-chains fatty acids such as C12:0 and C14:0 from the plastid fatty acid synthetic pathway can be expressed in a plant (e.g., a soy plant). See, for example, Hu, et al.. PLosS One, 12(2):e0172296 (2017), and Voelker, et al., Science, 257(5066):72-4 (1992).

In some embodiments, plants having a desired fatty acid profile (e.g., high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content such as the Plenish® soybeans, a high medium chain fatty acid content, or a high saturated fatty acid content) can be used as a source for textured vegetable protein (e.g., textured soy protein) to improve the flavor profile and/or functional characteristics of the protein (e.g., one or more of reduced off-flavors, altered gel-strength, altered viscosity, improved oxidative stability, and improved whiteness). Methods for making textured vegetable protein (e.g., textured soy protein) having reduced off-flavors can include isolating protein from soybeans that yield an oil having a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content and processing the soy protein to obtain textured soy protein (e.g., forming the soy protein into desired shape by extrusion, or cooking the soy protein and spray drying).

In some embodiments, a plant has a low polyunsaturated fatty acid (e.g., linolenic acid and/or linoleic acid) content. In some embodiments, a plant has a low linolenic acid content. In some embodiments, a plant has a high monounsaturated fatty acid content. In some embodiments, a plant has a high oleic acid content. In some embodiments, a plant has a low polyunsaturated fatty acid content and a high monounsaturated fatty acid content. In some embodiments, a plant has a low linolenic acid content and a high oleic acid content. In some embodiments, a plant has a low polyunsaturated fatty acid content and a high oleic acid content. In some embodiments, a plant has a low linolenic acid content and a high monounsaturated fatty acid content. In some embodiments, a plant has a low unsaturated fatty acid content. In some embodiments, a plant has a high saturated fatty acid content. Without being bound by any particular theory, it is believed that in some embodiments, a low linolenic acid content can yield a low flavor plant (and/or protein product produced therefrom). As used herein, “low flavor” with respect to a plant and/or protein product means that the plant and/or protein product has less flavor than the source of the plant and/or protein product (e.g., soy, if a soy plant and/or protein product is described). For example, less of one or more compounds that give rise to a distinguishing flavor associated with the source of the protein. In some embodiments, a low flavor plant and/or protein product can have little flavor of its own. In some instances, a low flavor plant and/or protein product has less flavor than a known plant and/or protein product (e.g., a commercial soy protein isolate, such as those described herein). Having less flavor can be determined, for example, by a trained human panelist, or, for example, by measurement of one or more volatile compounds commonly understood to impart flavor and/or aroma. In some embodiments, a different fatty acid may be increased, for example, a saturated fatty acid, such as stearic acid or palmitic acid, or a combination of one or more saturated fatty acids.

A transformed cell, callus, tissue, or plant can be identified and isolated by selecting or screening the engineered plant material for particular polypeptides or activities, e.g., those encoded by marker genes or antibiotic resistance genes. Such screening and selection methodologies are well known to those having ordinary skill in the art. In addition, physical and biochemical methods can be used to identify transformants. These include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, SI RNase protection, primer-extension, quantitative realtime PCR, or reverse transcriptase PCR (RT-PCR) amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are well known. After a polynucleotide is stably incorporated into a transgenic plant, it can be introduced into other plants using, for example, standard breeding techniques. A population of transgenic plants can be screened and/or selected for those members of the population that produce the target product. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of the target polypeptide or nucleic acid encoding the target polypeptide.

A plant or plant cell can be transformed by having a construct integrated into its genome, i.e., can be stably transformed. Stably transformed cells typically retain the introduced nucleic acid with each cell division. A plant or plant cell also can be transiently transformed such that the construct is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced nucleic acid construct with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.

Transgenic plant cells used in methods described herein can constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. As used herein, a transgenic plant also refers to progeny of an initial transgenic plant provided the progeny inherits the transgene. As used herein, a transgenic plant also refers to progeny of an initial transgenic plant provided the progeny inherits the transgene. "Progeny" includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on Fi, F2, F3, F4, F5, Fe and subsequent generation plants, or seeds formed on BCi, BC2, BC3, and subsequent generation plants, or seeds formed on F1BC1, F1BC2, F1BC3, and subsequent generation plants. The designation Fi refers to the progeny of a cross between two parents that are genetically distinct. The designations F2, F3, F4, F5, and Fe refer to subsequent generations of self- or sib-pollinated progeny of an Fi plant. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct.

Methods of Producing Heme-Loaded Heme Polypeptides

The transgenic plants or plant cells described herein can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field, and then the heme-loaded polypeptide can be isolated. In some embodiments, the heme-loading of recombinant heme-containing polypeptides can be increased using the methods and transgenic plants, cells, and seeds described herein. In some embodiments, overall heme-loaded heme protein concentration can be increased in the transgenic plants, cells, and seeds described herein. In some embodiments, increased heme loading caused by the upregulation of heme biosynthesis can increase overall heme-loaded protein accumulation in the transgenic plants, cells, and seeds described herein. In some embodiments, increased heme biosynthesis allows the accumulation of more heme-loaded protein than a corresponding control plant.

As used herein, a “corresponding control plant” is a similar plant except lacking the transgene or not expressing the transgene. For example, a corresponding control plant of a transgenic plant that has a gene encoding a heme-containing polypeptide expressed in seeds would be the same in all aspects (e.g., genetic makeup of plant and so forth), except that the transgenic plant would express a heme-containing polypeptide in the seeds that the corresponding control plant does not.

For example, using the methods described herein, the heme-loaded hemecontaining polypeptide can be present in one or more plant tissues, e.g., seeds, vegetative tissues, reproductive tissues, or root tissues, at increased levels relative to that of corresponding control plants that do not express the polypeptide that upregulates heme biosynthesis. For example, the heme-loading of a recombinant heme-containing polypeptide can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, as compared to the heme-loading of the heme-containing polypeptide in a corresponding control plant that does not express the polypeptide that upregulates heme biosynthesis. In some embodiments, the heme loaded heme-containing polypeptide can be at least 0.01%, 0.05%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of total seed protein in seeds of the transgenic plant. Similarly, in some embodiments, the heme loaded hemecontaining polypeptide can be at least 0.01%, 0.05%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of total protein from other plant tissues (e.g., roots, leaves, etc.).

In some embodiments in which an inducible promoter is used, the transgenic seeds can be germinated in a contained system in the presence of an inducer and the heme-loaded polypeptide can be isolated from the seedlings. As used herein, a “contained system” refers to a system for germinating transgenic seeds in bulk in a controlled environment (e.g., controlled temperature, light, moisture, fertilizer, and/or humidity) such as a malting or hydroponic system. Germinating seeds in a greenhouse or under field conditions is not considered to be a contained system. See, for example, U.S. Publication No. 2019/0292555 and International Publication No. WO 2018/102656.

To isolate the heme-loaded heme protein from the transgenic plants, cells, or seeds, the plant material can be processed using any appropriate measure including, for example, grinders, hammer mills, shredders, chippers, screwpress, or high pressure homogenization. The heme-loaded proteins can be separated on the basis of their molecular weight, for example, by size exclusion chromatography, ultrafiltration through membranes, or density centrifugation. In some embodiments, heme-loaded proteins can be separated based on their surface charge, for example, by isoelectric precipitation, anion exchange chromatography, or cation exchange chromatography. Heme-loaded proteins also can be separated on the basis of their solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents or solvent extraction. Heme-loaded proteins also can be separated by their affinity to another molecule, using, for example, hydrophobic interaction chromatography, reactive dyes, or hydroxyapatite. Isoelectric points of target proteins, such as heme-loaded proteins, can be modified by altering the charge exposed amino acid residues to facilitate separation (see, for example, U.S. Patent No. 9,828,429).

In some embodiments, heme loaded proteins can be extracted in native form as described in WO 2016/054375. For example, a heme-loaded protein can be extracted from the processed plant material with an aqueous solution containing polyethylene glycol (PEG) (e.g., PEG having a MW of 8000) and, optionally, a flocculant such as an alkylamine epichlorohydrin, to generate an extraction slurry that contains bulk solids and an extract; optionally adjusting the pH of the extraction slurry to a pH of 2 to 10; collecting the extract and adding salt such as magnesium sulfate to form a two-phase mixture, separating the two-phase mixture using, for example, gravity settling or centrifugation (e.g., using a disk stack centrifuge) to generate a PEG phase and a product phase; and collecting and filtering (e.g., microfiltering) the product phase to generate a filtered product phase that contains the protein. The filtered product phase can be concentrated and diafiltered to generate a target product concentrate. A product concentrate can be sterilized, e.g., by UV irradiation, pasteurization, or microfiltration, and dried, e.g., by spray drying or a freeze drying under mild conditions.

In some embodiments, methods of preparing a protein composition (e.g., a protein concentrate, a protein isolate, a low flavor protein composition, or a low color protein composition) can include (a) adding an aqueous solution to a source protein composition (e.g., a protein composition from plants, cells, seeds, or other plant parts, or from bacteria, fungi) to form a solution of solubilized protein; (b) optionally removing solids from the solution of solubilized protein; (c) adding an organic solvent (e.g., methanol or ethanol) to the solution of solubilized protein to form a solid phase and a liquid phase, and (d) separating the solid phase from the liquid phase to form a protein composition. See, for example, PCT/US2021/020356, entitled “Materials and Methods for Protein Production” and filed March 1, 2021, U.S. Provisional Application No. 62/993,675, entitled “Materials and Methods for Protein Production” and filed on March 23, 2020, and U.S. Provisional Application No. 62/983,558, entitled “Materials and Methods for Protein Production” and filed on February 28, 2020.

In some embodiments, methods for purifying the protein minimize the development of undesirable odors and flavors in the purified protein, enhance functionality and increase protein yield. Such methods can include, for example, lysing an aqueous suspension of a plurality of cells to obtain a cell lysate; clarifying the cell lysate, optionally in the presence of one or more flocculants, to obtain a clarified lysate; filtering the clarified lysate to obtain a filtered lysate; concentrating the filtered lysate to obtain a protein composition; and optionally pasteurizing the protein composition of protein to obtain a pasteurized protein composition, wherein the lysing, clarifying, and filtering steps, independently, are performed at a pH between about 8.5 and about 12.0. The method optionally can include any one or more of the following: filtering (e.g., microfiltration, ultrafiltration, diafiltration, or a combination thereof), clarifying (e.g., by centrifugation to less than about 20% dry solids), or washing an aqueous suspension of a plurality of cells at a pH between about 8.5 and about 12.0 before step a). See, U.S. Patent Application No. 16/138,876, entitled “Methods for Purifying Protein” and filed on September 21, 2018. In some methods for purifying proteins from a plurality of cells having cell walls, the method including: a) perforating the cell walls of the plurality of cells, b) separating an aqueous suspension of the plurality of cells to form a solids portion and a liquid portion, c) filtering the liquid portion to form a filtrate and a retentate, d) concentrating the retentate to form a protein composition, and e) optionally pasteurizing the protein composition, wherein each of a)-d), independently, are performed at a pH of about 8.5 and about 12.0, and/or wherein the method results in the liquid portion including at least about 50% by weight of the cytoplasmic proteins of the plurality of cells. See, for example, PCT/US2020/050774, entitled “Protein Methods and Compositions” and filed September 14, 2020.

Additional methods for producing protein compositions, e.g., soy protein products, having reduced off-flavors can include an extraction using a mixture of supercritical carbon dioxide, and optionally an organic solvent (see, for example, U.S. Patent No. 7,638,155 and U.S. Publication No. 2008/0145511), salting-out methods, using, for example, ammonium sulfate fractionation of phospholipid-bound proteins, or cyclodextrin, for example P-cyclodextrin, treatment methods than can optionally include physicochemical treatments (see, for example, Damodaran and Arora, Annu. Rev. Food Sci. Technol. 2013. 4:327-346).

This document also relates to food products that include proteins produced and/or purified as described herein (e.g., plant-based food compositions such as plant-based meat substitutes like plant-based chicken or beef substitutes). Exemplary food products are described in, for example, U.S. Publication Numbers 20140193547(Al) and 20150305361(Al), and U.S. Patent Nos. 10,039,306; 9,700,067; 10,172,380; and 9,011,949. For example, plants having a desired fatty acid profile (e.g., a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content such as the Plenish® soybeans (U.S. Publication No. 2008/0312082), a high medium chain fatty acid content, or a high saturated fatty acid content) or other desired characteristics (e.g., pea cultivar Snak Hero (SL3192), optionally with modified fatty acid metabolism (see, for example, U.S. Patent No. 10,660,297)) can be used as a source for textured vegetable protein (e.g., textured soy protein) or other protein products (e.g., protein concentrate or protein isolate) to improve the flavor profile and/or functional characteristics of the protein (e.g., one or more of reduced off-flavors, altered gelstrength, altered viscosity, improved oxidative stability, and improved whiteness). Methods for making textured vegetable protein (e.g., textured soy protein) having reduced off-flavors can include isolating protein from soybeans that yield an oil having a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content and processing the soy protein to obtain textured soy protein (e.g., forming the soy protein into desired shape by extrusion, or cooking the soy protein and spray drying). In some embodiments, the proteins are prepared by aqueous ethanol extraction, subjected to an initial heating and holding process to re-solubilize the proteins, and optionally an enzyme hydrolysis to further modify and functionalize the proteins. Protein isolates prepared in such a manner have very low fat content and are bland tasting and have low levels of volatile compounds. See, e.g., U.S. Publication No. 2008/0008815.

For example, plants having a desired fatty acid profile (e.g., a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, or a high saturated fatty acid content) can be used as a source for lipids (including, but not limited to, oils, fats, phospholipids, triglycerides, diglycerides, monoglycerides, and free fatty acids) with a desired profile. In some embodiments, lipids can be purified separately from proteins, for example, by trapping lipids in lipid bodies and isolating the lipid bodies.

In some embodiments, food products (e.g., plant-based food compositions such as plant-based meat substitutes like plant-based chicken or beef substitutes) can include one or more proteins and/or lipids from a plant having a desired fatty acid profile (e.g., a high monounsaturated fatty acid content and/or a low polyunsaturated fatty acid content, a high medium chain fatty acid content, or a high saturated fatty acid content) and/or decreased expression and/or activity of one or more lipases, lipoxygenases, and/or hydroperoxide lyases. In some embodiments, food products are configured for consumption by humans.

In some embodiments, food products (e.g., plant-based food compositions such as plant-based meat substitutes like plant-based chicken or beef substitutes) may include one or more of metal ions or minerals (e.g., sodium, potassium, calcium, magnesium), organic acids (e.g., acetic acid, ascorbic acid, citric acid, folic acid, fumaric acid, glycolic acid, lactic acid, malic acid, succinic acid, tartaric acid), free amino acids (e.g., cysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, valine, arginine, histidine, alanine, asparagine, aspartate, glutamate, glutamine, glycine, proline, serine, tyrosine, selenocysteine, citrulline, ornithine, beta-alanine, homoserine, non- proteinogenic amino acid, amino acid derivative), peptides (e.g., dipeptides, tripeptides, tetrapeptides, oligopeptides, polypeptides), polysaccharides (e.g., alginate, konjac, curdlan, gellan, carrageenan, locust bean gum, pectin, fecula, arrowroot, cornstarch, katakuri starch, potato starch, wheat starch, rice starch, modified food starch, maltodextrin, sago, tapioca, alginin, guar gum, xanthan gum, furcellaran, agar, cellulose, methylcellulose, hydroxymethylcellulose, acacia gum, amylopectin), sugars (e.g., monosaccharides, disaccharides, sucrose, glucose, fructose, maltose, ribose, arabinose, galactose, xylose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, inositol, nucleotide-bound sugars, molasses), sugar alcohols (e.g., erythritol, glycerol, isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol, hydrogenated starch hydrolysates), nucleotides (e.g., inosine, inosine monophosphate (IMP), guanosine, guanosine monophosphate (GMP), adenosine monophosphate (AMP)), provitamins, vitamins (e.g., A, B, C, D, E, K), antioxidants, antimicrobials, preservatives, hydrolysates (e.g., vegetable protein hydrolysate, soy protein hydrolysate, yeast protein hydrolysate, algal protein hydrolysate), yeast extracts, metabolites, natural flavors, natural pigments, emulsifiers, stabilizers, thickeners, and sulfur compounds (e.g., cysteine, acetylcysteine, cystine, taurine, thiamine, methionine, glutathione, alliin, biotin). In some embodiments, food products can contain one or more heme-containing polypeptides. In some embodiments, food products may be free of one or more ingredients selected from gluten (e.g., wheat gluten), wheat-derived allergens, peanut-derived allergens, tree-nut-derived allergens, milk-derived allergens, egg-derived allergens, shellfish-derived allergens, fish-derived allergens, soy-derived allergens, sesame-derived allergens, caramel coloring, artificial coloring, artificial flavors, artificial sweeteners, high-fructose corn syrup, sugar alcohols, cholesterol, trans fats, hydrogenated oils, nitrites, and nitrates, among other ingredients. In some embodiments, food products may be free of heme-containing polypeptides from an animal source. In some embodiments, food products may be free of animal products.

In some embodiments, plant-based food compositions such as plant-based meat substitutes like plant-based chicken or beef substitutes may contain one or more ingredients derived from algae, fungi (e.g., yeast or filamentous fungi), ciliates, and/or bacteria, such as proteins, lipids, hydrolysates, or extracts.

In some embodiments, a target protein other than a heme-containing protein can be used, such as a protein that modifies other proteins (e.g., a glutaminase). In some embodiments, a target protein can include one or more enzymes in the biosynthetic pathway of a molecule of interest (e.g., a pharmaceutical (e.g., a polyketide), a porphyrin or derivative thereof (e.g., a heme), or a carbohydrate (e.g., methylcellulose)).

As used herein, a “pharmaceutical” can be any compound and/or composition useful for preventing, treating, or ameliorating a disease or a symptom thereof. In some cases, a pharmaceutical can include a protein (e.g., an antibody or antigen-binding fragment thereof or a therapeutic enzyme, such as an 1-asparaginase). In some cases, a pharmaceutical can include an organic and/or inorganic compound (e.g., a polyketide such as erythromycin).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED:

1. A transgenic plant comprising at least one recombinant nucleic acid, wherein the recombinant nucleic acid comprises

(i) a first promoter operably linked to a nucleic acid encoding a heme-containing polypeptide and;

(ii) a second promoter operably linked to a nucleic acid encoding a polypeptide that upregulates heme biosynthesis, wherein heme-loading of the heme-containing polypeptide is increased in the transgenic plant relative to that of a corresponding control plant that does not comprise the second promoter operably linked to the nucleic acid encoding the polypeptide that upregulates heme biosynthesis, and wherein the transgenic plant has a fatty acid profile selected from the group consisting of a high monounsaturated and a low polyunsaturated fatty acid content, a high medium chain fatty acid content, and a high saturated fatty acid content.

2. The transgenic plant of claim 1, wherein the first and second promoters are the same.

3. The transgenic plant of any one of claims 1-2, wherein the first and second promoters are inducible.

4. The transgenic plant of any one of claims 1-3, wherein the first and second promoters are seed specific promoters.

5. The transgenic plant of claim 4, wherein the seed specific promoter is selected from the group consisting of a soy beta-conglycinin seed specific promoter, a Gl- Glycinin seed specific promoter, a Kunitz trypsin inhibitor (KTI) promoter, and an oleosin promoter.

6. The transgenic plant of any one of claims 1-5, wherein the recombinant nucleic acid further comprises a first targeting sequence operably linked to the nucleic acid encoding the heme-containing polypeptide, and a second targeting sequence operably linked to the nucleic acid encoding the polypeptide that upregulates heme biosynthesis.

7. The transgenic plant of claim 6, wherein the first and second targeting sequences target the polypeptides to the same intracellular location within the transgenic plant.

8. The transgenic plant of any one of claims 6-7, wherein the first and second targeting sequences are the same.

9. The transgenic plant of any one of claims 6-8, wherein the targeting sequence encodes a vacuole targeting signal peptide.

10. The transgenic plant of claim 9, wherein the vacuole targeting signal peptide is a soy conglycinin vacuole targeting signal peptide, a soy glycinin vacuole targeting signal peptide, or a plant seed storage protein vacuole targeting signal peptide.

11. The transgenic plant of any one of claims 6-8, wherein the targeting sequence encodes a plastid targeting signal peptide.

12. The transgenic plant of claim 11, wherein the plastid targeting signal peptide is a RuBisCO signal peptide.

13. The transgenic plant of any one of claims 6-9, wherein the targeting sequence is a soy beta-conglycinin targeting sequence.

14. The transgenic plant of any one of claims 6-9, wherein the targeting sequence is a G1 -Glycinin targeting sequence.

15. The transgenic plant of any one of claims 1-14, wherein the polypeptide that upregulates heme biosynthesis is a ferrochelatase, a glutamyl-tRNA reductase (GluTR) binding protein, a truncated glutamate tRNA reductase protein (GTR), an aminolevulinic acid synthase, or a combination of two or more of the polypeptides.

16. The transgenic plant of claim 15, wherein the polypeptide that upregulates heme biosynthesis is endogenous to the transgenic plant.

17. The transgenic plant of claim 15, where the polypeptide that upregulates heme biosynthesis is heterologous to the transgenic plant.

18. The transgenic plant of claim 15, wherein the ferrochelatase is a barley ferrochelatase, a tobacco ferrochelatase, a soy ferrochelatase, or a microbial ferrochelatase.

19. The transgenic plant of claim 18, wherein the microbial ferrochelatase is a Bradyrhizobium ferrochelatase or an Aspergillus ferrochelatase.

20. The transgenic plant of claim 15, wherein the aminolevulinic acid synthase is a bacterial aminolevulinic acid synthase.

21. The transgenic plant of any one of claims 1-20, wherein the heme-containing polypeptide is a globin polypeptide in Pfam 00042.

22. The transgenic plant of claim 21, wherein the globin polypeptide is a plant globin polypeptide.

23. The transgenic plant of claim 21, wherein the globin polypeptide is a leghemoglobin, a non-symbiotic hemoglobin, an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a myoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a neuroglobin, a chlorocruorin, a truncated hemoglobin, a truncated 2/2 globin, a hemoglobin 3, a cytochrome, or a peroxidase.

24. The transgenic plant of any one of claims 21-23, wherein the globin polypeptide is expressed in the cytosol of the seeds of the transgenic plant cells.

25. The transgenic plant of any one of claims 21-23, wherein the globin polypeptide is expressed in the vacuole of the seeds of transgenic plant cells.

26. The transgenic plant of any one of claims 1-25, wherein the transgenic plant is a soy plant.

27. The transgenic plant of any one of claims 1-25, wherein the transgenic plant is a rice plant.

28. The transgenic plant of any one of claims 1-25, wherein the transgenic plant is selected from the group consisting of: a barley, a wheat, a com, a rye, an oat, a beet, a sugar beet, a parsnip, a bean, a leafy vegetable, a tuber, and a grass.

29. The transgenic plant of claim 28, wherein the bean is an adzuki, a mung, a pea, a peanut, a lentil, or a garbanzo.

30. The transgenic plant of claim 28, wherein the leafy vegetable is an alfalfa, an arugula, a mustard, or a Brassica.

31. The transgenic plant of claim 28, wherein the grass is triticale or spelt.

32. The transgenic plant of claim 28, wherein the tuber is a potato, a sweet potato, or a cassava.

33. The transgenic plant of any one of claims 26-32, wherein the plant has a low polyunsaturated fatty acid content.

34. The transgenic plant of any one of claims 26-33, wherein the bean or the seed has a low linolenic acid content.

35. The transgenic plant of any one of claims 26-34, wherein the bean or the seed has a high monounsaturated fatty acid content.

36. The transgenic plant of any one of claims 26-35, wherein the bean or the seed has a high oleic acid content.

37. The transgenic plant of any one of claims 26-32, wherein the bean or the seed has a low unsaturated fatty acid content.

38. The transgenic plant of any one of claims 26-37, wherein the bean or the seed has a high saturated fatty acid content.

39. The transgenic plant of any one of claims 26-32, wherein the plant has a high monounsaturated fatty acid content and a low polyunsaturated fatty acid content.

40. The transgenic plant of any one of claims 26-32, wherein the plant has a high medium chain fatty acid content.

41. The transgenic plant of any one of claims 26-32, wherein the plant has a high saturated fatty acid content.

42. Seeds of the transgenic plant of any one of claims 1-41.

43. A method of making a textured soy protein having reduced off-flavors, the method comprising obtaining a soy protein from a soybean yielding a seed oil with a high monounsaturated fatty acid content and a low polyunsaturated fatty acid content and processing the soy protein to obtain the textured soy protein.

44. The method of claim 43, further comprising a step selected from the group consisting of: extracting the soy protein with supercritical CO2, extracting the soy protein with supercritical CO2 and a subsequent extraction with an organic solvent, fractioning the soy protein with ammonium-sulfate, and treating the soy protein with a cyclodextrin.

45. A method of making a textured soy protein having reduced off-flavors, the method comprising obtaining a soy protein from a soybean yielding a seed oil with a low linolenic acid content and processing the soy protein to obtain the textured soy protein.

46. The method of claim 45, further comprising a step selected from the group consisting of: extracting the soy protein with supercritical CO2, extracting the soy protein with supercritical CO2 and a subsequent extraction with an organic solvent, fractioning the soy protein with ammonium-sulfate, and treating the soy protein with a cyclodextrin.

47. A plant-based food composition comprising a heme-containing polypeptide from the seeds of claim 42.

48. The plant-based food composition of claim 47, wherein the plant-based food composition is a meat substitute.

49. The plant-based food composition of claim 48, wherein the meat substitute is a beef substitute.

50. The plant-based food composition of claim 48, wherein the meat substitute is a chicken substitute.

51. A plant-based food composition comprising a soybean protein, wherein the soybean protein is isolated from a soybean plant that yields a seed oil with a high monounsaturated fatty acid content and a low polyunsaturated fatty acid content.

52. A plant-based food composition comprising a soybean protein, wherein the soybean protein is isolated from a soybean plant that yields a seed oil with a low linolenic acid content.

53. The plant-based food composition of claim 51 or claim 52, wherein the soybean protein is a textured soybean protein.

54. The plant-based food composition of claim 51 or claim 52, further comprising a heme-containing polypeptide from the seeds of claim 42.

55. The plant-based food composition of any one of claims 51-54, wherein the plantbased food composition is a meat substitute.

56. The plant-based food composition of claim 55, wherein the meat substitute is a beef substitute.

57. The plant-based food composition of claim 55, wherein the meat substitute is a chicken substitute.

58. A genetically-modified plant comprising at least one genetic modification, wherein the genetic modification comprises an inversion of an endogenous nucleic acid, wherein the inversion results in

(i) a nucleic acid encoding a heme-containing polypeptide being operably linked to a nucleic acid encoding a seed-specific signal peptide and a seed-specific promoter of a seed-storage protein, and

(ii) a nucleic acid encoding the seed-storage protein being operably linked to a promoter of the heme-containing polypeptide, wherein the heme-containing polypeptide is expressed in seeds of the plant and the seed-storage protein is expressed in root nodules of the plant.

59. The genetically modified plant of claim 58, wherein the heme-containing polypeptide is a leghemoglobin.

60. The genetically modified plant of claim 58, wherein the nucleic acid encoding the heme-containing polypeptide has about 80% sequence identity to SEQ ID NO: 1.

61. The genetically modified plant of claim 58, wherein the nucleic acid encoding the seed-storage storage protein is a beta-conglycinin subunit.

62. The genetically modified plant of claim 61, wherein the beta-conglycinin subunit is the beta-conglycinin alpha prime subunit 2.

63. The genetically modified plant of claims 58-62, wherein the heme-containing polypeptide is expressed in the protein storage vacuole of the seeds of the genetically modified plant.

64. The genetically modified plant of claims 58-62, wherein the genetically modified plant is a soy plant.

65. The genetically modified plant of claims 58-62, wherein the genetically modified plant is a rice plant.

66. The genetically modified plant of claims 58-62, wherein the genetically modified plant is selected from the group consisting of a barley, a wheat, a corn, a rye, an oat, a beet, a sugar beet, a parsnip, a bean, a leafy vegetable, a tuber, and a grass.

67. The genetically modified plant of claim 66, wherein the bean is an adzuki, a mung, a pea, a peanut, a lentil, or a garbanzo. The genetically modified plant of claim 66, wherein the leafy vegetable is an alfalfa, an arugula, a mustard, or a Brassica. The genetically modified plant of claim 66, wherein the grass is triticale or spelt. The genetically modified plant of claim 66, wherein the tuber is a potato, a sweet potato, or a cassava. The genetically modified plant of any one of claims 66-70, wherein the plant has a low polyunsaturated fatty acid content. The genetically modified plant of any one of claims 66-71, wherein the bean or the seed has a low linolenic acid content. The genetically modified plant of any one of claims 66-72, wherein the bean or the seed has a high monounsaturated fatty acid content. The genetically modified plant of any one of claims 66-73, wherein the bean or the seed has a high oleic acid content. The genetically modified plant of any one of claims 66-70, wherein the bean or the seed has a low unsaturated fatty acid content. The genetically modified plant of any one of claims 66-75, wherein the bean or the seed has a high saturated fatty acid content. The genetically modified plant of any one of claims 66-70, wherein the plant has a high monounsaturated fatty acid content and a low polyunsaturated fatty acid content. The genetically modified plant of any one of claims 66-70, wherein the plant has a high medium chain fatty acid content. The genetically modified plant of any one of claims 66-70, wherein the plant has a high saturated fatty acid content. Seeds of the genetically modified plant of any one of claims 58-79. A method of making a genetically modified plant comprising: a) obtaining a plant cell comprising endogenously in the genome in the 5’ to 3’ direction: a first nucleic acid encoding a seed-storage polypeptide; a second nucleic acid comprising, in the 5’ to 3’ direction: i. a nucleic acid encoding a seed-specific signal peptide; ii. a seed-specific promoter, wherein the seed-specific promoter is operably liked to the nucleic acid encoding the seed-specific signal peptide and the first nucleic acid encoding the seed-storage polypeptide; and iii. a nodule-specific promoter sequence, and a third nucleic acid encoding a heme-containing polypeptide operably linked to the nodule-specific promoter sequence; b) cleaving, using a first site-specific nuclease, between the first nucleic acid encoding the seed-storage polypeptide and the second nucleic acid encoding the seed-specific signal peptide, and cleaving, using a second site-specific nuclease, between the nodule-specific promoter sequence and the third nucleic acid encoding the heme-containing polypeptide, c) selecting a genetically modified plant cell comprising an inversion of the second nucleic acid in its genome, wherein the genome of the genetically modified plant cell comprises in the 5’ to 3’ direction: the first nucleic acid encoding the seed-storage polypeptide; the second nucleic acid comprising, in the 5’ to 3’ direction, i. the nodule-specific promoter sequence; ii. the seed-specific promoter sequence; and iii. the seed-specific signal peptide, and the third nucleic acid encoding the heme-containing polypeptide operably linked to the seed-specific promoter sequence and the seed-specific signal peptide; and d) producing the genetically modified plant from the genetically modified plant cell. The method of claim 81, further comprising screening the genetically modified plant for expression of the first nucleic acid encoding the seed-storage protein in nodules and/or for expression of the third nucleic acid encoding the hemecontaining polypeptide in the seeds.

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