Recombinant phytoglobin

Abstract

The present invention relates to a recombinant heme or phytoglobin protein comprising C-helices and D-helices covalently linked to a CD loop region, and comprising a hexa-coordinate or penta-coordinate prosthetic heme group, wherein the heme or phytoglobin has an amino acid sequence that is at least 50% identical to the heme or phytoglobin contained in any one of SEQ ID NOs. 1 to 55, the CD loop region comprises one or more modifications compared to the parent CD loop region, thereby the modified CD loop region is more strongly bound to the prosthetic group than the parent unmodified CD loop region, the parent CD loop region is located at positions 52 to 78 of SEQ ID NOs. 1 in the recombinant heme or phytoglobin protein, optionally at positions 52 to 72 of SEQ ID NOs. The CD loop region has an amino acid sequence that is at least 50% identical to the CD loop region contained in any one of SEQ ID NOs. 56 to 147.

Description

[Technical Field]

[0001] The present invention relates to recombinant heme or phytoglobin or heme-binding proteins comprising C-helices and D-helices covalently linked to a CD loop region, and comprising six-coordinate or five-coordinate prosthetic heme groups. This includes six-coordinate globins and phytoglobins derived from the Caryophyllaceae family. The present invention also relates to compositions comprising the recombinant protein, non-therapeutic and therapeutic methods using the recombinant protein or the composition, methods for administering the recombinant protein, genes and gene constructs encoding the recombinant protein, host cells and cultures expressing the recombinant protein, and methods for producing the recombinant protein by culturing the host cells and cultures. Furthermore, the present invention relates to compositions comprising six-coordinate heme or phytoglobin proteins, and to various uses of compositions comprising six-coordinate heme or phytoglobin proteins and / or recombinant heme or phytoglobin proteins for humans and animals. [Background technology]

[0002] Iron is an essential element with vital functions such as oxygen transport, DNA synthesis, and muscle metabolism. According to the WHO, iron deficiency is a major nutritional deficiency affecting more than 2 billion people. It is the leading cause of anemia, which is the most prevalent nutritional deficiency in the world, affecting 33% of non-pregnant women, 40% of pregnant women, and 42% of children worldwide.

[0003] Iron deficiency results from the depletion of iron stores and occurs when iron absorption cannot keep up with the metabolic demands for iron necessary for growth maintenance and replenishment of iron loss (mainly associated with blood loss) over a long period of time. The main causes of iron deficiency include insufficient intake of highly bioavailable iron, increased iron requirements associated with rapid growth, pregnancy, and menstruation, and excessive blood loss due to illness or trauma.

[0004] When iron stores are depleted and sufficient iron is not supplied for red blood cell production, hemoglobin synthesis in red blood cell progenitor cells is inhibited, leading to hematological signs of iron deficiency and anemia (IDA).

[0005] Currently, IDA is treated with iron supplements, but the iron used in almost all of these products is inorganic iron, i.e., elemental iron. Elemental iron has low absorption rates, and most of it generates reactive oxygen species that damage cells, tissues, and organs as it passes through the digestive system, causing side effects such as digestive disorders, abdominal pain, nausea, and constipation. Furthermore, it leaves a metallic aftertaste in the patient's mouth. As a result, fewer than 4 out of 10 patients complete treatment, and the rest suffer from persistent iron deficiency or anemia. It is noteworthy that IDA is a recurrent disease prone to relapse even after recovery, making continuous iron supplementation necessary for these affected individuals.

[0006] The primary and best source of iron for humans is found in animal-derived foods, particularly red meat. There, iron exists in its organic form, heme iron, bound to proteins (hemoglobin and myoglobin). However, because heme iron has a loose binding relationship with apolipoproteins (due to its five-coordinate structure), it is easily released and can cause toxic tumorigenic reactions (explained in Example 1).

[0007] Five-coordinate phytoglobins readily lose heme and are rapidly oxidized in the oral cavity. This is the reason for the "meaty flavor," but it also has harmful effects on health. Heme, which becomes free heme in the early stages of digestion before reaching the iron absorption regions (duodenum and upper jejunum), is toxic and plays a central role in colorectal cancer. The processing of these meat products accelerates the release and accumulation of free heme in the final product, and these characteristics are the main reason why the WHO classifies red meat as "possibly carcinogenic" and processed red meat as "carcinogenic."

[0008] Bai et al. (2016) reported recombinant expression of the non-symbiotic hemoglobin family gene SoHb in Arabidopsis. However, heme-binding globin or phytoglobin is expressed in plants to mitigate cellular stress, such as abiotic nitrate stress. Therefore, plants are not a reliable source of phytoglobin, as it is only detected or absent in plants at very low concentrations (1-20 μmol per kg of fresh body weight in stressed plants). Furthermore, plants contain iron inhibitors such as phenolic compounds and phyticates, which are detrimental to phytoglobin. Thus, there remains a need for a safe and tolerable iron source suitable for fortification and / or supplementation, while avoiding the discomfort associated with elemental iron supplementation and the unhealthy effects of iron oxidation. [Overview of the Initiative]

[0009] One objective of the present invention is to provide an improved heme iron-containing protein that can enhance efficacy and convenience when administered to the target population.

[0010] Another objective of the present invention is to overcome the aforementioned problems associated with conventional dietary iron sources and to meet the demand for safe, tolerable dietary iron sources suitable for supplementation.

[0011] The recombinant and modified recombinant proteins containing heme iron provided herein exhibit improved stability and superior bioavailability compared to conventional iron supplements. Furthermore, the inventors have identified phytoglobins containing a hexa-coordinate heme group and having at least 86% identity with amino acid sequence number 1, or fragments, variants, or fusions thereof that retain a hexa-coordinate heme group, exhibiting bioavailability equivalent to myoglobin while simultaneously showing remarkably improved stability, as well as compositions containing such hexa-coordinate phytoglobins and their uses.

[0012] Thus, a first aspect of the present invention provides a recombinant heme or phytoglobin protein comprising a C-helix and a D-helix covalently linked in the CD loop region and comprising a hexacoordinate or pentacoordinate heme group lacking a molecule. Here, the heme or phytoglobin has an amino acid sequence that is at least 50% identical to the heme or phytoglobin contained in any one of SEQ ID NOs: 1 to 55.

[0013] In a further aspect, the present invention provides a composition comprising the recombinant protein disclosed herein and one or more carriers, drugs, additives and / or excipients.

[0014] In a further aspect, the present invention provides a non-therapeutic method for improving the endurance or hypoxia tolerance of a subject, the method comprising administering to the subject an effective amount of the recombinant protein or composition disclosed herein to improve the endurance or hypoxia tolerance of the subject.

[0015] In a further aspect, the present invention provides a method for treating, ameliorating or preventing a disease or deficiency in a subject or a recombinant protein or composition for use therein, the method comprising administering to the subject an effective amount of the recombinant protein or composition to ameliorate or prevent the disease or deficiency.

[0016] In a further aspect, the present invention provides a gene encoding the recombinant protein disclosed herein.

[0017] In a further aspect, the present invention provides a polynucleotide construct comprising the nucleotide sequence described herein operably linked to a control sequence that directs transcription and / or translation of the gene into the recombinant protein.

[0018] In a further aspect, the present invention provides a genetically modified host cell that expresses the gene or polynucleotide construct and recombinant protein disclosed herein.

[0019] In a further aspect, the present invention provides a method for producing a recombinant protein described herein, comprising the following steps: a) culturing a cell culture of the present disclosure under conditions that allow the production of the recombinant protein; and b) recovering and / or isolating the recombinant protein, if desired.

[0020] In a further aspect, the present invention provides a composition comprising a cell culture disclosed herein.

[0021] In a further aspect, the present invention relates to a recombinant heme or phytoglobin protein having a hexacoordinate heme group and having at least 86% identity with amino acid sequence number 1, or a fragment, variant, or fusion thereof that retains the hexacoordinate heme group.

[0022] In a further aspect, the present invention relates to a composition comprising at least one recombinant hexacoordinate heme or phytoglobin protein described herein.

[0023] A further aspect relates to the use of a recombinant hexacoordinate heme or phytoglobin protein according to the present invention, and / or a composition comprising said hexacoordinate heme or phytoglobin protein, for various applications, such as in the treatment, amelioration, or prevention of iron deficiency and / or anemia in a subject.

[0024] A further aspect of the present invention relates to a non-recombinant heme or phytoglobin protein comprising a hexacoordinate heme group and having at least 86% sequence identity with amino acid sequence number 1, or a fragment thereof that retains the hexacoordinate heme group, and compositions comprising said non-recombinant hexacoordinate heme or phytoglobin protein and their use. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] [Figure 1]It is a diagram of the production of recombinant plant hemoglobin. (A) Escherichia coli cells containing recombinant expressed hemoglobin. (B) Cell lysate after sonication. (C) Purified recombinant hemoglobin. (D) It is a diagram of SDS-PAGE showing the purification process. M: Protein size marker, 1: Cell lysate, 2: IEX purification, 3: HIC purification, 4: IEX purification. [Figure 2] It is a graph of the auto-oxidation of plant hemoglobin. It is a graph showing the change over time of the auto-oxidation of plant hemoglobin. The data shown corresponds to the change in absorbance monitored at 425 nm. Bv: Beet (Beta vulgaris), Chq: Quinoa (Chenopodium quinoa), so: Spinach (Spinacia oleracea). See Example 1. [Figure 3] It is a graph showing the melting temperature (Tm) of plant hemoglobin. Graphical display of Tm observed by the formation of a shoulder on the curve at the temperature where fluorescence increases due to protein denaturation scattering. So: Spinach, Bv: Beet, Zm: Maize, BtMg: Bovine myoglobin, Chq: Quinoa. See Example 2. [Figure 4] It is a graph showing DNA degradation by hemoglobin. A. An agarose gel showing the amount of degraded DNA. B. Shows the ratio of SC pDNA after incubation with oxidized hemoglobin. rHbA: Recombinant adult human hemoglobin, Bv: Beet (Beta vulgaris), Cq: Quinoa (Chenopodium quinoa), So: Spinach (Spinacia oleracea). See Example 3 and Table 3. [Figure 5] It is a graph showing the iron bioavailability of plant hemoglobin. Results of the Caco-2 cell assay are shown. A, Ferritin formation by pure hemoglobin. B, Ferritin formation in pure hemoglobin and food mixtures. So, Spinach; Chq, Quinoa; Zm: Maize; BtMg: Beef myoglobin; BV: Beet; CBF: Canned baby food; PBS: Plant-based beverage. n = 3, p < 0.05. See Example 4 and Table 4. [Figure 6]It shows that hemoglobin synthesis consists of two steps. The first step is the synthesis of the heme b group, and the second step is followed by the synthesis of an apoprotein (globin protein) that holds the heme group. The ring system in heme synthesis is that of protoporphyrin IX. This prosthetic molecular group is firmly but non-covalently bound within the globin (apoprotein), which is the host protein. [Figure 7] It is a diagram showing the structure of six-coordinate heme-bound globin. The CD loop is present within the circle. Iron is located at the center of the heme group coordinated by two histidine side chains. [Figure 8] It shows the phylogenetic tree of different types and classes of heme or phytoglobin proteins. (A) This phylogenetic tree was constructed using only the CD loop (SEQ ID NO: 56 - 110) of heme or phytoglobin proteins. (B) In this phylogenetic tree, the CD loops of heme and phytoglobin proteins are replaced with the parental CD loop SEQ ID NO: 56. [Figure 9] It shows the expression and production of heme-binding proteins each containing a modified CD loop. From the upper left in order from left to right, top to bottom, the heme-binding proteins are as follows: A1_H_01; A2_H_02; A3_H_03; A4_H_04; A5_H_05; A6_H_06; B1_H_07; B2_H_08; B3_H_09; B4_H_10; B5_H_11; B6_H_12; C_1_H_13; C2_H_14; C3_H_15; C4_H_16; C5_H_17; C6_18; D1_H_19; D2_H_20; D3_H_21; D4_H_22; D5_H_23; D6_H_24 (SEQ ID NO: 11 - 134).

Modes for Carrying Out the Invention

[0026] Definitions The term "phytoglobin" is used to mean the globular proteins of plants (algae and terrestrial plants) containing a heme-deficient molecular group. These proteins are also known as plant hemoglobins, and these terms are used interchangeably herein.

[0027] As used herein, the term “heme-binding protein” refers to a globular protein having a heme prosthetic group containing at least one iron atom.

[0028] Heme group: Heme is a porphyrin molecule that holds iron molecules. Phytoglobin and heme-binding proteins typically have a heme b prosthetic group.

[0029] Iron Deficiency: In this context, the term "iron deficiency" should be understood as a condition in which the physiological requirements for iron are not met by dietary iron absorption, or a condition in which iron absorption cannot keep up with the metabolic demands for iron necessary for growth or maintaining pregnancy, and primarily for blood loss due to menstruation, or for replenishing iron loss due to disease or trauma, over a long period of time. Symptoms are mild and may include fatigue, weakness, and headaches. If iron deficiency progresses, it can lead to iron deficiency anemia, which is the most common form of anemia.

[0030] The term "anemia" is defined as a hemoglobin concentration below a certain threshold, which varies depending on the age, sex, physiological status, smoking habits, and altitude of residence of the evaluation population. The WHO defines anemia in children under 5 years of age and pregnant women as a hemoglobin concentration <110 g / L at sea level, and anemia in non-pregnant women as a hemoglobin concentration <120 g / L. Anemia can be caused by nutritional deficiencies, but it can also be caused by other chronic or acute diseases or conditions. In the context of this application, the terms "iron deficiency," "iron deficiency anemia," and "anemia" are used interchangeably and all refer to a condition in which increased dietary iron intake is beneficial.

[0031] Sequence Identity: The relationship between two amino acid sequences or two nucleotide sequences is described by a parameter called “sequence identity.” In this context, the term “sequence identity” is defined as sequence identity at the amino acid level between proteins. Protein sequence identity can be determined by comparing the amino acid sequences at specific positions in each sequence when the sequences are aligned. To determine the identity percentage of two amino acid sequences, the sequences are aligned for optimal comparison. Then, the amino acid residues at the corresponding amino acid positions are compared. If the position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The identity percentage between two sequences is a function of the number of identical positions shared between the sequences (i.e., identity percentage = number of identical positions / total number of positions (e.g., duplicate positions) x 100). Sequence identity between two amino acid sequences can be determined, for example, using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453). This algorithm is implemented in the Needle program (preferably version 5.0.0 or later) of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277). The parameters used are a gap start penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The identity percentage, labeled as "longest identity" in Needle (obtained using the -no brief option), is used and calculated as follows: (Number of identical residues x 100) / (Alignment length - Total number of gaps within alignment). In this context, sorting was performed using NCBI's Protein Blast (blastp) and the matrix BLOSUM62.

[0032] Variant: The term "variant" means a polypeptide having a hexacoordinate heme group and including changes such as substitution, insertion, and / or deletion at one or more (e.g., several) positions.

[0033] Substitution means replacing the amino acid occupying a certain position with a different amino acid. Deletion means removing the amino acid occupying a certain position. Insertion means adding one or more (e.g., several) amino acids adjacent to and immediately following the amino acid occupying a certain position. In some embodiments of the present invention, the variant includes only conservative substitutions. Conservative substitution means the case where an amino acid residue is substituted with an amino acid having similar chemical properties. For example, when serine is substituted with threonine, or when leucine is substituted with isoleucine, both are regarded as conservative substitutions.

[0034] Fragment: The term "fragment" as used herein refers to a polypeptide in which one or more (e.g., several) amino acids are missing from the amino terminus and / or carboxyl terminus of any one of the mature polypeptides of any of the parent sequences disclosed herein, such as SEQ ID NO: 5, 7, 9, and 10, and the fragment has a hexacoordinate heme group. In one aspect, the fragment includes at least 200 consecutive amino acid residues of SEQ ID NO: 1 - 55, e.g., at least 300 consecutive amino acid residues of SEQ ID NO: 5, 7, 9, and 10, or at least 60 consecutive amino acid residues, or at least 80 consecutive amino acid residues, or at least 100 consecutive amino acid residues.

[0035] Fusion polypeptide: The term “fusion polypeptide” or “fusion” refers to a polypeptide in which one polypeptide is fused to the N-terminus or C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to the polynucleotide of the present invention. Techniques for producing fusion polypeptides are well known in this field and involve ligating polypeptide-encoding sequences so that they are in a frame and the expression of the fusion polypeptide is controlled by the same promoter and terminator. Fusion polypeptides can also be constructed using intern techniques in which the fusion polypeptide is produced post-translation (Cooper et al., 1993, EMBO J.12:2575-2583; Dawson et al., 1994, Science 266:776-779). A fusion polypeptide may further contain a cleavage site between the two polypeptides. Upon secretion of the fusion protein, this site is cleaved, releasing the two polypeptides. Examples of cleavage sites, though not limited to them, include Martin et al., 2003, J.Ind.Microbiol.Biotechnol.3:568-576; Svetina et al., 2000, J.Biotechnol.7Q:245-251; Rasmussen-Wilson et al., 1997, Appl.Environ.Microbiol.63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al. These include the sites disclosed in al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0036] Recombinant: The term "recombinant" as used with respect to cells, nucleic acids, proteins, or vectors means that they have been modified from their natural state. Thus, for example, a recombinant cell expresses a gene not found in a natural (non-recombinant) cell or expresses it at a different level or under different conditions than the natural gene. A recombinant nucleic acid has one or more nucleotides different compared to the natural sequence and / or is operably linked to a heterologous sequence (e.g., a heterologous promoter in an expression vector). A recombinant protein has one or more amino acids different compared to the natural sequence and / or is fused to a heterologous sequence. A vector containing a nucleic acid encoding a polypeptide is a recombinant vector. The term "recombinant" is synonymous with "genetically modified" and "transgenic". The recombinant polypeptides of the present invention are obtained using recombinant DNA technology. Such methods typically involve culturing host cells transformed with a recombinant DNA vector containing a DNA sequence encoding a heme or phytoglobin protein of the present invention, the DNA sequence being operably linked to appropriate expression signals and expressing the enzyme in a culture medium under conditions that allow for the expression of the heme or phytoglobin protein, and recovering the heme or phytoglobin protein from the culture broth. The DNA sequence may be integrated into the genome of the host cell. The DNA sequence may be of genomic origin, cDNA origin, synthetic origin, or any combination thereof and may be isolated or synthesized according to methods known in the prior art.

[0037] Pharmaceutically acceptable carrier: In this context, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are suitable for use in humans and / or animals without excessive harmful side effects (such as toxicity, irritation, allergic reactions, etc.) commensurate with a reasonable benefit / risk ratio.

[0038] The use of the terms "a", "an", "the", and similar reference terms in the description of the invention (especially in the following claims) should be construed to include both the singular and the plural unless otherwise stated herein or clearly contradicted by the context.

[0039] Terms such as "comprising", "comprises", "consisting", "having", "including", "at least", and "containing" are to be construed as open-ended terms (meaning "including, but not limited to") unless otherwise specified. The description of a range of values herein is merely intended as a shorthand way of referring individually to each of the individual values within the range and each individual value is incorporated into the specification as if it were individually set forth herein. All of the methods described herein can be performed in any suitable order unless otherwise specified or clearly inconsistent from the context. The use of any example or exemplary language (e.g., "such as") in this specification is merely intended to clarify the invention and does not constitute a limitation on the scope of the invention unless otherwise claimed. No representation in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0040] References to "about" a value or parameter herein include embodiments that are directed to the value or parameter itself. For example, a description of "about X" includes the embodiment "X". When used in combination with a measured value, "about" includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value and may include a range of plus or minus two standard deviations from the stated value.

[0041] Similarly, a reference to a gene or polypeptide "derived from" another gene or polypeptide X includes the gene or polypeptide X itself.

[0042] In this specification and the appended claims, the singular forms "a", "or", and "the" include plural referents unless the context clearly dictates otherwise.

[0043] The embodiments described herein are to be understood as including "consisting of" and / or "consisting essentially of" embodiments. As used herein, the term "comprises" or "comprising", and variations thereof, are used in an inclusive sense, unless the context clearly dictates otherwise or requires a contrary meaning by express language or necessary implication. That is, they identify the presence of the recited features, but do not preclude the presence or addition of further features in various embodiments.

[0044] As used herein, the terms "CD loop region", "CD variable loop region", or "variable loop region" refer to the region in a heme or a phytoglobin that extends from the end of the C-helix to the start of the E-helix, and these terms are used interchangeably.

[0045] As used herein, the terms "six-coordinate" or "five-coordinate" refer to the coordination of the heme iron in a heme or a phytoglobin with one histidine (five-coordinate) or two histidine amino acids, or another amino acid that binds the heme group to the apoprotein (six-coordinate).

[0046] As used interchangeably herein with respect to nucleotides, polypeptides, and cells, the terms "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents refer to entities "derived from a different species or cell." For example, a heterologous or recombinant polynucleotide gene is a gene within a host cell that does not naturally contain that gene, i.e., the gene is derived from a species or cell type different from the host cell. A heterologous or recombinant polypeptide refers to a polypeptide produced in a host cell that does not naturally contain the polypeptide, i.e., the polypeptide is derived from a species or cell type different from the host cell. When these terms are used herein with respect to a host cell, they refer to a host cell that contains and expresses a heterologous or recombinant polynucleotide. In some embodiments, the term "recombinant" or "non-native" when used, for example, with respect to a host cell, nucleic acid, or polypeptide, means a substance that has been modified in a way not found in nature or is equivalent thereto but produced or derived from synthetic materials and / or obtained by manipulation using recombinant techniques, or a substance corresponding to its native or natural form. Non-limiting examples include, inter alia, a recombinant host cell that expresses a gene not present in the natural (non-recombinant) form of the cell, or a recombinant host cell that expresses a native gene at different expression levels. As used herein, "heterologous" means that a polypeptide is not normally present within, and is not produced (i.e., expressed) by, the host organism, but is derived from a different species.

[0047] The term "expression" includes all steps involved in the production of a polypeptide (e.g., an encoded enzyme), including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0048] The term “expression vector” refers to a single-stranded or double-stranded, linear or circular DNA molecule containing a polynucleotide encoding a polypeptide and operably ligated to a regulatory sequence that provides its expression. Expression vectors include expression cassettes for the integration of a gene into a host cell, as well as plasmids and / or chromosomes containing the gene. A vector capable of inducing the expression of an operably ligated gene is referred to herein as an expression vector. The term “vector” may also refer to a nucleic acid molecule capable of transporting another ligated nucleic acid molecule. One type of vector is a “plasmid,” which refers to a circular double-stranded nucleic acid loop to which additional nucleic acid fragments can be ligated. Certain other vectors can facilitate the insertion of foreign nucleic acid molecules into the bacterial genome. In this specification, such vectors are referred to as “transformation vectors.” Generally, vectors useful in recombinant nucleic acid technology often take the form of plasmids. In this specification, “plasmid” and “vector” are interchangeable because plasmids are one of the most common forms of vectors. Numerous suitable vectors are known, well-known to those skilled in the art, and commercially available.

[0049] The term "host cell" refers to any cell type that is susceptible to transformation, gene transfer, transduction, etc., by nucleic acid constructs or expression vectors containing polynucleotides expressed within the host cell. Host cells include the offspring cells of the parent cell, including those that are not identical to the parent cell due to mutations that occur during replication.

[0050] The terms “nucleic acid” or “polynucleotide” are used interchangeably herein to refer to polymers in which at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) are covalently linked by phosphate diester bonds, regardless of length or base modification.

[0051] The term "polynucleotide construct" refers to a single-stranded or double-stranded polynucleotide comprising a polypeptide-encoding polynucleotide and one or more regulatory sequences, isolated from naturally occurring genes, or modified or synthesized to include nucleic acid segments in a manner not found in nature.

[0052] The term "operably ligated" refers to a configuration in which a control sequence is positioned appropriately relative to a coding polynucleotide, and that control sequence directs the expression of the coding polynucleotide. More generally, "operably ligated" refers to a juxtaposition in which the described components are related in a way that allows them to function as intended. A control sequence "operably ligated" to a coding sequence is ligated in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. A promoter sequence is "operably ligated" to a gene if it is positioned close enough to the transcription start site of that gene to regulate the transcription of that gene.

[0053] In this specification, “promoter” typically refers to a DNA sequence located upstream (5' end) of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors necessary for transcription initiation. The choice of promoter depends on the nucleic acid sequence in question. A suitable “promoter” is generally one that has the ability to support transcription initiation and induce the production of mRNA molecules in the bacteria of the present invention.

[0054] "Polypeptide" and "protein" are used interchangeably herein to refer to polymers in which at least two amino acids are covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidization, myristylation, ubiquitination, etc.). This definition includes D-amino acids, L-amino acids, and mixtures of D-amino acids and L-amino acids.

[0055] The terms "nucleotide sequence" and "polynucleotide" are used interchangeably herein.

[0056] The terms “preferably,” “commonly,” “particularly,” and “typically” are not used herein to limit the scope of the described invention or to suggest that particular features are important, essential, or significant to the structure or function of the described invention. Rather, these terms are intended to simply highlight alternative or additional features that may or may not be available in particular embodiments of the invention.

[0057] As used herein, the term “cell culture” refers to a culture medium containing multiple host cells as described herein. A cell culture may contain a single host cell line or two or more different host cell lines. A culture medium is any medium containing a recombinant host (e.g., a liquid medium (i.e., culture medium) or a semi-solid medium) and may contain additional components (e.g., a carbon source, a nitrogen source, a phosphate source, vitamins, trace elements, salts, amino acids, nucleic acid bases, etc.).

[0058] As used herein, the terms “endogenous” or “native” refer to genes or polypeptides within a host cell that originate from the same host cell.

[0059] As used herein, the terms “substantially,” “approximately,” or “about” refer to a reasonable deviation within a range in which the value or parameter does not change significantly. These terms, in relation to deviations from a value, should be interpreted to include deviations of the value in which the deviation does not negate the meaning of the deviated value. For example, in relation to a reference number, the term “degree” can include values ​​within a range of ±10% from that value. For example, a deviation from a value can include values ​​that are plus or minus a specific percentage from that value, such as ±9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0060] When numerical limits or ranges are recited in this specification, the endpoints are included. Also, all values and subranges within the numerical limits or ranges are specifically included as if expressly recited.

[0061] As used herein, the term "and / or" is intended to represent an inclusive "or." The expression "X and / or Y" means X or Y, as well as both X and Y. Further, the expression "X, Y, and / or Z" means X, Y, and Z individually, or any combination of X, Y, and Z.

[0062] As used herein with respect to a compound, the term “isolated” refers to any compound that, through artificial intervention, has been placed in a form or environment different from that which exists in nature. Isolated compounds include, but are not limited to, compounds disclosed herein in which the ratio to other components associated with the compound in nature has been increased or decreased. In key embodiments, the amount of the compound is increased relative to other components associated with the compound in nature. In some embodiments, the compounds herein may be isolated in a pure or substantially pure form. In this context, a substantially pure compound means a compound isolated from other foreign or unwanted substances that were present from the start of the compound's production or generated during the production process. Such a substantially pure compound preparation contains less than 10% by weight, e.g., less than 8% by weight, e.g., less than 6% by weight, e.g., less than 5% by weight, e.g., less than 4% by weight, e.g., less than 3% by weight, e.g., less than 2% by weight, e.g., less than 1% by weight, e.g., less than 0.5% by weight, of other foreign or unwanted substances that would normally be associated with the compound when expressed in its natural or recombinant form. In one embodiment, the separated compound is at least 90% by weight pure, for example, at least 91% by weight pure, for example, at least 92% by weight pure, for example, at least 93% by weight pure, for example, at least 94% by weight pure, for example, at least 95% by weight pure, for example, at least 96% by weight pure, for example, at least 97% by weight pure, for example, at least 98% by weight pure, for example, at least 99% by weight pure, for example, at least 99.5% by weight pure, for example, 100% by weight pure.

[0063] As used herein with respect to polynucleotides and genes, the term “deletion” refers to the manipulation of a gene so that it is no longer expressed in a host cell. When the terms “deletion” or “deleted” are used with respect to polypeptides, it refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids constituting the polypeptide. In this case, the enzyme activity and / or improved properties of the modified enzyme are retained. Deletions can be induced in the internal and / or terminal regions of the polypeptide, and in various embodiments, deletions may include continuous or discontinuous segments.

[0064] The terms “insertion,” “inserted,” or “addition” used in relation to polypeptides refer to modifying a polypeptide by adding one or more amino acids to a reference polypeptide. Insertions can include the addition of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids. Insertions can be made to the internal region of the polypeptide, or to the carboxyl or amino terminus. Insertions may be a continuous amino acid segment or may be separated by one or more amino acids in the reference polypeptide.

[0065] In addition to the above, "substituted" or "substitution" when referring to a polypeptide means modifying the polypeptide by replacing one amino acid residue with another residue. For example, replacing a serine residue with a glycine or alanine residue in a polypeptide sequence is an amino acid substitution. When used with respect to a polynucleotide, "substituted" or "substitution" means modifying the polynucleotide by replacing one nucleotide with another nucleotide. For example, replacing cytosine with thymine in a polynucleotide sequence is a nucleotide substitution.

[0066] In addition to the above, "conservative substitution" when used with respect to a polypeptide means replacing an amino acid residue with a different residue having a similar side chain, and thus generally involves replacing an amino acid within the polypeptide with an amino acid within the same or a similar amino acid class. Examples include, but are not limited to, amino acids having aliphatic side chains can be replaced with other aliphatic amino acids (e.g., alanine, valine, leucine, isoleucine); amino acids having hydroxyl side chains can be replaced with other amino acids having hydroxyl side chains (e.g., serine, threonine); amino acids having aromatic side chains can be replaced with other amino acids having aromatic side chains (e.g., phenylalanine, tyrosine, tryptophan, histidine); amino acids having basic side chains can be replaced with other amino acids having basic side chains (e.g., lysine, arginine); amino acids having acidic side chains can be replaced with other amino acids having acidic side chains (e.g., aspartic acid or glutamic acid); and hydrophobic or hydrophilic amino acids can be replaced with other hydrophobic or hydrophilic amino acids, respectively.

[0067] In addition to the "non-conservative substitutions" described above, when used in relation to polypeptides, it refers to the substitution of an amino acid in a polypeptide with an amino acid whose side chain properties are significantly different. Non-conservative substitutions may involve amino acids from other groups rather than within the defined group, and affect (a) the structure of the peptide backbone in the substitution region (e.g., substitution of glycine with serine), (b) the charge or hydrophobicity, or (c) the bulkiness of the side chain. Examples of non-conservative substitutions, though not limited to these, include the substitution of an acidic amino acid with a basic or aliphatic amino acid, an aromatic amino acid with a small molecule amino acid, and a hydrophilic amino acid with a hydrophobic amino acid.

[0068] Recombinant heme or phytoglobin protein The first embodiment provided herein is a recombinant heme or phytoglobin protein comprising C-helices and D-helices covalently linked to a CD loop region, and comprising a hexa-coordinate or penta-coordinate prosthetic heme group, wherein the heme or phytoglobin has an amino acid sequence that is at least 50% identical to the heme or phytoglobin contained in any one of SEQ ID NOs. 1 to 55. Preferably, the amino acid sequence of the heme or phytoglobin is at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 98%, for example, at least 99%, for example, 100% identical to the heme or phytoglobin contained in any one of SEQ ID NOs. 1 to 55.

[0069] The heme prosthetic group in hemoglobin is, in most cases, bound to the globin apolipoprotein by coordination bonds via one or two histidine side chains, or other amino acids that keep the heme group attached to the apolipoprotein. Proteins to which heme iron is coordinated by one histidine are called "pentocoordinate" hemoglobin, and most red blood cell hemoglobin and other oxygen transporters belong to this group. Heme iron is coordinated via two histidine amino acid chains, or other amino acids that maintain heme group attachment to the apolipoprotein, and is called "hexocoordinate" hemoglobin or hexocoordinate phytoglobin, and has been found to be more stable than pentocoordinate phytoglobin. The hexocoordinate structure contributes to the stabilization of iron binding, retaining iron groups within the protein and protecting them from oxidation and premature release.

[0070] The recombinant heme or phytoglobin proteins described herein have been shown to have improved bioavailability compared to conventional iron supplements. Furthermore, the amino acid sequence of the CD loop region has been found to significantly influence the strength with which the prosthetic heme group coordinates and binds to the protein, the degree to which the heme group is stably positioned within the complex, and the degree to which the heme group is protected from auto-oxidation. Auto-oxidation in hemoglobin leads to the production of superoxide and hydrogen peroxide (H2O2). The resulting H2O2 reacts with the iron (Fe(II)) in ferrous oxyhemoglobin, undergoing the Fenton reaction to produce a highly reactive hydroxyl radical (Fe(III)). This radical causes cell and tissue damage (Sadrzadeh et al. (1984)). 4(See reference). This is "spontaneous oxidation," and its rate is an indicator of the intrinsic reactivity of the hemoglobin molecule. In addition to generating harmful radicals, auto-oxidation results in the release of heme groups from the protein. Heme is an iron-coordinated porphyrin and is considered a major molecule contributing to tumorigenesis. Heme is found primarily in red and processed meats, in the form of hemoglobin and myoglobin. The involvement of dietary heme in cancer has been highlighted in various types of cancer. In fact, excessive consumption of red and processed meats is associated with an increased incidence of esophageal cancer, gastric cancer, breast cancer, endometrial cancer, pancreatic cancer, and lung cancer. However, most research has focused on the role of dietary heme in the pathogenesis of colorectal cancer (CRC), a leading cause of cancer death in Western countries (Fiorito et al. (2020)). 5 ).

[0071] Accordingly, modifications to the CD loop region have been found to affect the expression and stability of heme or phytoglobin. Furthermore, modifications have been identified that enhance the binding strength and stability of heme within the protein and reduce the harmful effects of auto-oxidation and heme release from the protein. Therefore, in one embodiment, the CD loop region in the recombinant protein contains one or more modifications compared to the parent CD loop region, thereby allowing the modified CD loop region to bind more strongly to the prosthetic group than the unmodified CD loop region of the parent. The parent CD loop region is preferably specific to the parent protein. Furthermore, the parent CD loop region is preferably located in the recombinant heme or phytoglobin protein at positions 52 to 78 of SEQ ID NO: 1. Since this region includes the initial region extending from the CD loop, in some embodiments, the parent CD loop region is preferably located in the recombinant heme or phytoglobin protein at positions 52 to 72 of SEQ ID NO: 1. In further embodiments, the CD loop region of the recombinant protein has at least 50% identical amino acid sequence to the CD loop region contained in any one of SEQ ID NOs. 56 to 147. Preferably, the CD loop region of the recombinant protein is at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 98%, for example, at least 99%, for example, 100% identical to the CD loop region contained in any one of SEQ ID NOs. 56-110 are CD loop regions identified in phytoglobin SEQ ID NOs. 123-147 are genetically modified CD loop regions. SEQ ID NOs. 111-123 represent CD loop regions also present in SEQ ID NOs. 56-110, but also include some amino acid residues before and / or after the CD loop region.

[0072] Modifications in the CD loop region can be any modifications that increase the binding strength or coordination strength of the heme group, and include deletions, substitutions and / or additions of one or more amino acids. In some embodiments, the modification changes the flexibility, becoming more rigid or more flexible compared to the parental CD loop region. In some embodiments, the modification of the CD loop region includes substitution of flexible amino acids and / or small amino acids (G, A, V, S, I, L, M) with rigid and / or bulky amino acids (such as P, F, W, Y), and / or the reverse substitution thereof. The CD loop region may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications such as substitutions compared to the parental CD loop region. Referring to the corresponding amino acid positions in the CD loop region of quinoa (Chenopodium quinoa), substituting S13 with G improves the expression level and production amount of the recombinant protein. Maintaining the position of P21 in the parental CD loop region or substituting it with P if it is not P also improves the expression level and production amount of the recombinant protein. Position V15 can be substituted with G, especially when used in combination with the substitution of S13 with G, which has a beneficial effect on the expression level and production amount of the recombinant protein. Position A25 can be substituted with G or P. Position A27 can be substituted with G. However, in the hexacoordinate recombinant protein, some positions should not be substituted or only conservative substitutions should be made. Position P1 should not be substituted. The reason is that substitution may increase the instability of the protein and affect its expression. The same applies to positions A3 and F7. Position S8 should not be substituted unless it is accompanied by a second substitution (substituting A25 and / or A27 with G) around His26. The effect of the substitution is offset by this second substitution. P21 should be maintained or substituted only with conservative amino acids. A25 should not be substituted with G or other conservative amino acids. Therefore, in some embodiments, the amino acid modification in the CD loop region is a modification corresponding to one or more modifications selected from S13G, S13P, V15G, A25P, A27G of SEQ ID NO: 56.In other embodiments, the amino acids within the CD loop corresponding to the positions of P1, A3, F7, S8, S13, P16, P21 and / or P25 of SEQ ID NO: 56 are conserved or only conservative substitutions are made. In further embodiments, the CD loop region comprises an amino acid sequence represented by any one of SEQ ID NOs: 123, 127, 128, 129, 130, 131, 133, 134, 135, 137, 139, 141, 142, or 145. In particular, the CD loop region comprises an amino acid sequence represented by any one of SEQ ID NOs: 127, 128, 130, 131, 135, 139, 141, 142, or 145.

[0073] In further embodiments, the recombinant protein comprises a signal peptide that directs secretion of the recombinant protein from the host cell in which it is produced. The signal peptide appropriately directs expression in microbial cells. In some embodiments, the signal peptide is heterologous to the recombinant protein and is selected as needed for optimized functionality in the selected host cell (eukaryotic cell, bacterial cell, or archaeal cell, preferably a bacterial cell or fungal cell). The signal peptide preferably has an amino acid sequence that is at least 50% identical to the signal peptide contained in any one of SEQ ID NOs: 203 - 213. Preferably, the signal peptide has an amino acid sequence that is at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99%, such as 100% identical to the signal peptide contained in any one of SEQ ID NOs: 203 - 213.

[0074] In further embodiments, the recombinant protein is non - natural or synthetic and is further modified as needed, for example, it is a fusion protein. The fusion protein can, for example, contain two or more prosthetic heme groups.

[0075] In a further embodiment, the recombinant protein is a class 1 non-symbiotic hemoglobin. Furthermore, the recombinant protein may be derived from plants of the Gypsophila genus. Phytoglobins isolated from this genus, particularly those isolated from spinach or quinoa, have been found to be particularly stable.

[0076] The recombinant protein may further include one or more post-translational modifications, such as glycosylation and / or phosphorylation, compared to the corresponding native protein.

[0077] As described above, the inventors of the present invention have surprisingly succeeded in developing hexacoordinate phytoglobins that exhibit good biological availability while also showing improved stability. Due to these properties, they are particularly suitable for use in dietary iron supplementation and / or fortification, as well as for the prevention or treatment of iron deficiency and / or anemia in organisms.

[0078] Thus, in a further embodiment, the present invention relates to a recombinant heme or phytoglobin containing a hexacoordinate heme group for use in the treatment or prevention of iron deficiency and anemia in a subject, wherein the heme or phytoglobin protein has at least 86% identity with amino acid sequence number 1, or a fragment, variant, or fusion thereof that retains the hexacoordinate heme group. A further embodiment relates to a recombinant heme or phytoglobin protein according to the present invention, wherein the heme or phytoglobin has at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity with sequence number 1.

[0079] Further embodiments relate to recombinant heme or phytoglobin proteins according to the invention, said heme or phytoglobin proteins being class 1 and / or class 2 non-symbiotic hemoglobins. Further embodiments relate to recombinant heme or phytoglobin proteins according to any of the invention, said recombinant heme or phytoglobin proteins being derived from plants of the Caryophyllaceae family, such as quinoa and / or spinach.

[0080] Thus, one embodiment of the invention relates to a recombinant heme or phytoglobin protein according to the invention, said heme or phytoglobin protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-55, particularly SEQ ID NOs: 1, 3, 4, 5, 7, 9, and 10 and having at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity.

[0081] Further embodiments relate to recombinant heme or phytoglobin proteins according to the invention comprising, or consisting of, an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 1-55, particularly SEQ ID NOs: 1, 3, 4, 5, 7, 9, and 10, or to any fragment, variant, or fusion thereof that retains a six-coordinate heme group. SEQ ID NO: 1 is a six-coordinate phytoglobin from quinoa and SEQ ID NO: 5 is a six-coordinate phytoglobin from spinach.

[0082] Further embodiments relate to recombinant phytoglobin proteins according to the invention comprising, or consisting of, amino acid sequence SEQ ID NO: 1 or any fragment, variant, or fusion thereof that retains a six-coordinate heme group.

[0083] In one embodiment, the present invention relates to a heme or phytochrome according to the present invention, wherein the phytochrome has an auto-oxidation rate of less than 0.2, for example, less than 0.15, for example, in the range of 0.01 to 0.18, or in the range of 0.01 to 0.15, for example, about 0.1h -1 and has an auto-oxidation rate of.

[0084] In one embodiment, the present invention relates to a heme or phytochrome according to the present invention, wherein the phytochrome has no metallic taste or has a neutral taste.

[0085] In one embodiment, the present invention relates to a phytochrome according to the present invention, wherein the phytochrome has a melting temperature exceeding 68°C, for example, in the range of 68°C to 75°C, for example, in the range of 70°C to 76°C. In some embodiments, the melting temperature exceeds 76°C, for example, exceeds 77°C, for example, exceeds 78°C, for example, exceeds 79°C, for example, exceeds 80°C, for example, exceeds 81°C, for example, exceeds 82°C, for example, exceeds 83°C, and can exceed about 84°C. The improvement in stability is associated with the improvement of taste.

[0086] As described above, the variable loop region or CD loop region between α-helices contributes to the flexibility of the molecule and determines to some extent the access to the heme group and its stability. Heme and phytochrome proteins may exhibit great sequence diversity, but the variable loop (CD variable loop or CD loop) between α-helices B and E is characterized by high conservation in some cases, indicating the relative importance of these residues. In other cases, the CD loop may be characterized by great variability. See the alignment of the CD variable loop in Example 6.

[0087] Therefore, one aspect of the present invention relates to a recombinant α-heme or phytochrome protein containing a six-coordinate heme group, wherein the α-heme or phytochrome contains a CD variable loop region having at least 50% or at least 60% identity with amino acid sequence numbers 56 to 147.

[0088] A further embodiment of the present invention relates to a recombinant α - heme or phytoglobin protein according to the present invention, wherein the α - heme or phytoglobin protein comprises a CD variable loop region having at least 65%, for example at least 70%, for example at least 78%, for example at least 80%, for example at least 84%, for example at least 86%, for example at least 90%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% sequence identity with amino acid sequence numbers 56 - 147.

[0089] A particular embodiment relates to a recombinant heme or phytoglobin protein according to the present invention, wherein the heme or phytoglobin comprises a CD variable loop region having at least 50%, for example at least 60%, for example at least 65%, for example at least 70%, for example at least 80%, or for example at least 90% sequence identity with amino acid sequence number 56.

[0090] Without wishing to be bound by theory, the inventors have identified a plurality of residues that appear to be key to improving the stability of the heme or phytoglobin proteins of the present invention. One embodiment relates to a recombinant heme or phytoglobin protein according to the present invention, wherein the heme or phytoglobin protein has at least 65%, for example at least 70%, for example at least 78%, for example at least 80%, for example at least 84%, for example at least 86%, for example at least 90%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% sequence identity with amino acid sequence numbers 1 - 55.

[0091] A further embodiment relates to a recombinant heme or phytoglobin protein comprising a hexacoordinate heme group, wherein the heme or phytoglobin protein comprises a region having the amino acid sequence of sequence numbers 56 - 147.

[0092] This region shows high variability while showing high conservation at specific points, and seems to indicate the relative importance of these conserved residues.

[0093] Even very small changes in highly conserved residues result in changes in stability. For example, the variable loop from Arabidopsis Thaliana (SEQ ID NO: 119) differs from spinach (SEQ ID NO: 111) in having two non-conservative substitutions. However, when alanine is substituted with threonine and aspartic acid is substituted with proline, this phytoglobin seems to be destabilized. Furthermore, the variable loop of sugar beet phytoglobin (SEQ ID NO: 62) differs only in having two non-conservative substitutions compared to SEQ ID NO: 125. However, substitution of P with G at positions 01 and 16 based on the parental CD loop (SEQ ID NO: 56) seems to result in destabilization of this phytoglobin. SEQ ID NO: 111: FLRIFEIAPT AKKMFSFLRD SNVPLEQNPK LKAHAMSV

[0094] The variable loop region of maize has only 79% identity with SEQ ID NO: 13, which seems to correlate with the reduced low-temperature stability of maize phytoglobin. The variable loop region of maize has only 70% identity with SEQ ID NO: 56, which also seems to correlate with the reduced low-temperature stability of maize phytoglobin.

[0095] Conversely, the variable loop region of sugar beet BvHB1.2 has SEQ ID NO: 116 or 62, and an improvement in stability and biological availability is observed. Accordingly, a preferred embodiment relates to a recombinant heme or phytoglobin protein containing a six-coordinate heme group, and the phytoglobin contains SEQ ID NO: 56 or 116. The improvement in stability of this embodiment is surprising since it involves substitution of a highly conserved proline with glycine. Proline is bulky and causes a bend in the molecule. Also, when conserved, it is usually extremely important for structure conservation. Therefore, it is surprising that non-conservative substitutions result in improved function.

[0096] A further embodiment of the present invention relates to a recombinant heme or phytoglobin protein comprising a hexacoordinate heme group, said heme or phytoglobin protein comprising a region having an amino acid sequence similar to SEQ ID NO: 123, 127, 128, 129, 130, 131, 133, 134, 135, 137, 139, 141, 142, or 145, particularly SEQ ID NO: 127, 128, 130, 131, 135, 139, 141, 142, or 145.

[0097] A further embodiment relates to any of the aforementioned recombinant heme or phytoglobin proteins comprising a hexacoordinate heme group for use in the treatment of iron deficiency and / or anemia and / or for use in food, said phytoglobin comprising a CD variable loop region as described herein.

[0098] Composition In a further aspect, compositions are provided herein comprising a recombinant protein disclosed herein and one or more carriers, agents, additives and / or excipients. In particular, this aspect of the invention relates to compositions comprising at least one recombinant hexacoordinate heme or phytoglobin protein as described herein.

[0099] In some embodiments, one or more carriers, agents, additives and / or excipients are selected from salts, antioxidants and / or reducing agents. Salts are appropriately selected from NaCl, ammonium sulfate, CaCl2, KCl, and MgCl2, and in some embodiments, are added to the composition in amounts between 10 μM and 2 M or between 10 μmol and 2 moles / kg. Antioxidants are appropriately selected from ascorbic acid, 2-mercaptoethanol, dithiothreitol (DTT), superoxide dismutase (SOD), catalase, β-carotene, lycopene, glutathione, melatonin, estrogen, and biquinol-10, N-acetylcysteine, lipoic acid, zinc, selenium, and / or copper salts, quercetin, catechin, cortisone, estradiol, estriol, and α-tocopherol, and in some embodiments, are added to the composition between 10 μM and 1 M or between 10 mmol and 2 moles / kg. The reducing agent is appropriately selected from ascorbic acid (vitamin C), tocopherol, carotenoids, flavonoids, and glutathione, and in some embodiments, is added to the composition in an amount between 0.01 and 10 mg / g.

[0100] This composition may further contain at least one vitamin and / or mineral selected from vitamin C, vitamin D3, vitamin E, vitamin B6, vitamin B12, folate, folic acid, biotin, zinc, copper, magnesium, selenium and / or combinations thereof.

[0101] In further embodiments, the composition contains at least 0.002% (weight / weight) of recombinant protein, for example, 0.1 to 100% by weight, or at least 0.1 μM of recombinant protein, for example, 0.1 μM to 8 M of recombinant protein. The compositions may, in some embodiments, be food products, feed products, beverages, food ingredients, health supplements or nutritional supplements, and / or pharmaceutical products. In some embodiments, the compositions are nutritional supplements, and in other embodiments, the compositions are food ingredients. In some embodiments, the food compositions are nutritionally balanced and suitable for children, the elderly, or subjects undergoing medical or surgical treatment. The recombinant proteins described herein have a low tendency to release heme and iron, and therefore have improved properties in reducing or eliminating metallic odor or taste; thus, in some embodiments, the compositions reduce or eliminate metallic odor or taste.

[0102] The composition may be formulated as a dry preparation, a liquid preparation, or a slurry or dispersion. The composition may also be formulated as tablets, capsules, liquids, drops, concentrates, powders, granules, and / or combinations thereof.

[0103] In further embodiments, the composition carrier is a pharmaceutically acceptable carrier, and additionally or instead, the composition is a pharmaceutical composition selected from the group consisting of drugs, vaccines, personal care compositions and combinations thereof.

[0104] The compositions according to the present invention are neutral in taste and have no metallic taste, and therefore can be formulated with high concentrations of iron. Accordingly, some embodiments of the present invention relate to compositions containing, for example, 0.1 μM to 8 M of recombinant heme or phytoglobin protein (corresponding to 0.017 to 170,000 mg / g).

[0105] As described above, the compositions of the present invention are dietary compositions such as feed, food, beverage, nutritional supplement, or food ingredient, and can be formulated in any suitable way. For example, the compositions of the present invention have dosage forms selected from the group consisting of tablets, capsules, liquids, drops, concentrates, powders, granules, and combinations thereof.

[0106] As described above, the compositions of the present invention can be formulated as pharmaceuticals using conventional pharmaceutically acceptable excipients and / or carriers.

[0107] As described above, the compositions according to the present invention may comprise one or more further components, such as antioxidants, reducing agents, nutrients, or compounds suitable for supporting iron supplementation and / or intake. For example, some embodiments of the present invention relate to compositions of the present invention further comprising at least one vitamin and / or mineral, for example, one or more selected from the group consisting of vitamin C, vitamin D3, vitamin E, vitamin B6, vitamin B12, folate, folic acid, biotin, zinc, copper, magnesium, selenium, and combinations thereof. Furthermore, embodiments of the present invention relate to compositions of the present invention further comprising one or more reducing agents, for example, ascorbic acid (vitamin C), tocopherol, carotenoids, flavonoids, and glutathione.

[0108] The compositions of the present invention may further comprise any suitable food-grade preservatives, excipients, thickeners, flavorings, or colorings deemed useful.

[0109] In Example 4, phytoglobins derived from sugar beets, spinach, and quinoa were demonstrated to be bioavailable iron sources. Therefore, these and molecules, when used as organic, bio-based iron supplements and food ingredients or feed, can be used to prevent and treat iron deficiency and anemia (IDA) without affecting the taste of the food to which they are added.

[0110] One particular embodiment relates to a liquid composition comprising a non-recombinant and / or non-recombinant heme or phytoglobin protein containing a hexa-coordinate heme group. The heme or phytoglobin protein has at least 86% identity with amino acid sequence number 1; or its fragments, variants, or fusions that retain the hexa-coordinate heme group. Such a liquid composition may be used for the treatment or prevention of iron deficiency.

[0111] The liquid composition according to the present invention has the advantage of being more easily absorbed than, for example, encapsulated formulations. The neutrality of the heme or phytoglobin protein of the present invention makes it possible to provide liquid formulations.

[0112] The liquid composition may further contain one or more stabilizers, excipients, antioxidants, or other known compounding agents.

[0113] Terms of Use. Due to their stability and bioavailability, the recombinant heme or phytoglobin proteins described herein and compositions containing them are suitable for multiple applications.

[0114] Accordingly, further embodiments provided herein are non-therapeutic methods for improving the endurance or hypoxia tolerance of a subject, which include administering to the subject an amount effective in improving the endurance or hypoxia tolerance of the subject, one of the recombinant proteins or compositions disclosed herein. In some embodiments, the subject is, optionally, performing or preparing to perform physical activity under hypoxic pressure, such as mountain climbing or hiking. In further embodiments, the non-therapeutic method includes administering to the subject's skin an amount effective in altering the color or texture of the skin for cosmetic purposes.

[0115] In a further embodiment, another embodiment provides a method comprising a recombinant heme protein or phytoglobin protein or composition described herein for use in the treatment, improvement, or prevention of a disease or deficiency and / or anemia in a subject, comprising administering to a subject an amount effective to treat, improve, or prevent the disease or deficiency of the recombinant heme protein or phytoglobin protein or composition.

[0116] This application describes both methods for the use of recombinant proteins for therapeutic purposes and recombinant proteins for use in therapeutic applications, and it should be understood that the alternative disclosures are interchangeable in expressing the same subject matter in an alternative form of description.

[0117] The disease or deficiency to be treated is preferably iron deficiency or any disease resulting from such iron deficiency. In the treatment in some embodiments, the recombinant protein is preferably a heme or phytoglobin protein, particularly a heme or phytoglobin protein comprising a hexa-coordinate heme group and having at least 86% the same amino acid sequence as the phytoglobin contained in SEQ ID NO: 1, and in particular, the recombinant phytoglobin has at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% the same amino acid sequence as the phytoglobin contained in SEQ ID NO: 1. The recombinant protein used for the treatment, improvement or prevention of deficiencies and / or diseases is preferably non-symbiotic hemoglobin or class 2. Additionally, recombinant phytoglobin may be derived from a subfamily of carnations, such as spinach or quinoa. Recombinant phytoglobin for therapeutic purposes preferably has an amino acid sequence contained in or comprising SEQ ID NO: 1 or 4.

[0118] In further embodiments, recombinant proteins contain six-coordinate heme groups and exhibit improved stability compared to five-coordinate hemoglobin. In particular, therapeutic recombinant proteins have a lifespan of less than 0.2, e.g., less than 0.15, e.g., 0.01h. -1 ~0.18h -1 The range, or 0.01h -1 ~0.15h -1 The range, for example, about 0.1h -1It has an auto-oxidation rate. For therapeutic purposes, the recombinant protein reduces or eliminates metallic taste, or has a neutral taste.

[0119] In some embodiments for therapeutic use, the recombinant protein has a melting temperature greater than 68°C, for example, in the range of 68°C to 75°C, for example, in the range of 70°C to 76°C. In some embodiments for therapeutic use, the melting temperature may be greater than 76°C, for example, greater than 77°C, for example, greater than 78°C, for example, greater than 79°C, for example, greater than 80°C, for example, greater than 81°C, for example, greater than 82°C, for example, greater than 83°C, and about 84°C.

[0120] In further embodiments, the recombinant heme or phytoglobin protein comprises a hexa-coordinate heme group and further comprises a CD loop region having an amino acid sequence with at least 60% sequence identity to the CD loop region included in SEQ ID NO: 111 or 56, preferably with at least 90% sequence identity to the CD loop region included in SEQ ID NO: 111 or 56.

[0121] In other embodiments, the CD loop region in the recombinant protein has an amino acid sequence having one or more, two or more, three or more, or four or more amino acid substitutions in the CD loop region included in SEQ ID NO: 111 or 56. In addition, or instead, the CD loop region has an amino acid sequence including or consisting of SEQ ID NOs: 56 to 147.

[0122] In some embodiments, the subjects are blood donors. In other embodiments, the subjects are vegetarians, vegans, and / or flexitarians. The subjects may also be animals, such as pets, poultry, or livestock. In particular, the subjects are women, optionally selected from the groups of pregnant women, lactating women, and women of reproductive age.

[0123] Diseases that can be treated include endometriosis, dysmenorrhea, menorrhagia, cancer, kidney disease, diabetes, obesity, metabolic syndromes, gastrointestinal disorders, mental disorders, genetic disorders, age-related diseases, acute diseases, and / or infections. Gastrointestinal disorders include celiac disease, inflammatory bowel disease, and / or peptic ulcers. Genetic disorders are selected from hemolytic anemia and / or autoimmune diseases. Mental disorders include bulimia and / or anorexia nervosa. Infections include malaria and / or streptococcal infections. Diseases may be chronic or acute. Acute diseases include trauma, gastrointestinal surgery, gastrointestinal bleeding, renal failure, and / or acute toxicity. Further embodiments relate to recombinant hexa-coordinate heme or phytoglobin protein and / or compositions comprising said hexa-coordinate phytoglobin according to the present invention, including use in various applications, e.g., in the treatment, improvement, or prevention of iron deficiency and / or anemia in subjects.

[0124] For example, the heme or phytoglobin proteins and compositions containing them described herein are suitable for use in food, for supplementing dietary iron intake (food supplements, functional foods), for fortifying food, for treating, preventing or improving iron deficiency, for treating, preventing or improving anemia, and / or for use in pharmaceuticals.

[0125] In one embodiment, the present invention relates to a composition comprising recombinant hexa-coordinate phytoglobin as described herein for use as a drug.

[0126] In particular, the heme or phytoglobin proteins and compositions according to the present invention are suitable, for example, for the treatment, improvement, or prevention of iron deficiency and / or anemia in subjects with chronic and / or acute diseases who are at risk of iron deficiency. Certain populations are at particularly high risk of iron deficiency, and the heme or phytoglobin proteins and compositions containing them according to the present invention are suitable for use by these populations. For example, because they have a neutral taste, they are particularly suitable for use in children's foods or nutritional supplements.

[0127] Further embodiments relate to a heme or phytoglobin protein containing a hexa-coordinate heme group for use in the treatment or prevention of iron deficiency in a subject, wherein the heme or phytoglobin protein has at least 86% identity with amino acid sequence number 1, or a fragment, variant, or fusion thereof that retains a hexa-coordinate heme group.

[0128] The phytoglobin according to the present invention may be produced by recombinant or non-recombinant methods.

[0129] The recombinant heme or phytoglobin proteins described herein have the advantage of being able to be produced in large quantities and with high purity. Furthermore, recombinant phytoglobin exhibits little batch-to-batch variability. The phytoglobin content in plant tissues fluctuates with plant distribution and over time, making it difficult to produce commercially viable quantities. In addition, plants may contain other compounds that inhibit iron uptake.

[0130] Nevertheless, the present invention also relates to a non-recombinant heme or phytoglobin protein containing a hexa-coordinate heme group for use in food, wherein the phytoglobin has at least 86% identity with amino acid sequence number 1, or its fragment, variant, or fusion that retains a hexa-coordinate heme group.

[0131] The non-recombinant heme or phytoglobin protein according to the present invention has all the advantages described herein with respect to the recombinant form, and thereafter, unless otherwise specified, all embodiments described with respect to the recombinant form also apply to the non-recombinant heme or phytoglobin protein according to the present invention.

[0132] Recombinant protein is appropriately administered to the subject in a range of 0.1 mg to 350 mg per kg of body weight, for example, 0.25 mg to 100 mg, for example, 0.5 mg to 50 mg, for example, 0.75 mg to 10 mg, or for example, 0.75 mg to 3 mg. In one embodiment, the subject is male, and the amount of recombinant protein administered to the subject as needed is 0.75 mg to 1.25 mg per kg of body weight, for example, about 1 mg. In another embodiment, the subject is female, and the amount of recombinant protein administered to the subject as needed is 1.3 mg to 1.5 mg per kg of body weight, for example, about 1.4 mg.

[0133] Genes and constructs In a further embodiment, a gene encoding any of the recombinant proteins disclosed herein is provided. The gene preferably has a nucleotide sequence that is at least 50% identical to the nucleotide sequence contained in any of the SEQ ID NOs: 148-202 encoding the corresponding recombinant protein of SEQ ID NOs: 1-55.

[0134] In further embodiments, polynucleotide constructs comprising the genes described herein, operably ligated to a regulatory sequence that directs the transcription and / or translation of the genes within a host cell, are provided herein. The regulatory sequence may be a promoter, which is a polynucleotide recognized by the host cell for the expression of the polynucleotide. The promoter comprises a transcriptional regulatory sequence that mediates the expression of the polypeptide. The promoter is any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and is obtained from a gene encoding an extracellular or intracellular polypeptide of a congeneral or heterogeneous species relative to the host cell. The promoter is an inducible or constitutive promoter and, if necessary, has at least 50% identical nucleotide sequence to the promoters included in SEQ ID NOs. 225-235. Furthermore, the polynucleotide construct is an expression vector. The expression vector is any vector (e.g., plasmid, viral, or chromosomal vector) that can be conveniently used in a recombinant DNA procedure and can result in the expression of a gene encoding a recombinant protein. The choice of vector usually depends on its compatibility with the host cell into which the vector is introduced. The vector may be an autonomous replicating vector, that is, a vector that exists as an extrachromosomal entity that replicates independently of chromosome replication (e.g., plasmids (linear or closed circular plasmids), extrachromosomal elements, minichromosomes, artificial chromosomes). The vector may include means to ensure self-replication. Alternatively, the vector may be integrated into the genome upon introduction into a host cell and replicate together with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids containing the entire DNA to be introduced into the host cell's genome, or transposons may be used.

[0135] A vector may contain one or more selection markers that facilitate the selection of cells for transformation, gene transfer, transduction, etc. Selectable markers are genes whose products provide biocide or virus resistance, heavy metal resistance, prototrophicity to trophic requirements, etc. Useful selection markers for fungal host cells include amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinotricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenylate transferase), trpC (anthranilate synthase), and their equivalents. Useful selection markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0136] The vector may further contain elements that enable integration into the host cell genome, or elements that autonomously replicate within the cell, independently of the genome. For integration into the host cell genome, the vector may undergo homologous or non-homologous recombination, depending on a gene encoding a recombinant protein, or other elements of the vector. Alternatively, the vector may contain additional polynucleotides to direct integration into the host cell genome by homologous recombination to a precise location within the chromosome. To increase the probability of precise location integration, the integration element should contain a sufficient number of nucleic acids, e.g., 100–10,000 base pairs, e.g., 400–10,000 base pairs, e.g., 800–10,000 base pairs, etc., that have high sequence identity with the corresponding target sequence to increase the probability of homologous recombination. The integration element can be any sequence homologous to the target sequence in the host cell genome. Furthermore, the integration element can be a non-coding or coding polynucleotide. On the other hand, the vector may be integrated into the host cell genome by non-homologous recombination. For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously within the host cell. An origin of replication is any plasmid self-replicating machinery that mediates autonomous replication within the cell. The terms “origin of replication” or “plasmid self-replicating machinery” refer to polynucleotides that enable a plasmid or vector to replicate in vivo. Useful origins of replication for fungal cells include AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00 / 24883). Isolation of the AMA1 sequence and construction of a plasmid or vector containing this gene can be achieved using the method disclosed in WO2000 / 24883. Useful origins of replication for yeast host cells include the 2-micron origin, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0137] The production of recombinant protein can be increased by inserting multiple copies of the polynucleotide encoding the recombinant protein of the present invention into host cells. The increase in copy number can be achieved by incorporating one or more additional copies of the protein-coding sequence into the host cell genome, or by including an amplified selectable marker gene in the polynucleotide. This allows for the selection of cells containing amplified copies of the selectable marker gene (and additional copies of the polynucleotide) by culturing them in the presence of a suitable selector. Procedures for constructing the recombinant expression vector of this disclosure by ligating these elements are well known to those skilled in the art (see, for example, Green & Sambrook, 2012, Molecular cloning: A laboratory manual, Fourth Edition, Cold Spring Harbor Laboratory, New York, USA).

[0138] In line with the foregoing, this specification also discloses host cells containing polynucleotide constructs as described above.

[0139] Host cells expressing recombinant proteins In a further embodiment, genetically modified host cells expressing a gene or polynucleotide construct and the recombinant protein described herein are provided herein. The host cell can be a eukaryotic cell, a bacterial cell, or an archaeal cell. Among eukaryotic cells, fungal cells, plant cells, mammalian cells, or insect cells are useful. Among plant cells, those derived from the genera Arabidopsis, Lepidium, Nicotiana, Triticum, Hordeum, Oryza, Chenopodium, Beta, or Glycine are particularly useful. For example, Arabidopsis thaliana, Lepidium campestre, Nicotiana tabacum, Triticum aestivum, Hordeum vulgare, Oryza sativa, Chenopodium quinoa, Beta vulgaris, or Glycine max are particularly useful. As fungal cells, yeast or filamentous fungi are preferred. Preferred filamentous fungi are those belonging to the genera Aspergillus, Trichoderma, or Rhizopus, such as species of Aspergillus, Aspergillus oryzae, Trichoderma, or Rhizopus.Preferred yeasts are derived from the genera Pichia, Saccharomyces, Yarrowia, Kluveromyces, Ashbya, or Hansenula, such as methanol-utilizing yeast (P. pastoris), Pichia sp., budding yeast (S. cerevisiae), Yarrowia sp., alkane-utilizing yeast (Y. lipolytica), Kluyveromyces lactis, brewer's yeast (Saccharomyces carlsbergensis), budding yeast (Saccharomyces cerevisiae), Saccharomyces diastaticus, and Saccharomyces douglasi. These include species such as *Saccharomyces douglasii*, *Saccharomyces kluyveri*, *Saccharomyces bensis*, *Saccharomyces oviformis*, *Ashbya gossypii*, *H. polymorpha*, or species of the genus *Hansenula*.Among bacteria, preferred species include Escherichia, Bacillus, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, and Acinetobacter. These are bacteria that infest the genera Acetobacter or Pseudomonas, such as species E. coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas marcescens, Pseudomonas putida, Pseudomonas aeruginosa, and / or P. mutabilis. Algae are among the host cells of archaea. ClearColi (endotoxin-free E. coli) and E. coli Nissle strain 1917 (Schultz and Burton 2017) are particularly useful.

[0140] cell cultures containing host cells In further embodiments, cell cultures comprising host cells and culture media described herein are provided herein. Culture media suitable for host cells such as mammalian, insect, plant, fungal, and / or yeast cells are all well known in the art.

[0141] Methods for producing recombinant proteins In a further embodiment, a method for producing the recombinant protein described herein is provided herein, the method comprising the following steps: a) The step of culturing the cell culture described herein under conditions in which the cells can produce recombinant proteins; and b) A step of recovering and / or separating the recombinant protein, if necessary.

[0142] Cell cultures can be cultured using methods known in the art in nutrient media and conditions suitable for the production of recombinant proteins and / or cell proliferation as described herein. For example, cultures can be carried out in a suitable medium under conditions that allow host cells to grow and / or proliferate, by shaking flask culture or small or large-scale fermentation in laboratory or industrial fermenters (including continuous culture, batch culture, fed-batch culture, feed-and-draw culture, and solid-state fermentation), and then recovered and / or separated as necessary.

[0143] Culturing can be carried out using a suitable nutrient medium containing a carbon source, a nitrogen source, and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or can be prepared according to published recipes (e.g., catalog of the United States Cell Culture Lineage Preservation Service). The selection of a suitable medium may be based on the selection of host cells and / or regulatory requirements for the host cells. Such media are available in the art. If necessary, the medium may include additional components to prioritize the transformed expression host over other potential contaminating microorganisms. Thus, in one embodiment, a suitable nutrient medium includes a carbon source (e.g., glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellulosic biomass hydrolysates, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g., yeast extract, malt extract, peptone, etc.), and an inorganic nutrient source (e.g., phosphates, magnesium, potassium, zinc, iron, etc.). In some embodiments, the medium contains a high concentration of iron to enable the production of iron-containing recombinant proteins. Hemoglobin synthesis in host cells consists of two parts, as shown in Figure 6: the synthesis of heme b and the synthesis of apolipoprotein (globin protein). The heme b ring system is the protoporphyrin IX ring system. This prosthetic group is firmly but noncovalently bound within the host protein (apolipoprotein). To produce recombinant heme or phytoglobin protein with high titer, it is advantageous to supply high levels of iron in the culture medium. Iron can be supplied to the medium in the form of, for example, FeSO4, FeO, or Fe2O3, and the amount is preferably greater than [0.01 g / L]. Furthermore, the glucose concentration in the culture medium may be kept low (to avoid protein glycosylation), preferably at 50 mM glucose, for example, less than 40 mM, for example less than 30 mM, for example less than 20 mM, for example less than 10 mM, for example less than 5 mM, for example less than 4 mM, for example less than 3 mM, for example less than 2 mM, for example less than 1 mM. To further optimize the culture, expression, and production of recombinant heme or phytoglobin protein, δ-aminolevulinic acid (D-ALA) may be added if the production of ALA (see Figure 6) by ALA synthase is the enzymatic rate-limiting step in heme synthesis. To further optimize the culture, expression, and production of recombinant protein, carbon monoxide may be added to the culture medium by supplying carbon monoxide gas to the medium. To further optimize the culture, expression, and production of recombinant proteins, the dissolved oxygen (DO) level is preferably maintained at less than 20%, for example, less than 15%, for example, less than 10% by weight, for example, less than 9% by weight, for example, less than 8% by weight, for example, less than 7% by weight, for example, less than 6% by weight, for example, less than 5% by weight, for example, less than 4% by weight, for example, less than 3% by weight, for example, less than 2% by weight, for example, less than 1% by weight, for example, less than 0.1% by weight, for example, less than 0.01% by weight, for example, less than 0.001% by weight.

[0144] Host cell culture can be carried out over a period of about 0.5 to about 30 days. The culture process may be batch, continuous or feed-batch, and is preferably carried out at a temperature in the range of 0 to 100°C or 0 to 80°C, for example, about 0°C to about 50°C and / or at a pH of, for example, about 2 to about 10. Preferred fermentation conditions for yeast and filamentous fungi are a temperature in the range of about 25°C to about 55°C and a pH in the range of about 3 to about 9. Preferred fermentation conditions for bacteria such as E. coli are a temperature of 17 to 38°C and a pH of 5 to 8.

[0145] Appropriate conditions are typically selected based on the choice of host cells. Therefore, in one embodiment, the method described herein further includes one or more elements selected from the following: a) Culturing cell cultures in vegetative growth medium; b) Culturing cell cultures under aerobic or anaerobic conditions; c) Culture the cell culture under agitation; d) Culturing the cell culture at a temperature of 25-50°C; e) Culture the cell culture at a pH of 3-9; f) Culturing the cell culture for 10 hours to 30 days.

[0146] Recombinant proteins can be recovered and / or separated using methods known in the art, including but not limited to centrifugation, filtration, spray drying, or freeze-drying. For example, in certain embodiments, the method includes a recovery and / or separation step comprising separating the liquid phase of cells or cell culture from the solid phase of cells or cell culture to obtain a supernatant containing recombinant proteins, and subjecting the supernatant to one or more steps selected from the following: a) A step of separating the supernatant from the solid phase of the cell culture by filtration or gravity separation; b) The supernatant is brought into contact with one or more adsorbent resins to obtain at least a portion of the recombinant protein produced; c) The supernatant is brought into contact with one or more ion-exchange or reverse-phase chromatography columns, or with a filtration or ultrafiltration apparatus to obtain at least a portion of the recombinant protein; d) A step of extracting recombinant protein; and / or e) Precipitating the recombinant protein by crystallization or evaporation of the liquid-phase solvent (spray drying or freeze-drying if necessary); separating the recombinant protein by filtration or gravity separation if necessary; This is followed by the step of recovering and / or separating the recombinant protein.

[0147] In further embodiments, the composition may also be an edible composition containing a cell culture as described herein, and comprising recombinant protein, particularly for iron supplementation, suitable for ingestion by subjects requiring it. In this composition, the cell culture may be in the form of edible mycelium dispersed in a matrix such as a solid substrate, and suitable for ingestion by humans or animals.

[0148] The descriptions or discussions of prior art in this specification do not necessarily constitute an endorsement of such prior art as part of the current state of the art or as publicly known general knowledge.

[0149] Priorities, selections, and embodiments relating to specific aspects, features, or parameters of the invention should be considered in conjunction with all priorities, selections, and embodiments relating to all other aspects, features, and parameters of the invention, unless the context indicates otherwise. This applies particularly to the descriptions of recombinant and non-recombinant hexa-coordinate heme or phytoglobin proteins and all their features, which may form part of the final compositions or uses described herein. Embodiments and features of the invention are also outlined in the following sections and illustrated by the following non-limiting examples.

[0150] Examples material and method array This application includes a sequence listing prepared in WIPO Sequence 2.2.0 or later, which includes the following sequences, submitted electronically in ST26 format. This listing is incorporated in its entirety by reference. [Table 1] TIFF2026513230000002.tif240159TIFF2026513230000003.tif240159TIFF20265132300 00004.tif237159TIFF2026513230000005.tif234159TIFF2026513230000006.tif116159

[0151] cloning The protein-coding genes were found using the BLASTP online tool (https: / / blast.ncbi.nlm.nih.gov) with the sequence of GenBank acceptance number KF549981 (beet hemoglobin BvHb1.2) as a template. The following genes with sequence identity of 80% or higher from edible plants were selected: three class 1 hemoglobins from quinoa (Chenopodium quinoa) (acceptance numbers XP_021738545.1, XP_021738543.1, and XP_021731656.1) and one class 1 hemoglobin from spinach (Spinacia oleracea) (acceptance number XP_021850852.1). The genes for these selected sequences were custom synthesized by IDT technologies (www.idtdna.com) according to the supplier's instructions. Recombination-based cloning and gene transformation were performed according to the manufacturer's instructions (Gateway®, Invitrogen). Each gene was individually cloned into the pET-DEST42 plasmid, and the final expression vectors were transformed into BL21-DE3 E. coli cells.

[0152] Growth conditions for expression Cells carrying the expression vector pET-DEST42 containing the corresponding hemoglobin gene were grown in sterile TB medium containing 100 mg / ml carbenicillin at 37 °C and 150 rpm until OD 600 ≥ 2. Expression of the recombinant protein was induced by adding 0.5 mM IPTG and 0.3 mM δ-aminolevulinic acid. Also, to obtain a stable carbon monoxide (CO) hemoglobin compound, the cells were bubbled with carbon monoxide (CO) for a short time. After induction, the cells were cultured overnight at 22 °C and 150 rpm. Cells expressing recombinant hemoglobin were collected by centrifugation, rapidly frozen in liquid nitrogen, and stored at -80 °C until use.

[0153] Purification of Recombinant Hemoglobin and / or Phytoglobin All procedures were carried out at room temperature and all buffers were pH-adjusted. Purification was performed according to the report of Leiva Eriksson et al. in 2019 2 . The recombinant cells were resuspended in extraction buffer (50 mM Tris-HCl, pH 8.5, 50 mM NaCl, 25 mM glucose, 1 mM EDTA, 10 mM ascorbic acid, 5% glycerol) at a buffer concentration of 2 ml per gram of cells. The resuspended cells were disrupted by sonication (Q500 Qsonica), and the lysate was clarified by centrifugation at 27,000 g for two times (45 minutes for the first time and then 30 minutes).

[0154] To purify recombinant hemoglobin from the lysis solution, an AKTA purification system (Cytiva) was used. First, the recombinant protein was packed into a Q-Sepharose FF column and eluted with starting buffer (50 mM Tris-HCl, pH 8.5) and 50 mM NaCl. The red fraction was concentrated using a 10 kDa Viva-Spin column (Vivascience). Next, ammonium sulfate was added to the pooled sample to a final concentration of 0.8 M. Subsequently, the sample was loaded onto a Butyl-Sepharose HP column that had been pre-equilibriumized with 0.8 M ammonium sulfate in a 50 mM Tris-HCl, pH 8.0 buffer. The protein was eluted with a linear gradient of 10 CV (column volume) of ammonium sulfate in the range of 0.8 M to 0.0 M in a 50 mM Tris-HCl, pH 8.0 buffer. Finally, the protein was loaded onto a Q-Sepharose HP column and eluted in 50 mM Tris buffer at pH 8.5 using a linear gradient with 0–100 mM NaCl. The purified protein was analyzed by SDS-PAGE (see Figure 1-D), and the total heme amount was determined by the pyridine hemacromolecule method (Sinclair et al., (2001)). 3 The measurements were taken using the reference method. The samples were then rapidly frozen in liquid nitrogen and stored at -80°C until use.

[0155] Molecular weight of recombinant hemoglobin The native molecular weight of the purified recombinant protein was determined by gel filtration using a pre-packed HiLoad 16 / 60 Superdex 200pg (GE Healthcare) column with column buffers of 50 mM sodium phosphate, pH 7.2, and 150 mM NaCl. Standard proteins for mass spectrometry were prepared according to the instructions of the Gel Filtration Calibration Kit (Cytiva) using native protein standards: ferritin (444 kDa), aldolase (158 kDa), corn albumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), RNase (13.7 kDa), and aprotinin (6.5 kDa).

[0156] The purified protein was determined to be a homodimer with a molecular weight of approximately 35-40 kDa.

[0157] Example 1. Evaluation of intrinsic reactivity - Automated oxidation assay To investigate the auto-oxidation of recombinant proteins, a 400 μM stock solution of each protein was reduced with a slightly excess sodium dithionite to obtain the deoxyferrous form of the protein. The oxyferrous form of the protein was generated by passing the deoxyferrous protein through a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) using 0.1 M Tris-HCl buffer (pH 8.5) as the mobile phase. Subsequently, a portion of the sample was diluted to a final concentration of 5 μM with the same buffer. Oxidation of the sample was tracked by recording the entire spectrum (400–600 nm) every 15 minutes for the first 2 hours and every 60 minutes for the following 72 hours. The time course was obtained from the spectral change at 421 nm. Absorption spectra were recorded over time. The reaction was tracked with a Cary60 UV-VIS spectrophotometer (Agilent) equipped with an 18-cell holder (set to 20°C) with a water temperature thermostat. The OriginPro 8.6 program (OriginLab) was used for data analysis and figure creation (see Figure 2).

[0158] Auto-oxidation in hemoglobin leads to the production of superoxide and hydrogen peroxide (H2O2). The resulting H2O2 reacts with the iron (Fe(II)) in ferrous oxyhemoglobin, undergoing the Fenton reaction to produce a highly reactive hydroxyl radical (Fe(III)). This radical causes cell and tissue damage (Sadrzadeh et al. (1984)). 4 (See reference). This is "spontaneous oxidation," and its rate serves as an indicator of the intrinsic reactivity of the hemoglobin molecule.

[0159] In addition to generating harmful radicals, auto-oxidation results in the release of heme groups from proteins. Heme is an iron-coordinated porphyrin and is considered a major molecule contributing to tumorigenesis. Heme is found primarily in red and processed meats, in the form of hemoglobin and myoglobin. The involvement of dietary heme in cancer has been highlighted in various types of cancer. In fact, excessive consumption of red and processed meats is associated with an increased incidence of esophageal cancer, gastric cancer, breast cancer, endometrial cancer, pancreatic cancer, and lung cancer. However, most research has focused on the role of dietary heme in the pathogenesis of colorectal cancer (CRC), a leading cause of cancer death in Western countries (Fiorito et al. (2020)). 5 ).

[0160] In conclusion, measuring the auto-oxidation rate of hemoglobin determines its stability and inherent reactivity. A high auto-oxidation rate indicates that hemoglobin is highly toxic, especially when ingested in large quantities. Conversely, a low auto-oxidation rate means that the protein is stable and safe.

[0161] method Auto-oxidation was measured as described above. Hemoglobin proteins from sugar beet, quinoa, and spinach were incubated in ferrous oxystate (5 μM) at pH 8.0 and 37°C for 72 hours (Figure 2). The auto-oxidation rate was determined by fitting the time course (400-600 nm) to a single exponential function.

[0162] Results and conclusions Auto-oxidation rate of hemoglobin in sugar beet, quinoa, and spinach (k autoox ) was found to be at pH 8.0 and 37°C for 0.1 hours. -1This was determined. This result means that these proteins auto-oxidize at a lower rate than other common hemoglobins (Table 1). Therefore, hemoglobins from sugar beets, quinoa, and spinach are stable and have low intrinsic reactivity. This stability is achieved because they are hexa-coordinate proteins (the heme iron group is coordinated by two histidine side chains). On the other hand, iron proteins found in meat (hemoglobin and myoglobin) are penta-coordinate proteins. This means that the heme iron is coordinated by a single histidine side chain, leaving the other axial sites open, which makes them highly reactive and easily detached from apolipoproteins. In fact, the International Agency for Research on Cancer (IARC), after evaluating more than 800 studies conducted worldwide, classified processed meat as Group 1 "carcinogenic to humans" and fresh red meat as Group 2A "potentially carcinogenic to humans." This characteristic is also common to leghemoglobin derived from soy. Soy leghemoglobin is also a five-coordinate hemoglobin and is used in meat substitutes for color and flavor purposes (FAQ Impossible Foods). 6 Soy leghemoglobin exhibits a heme dissociation rate 600 times faster than sperm whale myoglobin under the same conditions, and, combined with its high auto-oxidation rate, becomes a far more unstable holoprotein (Hargrove and Olson (1996)). 7 ).

[0163] Both temperature and pH have been found to influence auto-oxidation and have a direct effect. The rate of auto-oxidation is usually accelerated at more acidic pH and higher temperatures. Because these hemoglobins may be used for human intake, auto-oxidation is being tested under or near physiological conditions (pH 7, 37°C). [Table 2]

[0164] Example 2: Evaluation of temperature stability Proteins are amphoteric molecules with positive and negative charges, and electrostatic repulsion occurs between groups at temperatures below or above their isoelectric point. This intramolecular repulsive interaction between charged groups is enhanced at high temperatures, leading to destabilization. The melting temperature (Tm) of a protein is defined as the temperature at which the concentration of the protein in its folded state equals the concentration in its unfolded state. Below Tm, the size and scattering intensity of the protein are constant, suggesting a stable tertiary structure. Above Tm, both the size and scattering intensity increase exponentially, indicating the presence of denatured proteins and aggregates.

[0165] Thermal stability is typically determined by solubility curve analysis. This analysis assesses the integrity of a protein while gradually increasing its temperature over a predetermined period. A protein is classified as meso-stable or thermally stable if its Tm (temperature) is below 70°C or above 70°C, respectively.

[0166] Hemoglobin is a protein that exists as monomers (e.g., human bovine myoglobin, soybean leghemoglobin), dimers (e.g., non-symbiotic hemoglobins derived from sugar beet, spinach, and quinoa), and tetramers (e.g., human adult and fetal hemoglobin). Therefore, the Tm differs depending on the hemoglobin species. In the case of hemoglobin proteins, Tm indicates not only protein denaturation but also the loss of heme iron. As explained in Example 1, heme iron is recognized as a carcinogenic molecule. Therefore, a high Tm is preferable because it allows the protein to be stable over a wider temperature range and withstand rapid temperature changes.

[0167] method The thermal stability of proteins (10-20 μL of sample at concentrations of 10-50 μM in 50 mM Tris-HCl, pH 8.5, n>3) was analyzed by nano-differential scanning fluorescence (nanoDSF) using a Prometheus NT.48 instrument (NanoTemper Technologies) equipped with an aggregation optical system. The excitation LED intensity (wavelength 280 nm) was set to 20%. The fluorescence intensity of the samples (wavelength 350 nm: F350 and 330 nm: F330) was recorded over a temperature gradient of 20-95°C (heating rate 1°C / min) (Figure 3). The melting point (Tm) of the proteins was obtained from the first derivative of the F350 / F330 fluorescence ratio plotted against temperature. The standard deviation was calculated using Microsoft Excel.

[0168] Results and Conclusions Hemoglobin derived from sugar beets, spinach, and quinoa has an average melting temperature of T o The (Tm) is above 70°C (approximately 72°C), indicating that it is a heat-stable protein (Table 2). In contrast, the melting temperature of bovine myoglobin is 67.4°C. This is expected because bovine myoglobin is a five-coordinate molecule and is inherently unstable. See Figure 2 and Table 2. [Table 3]

[0169] This example demonstrates that the recombinant hexa-coordinate heme or phytoglobin proteins of the present invention (derived from spinach, quinoa, and sugar beet) exhibit similar stability in accordance with these hexa-coordinate structures.

[0170] The stability of recombinant hexa-coordinate heme or phytoglobin proteins has been found to be due to their hexa-coordinate structure. In this form, the heme iron is stabilized within the apolipoprotein, reducing its vulnerability to temperature changes while maintaining the holoprotein structure. Previously, it was demonstrated that heme-free globin is unstable and rapidly precipitates at room temperature. When cyanide is added to and bound to the heme group of penta-coordinate hemoglobin, a significant reduction in hemoglobin precipitation is observed even after exposure to 50°C for 20 hours. The ability of ligands to stabilize hemoglobin under severe and prolonged thermal stress by immobilizing the sixth heme iron bond has been demonstrated by the effects of carbon monoxide and cyanide on a hemoglobin solution heated at 65°C for 2.5 hours (Rossi-Fanelli et al. 1958). 11 In the case of hexa-coordinate hemoglobin, the sixth heme iron bond is already "fixed" because it forms a covalent bond with the apolipoprotein, making these proteins more stable while securely holding the heme group within the protein.

[0171] Furthermore, as expected, five-coordinate myoglobin derived from beef has low stability.

[0172] However, hexa-coordinate phytoglobins derived from maize (SEQ ID NOs. 28 and 29) exhibit low stability comparable to that of myoglobin. This indicates that stability differs even within hexa-coordinate heme or phytoglobin proteins, and that the three phytoglobins derived from the Caryophyllaceae family have similarly improved stability.

[0173] In contrast, five-coordinate soy leghemoglobin (SEQ ID NO: 35) denatured at 64°C by UV-Vis spectroscopy, releasing heme iron. A similar phenomenon was observed with bovine hemoglobin (SEQ ID NO: 48), another five-coordinate hemoglobin derived from meat. This hemoglobin showed significantly higher storage stability at 4°C than at 23°C, but denatured after 40 days regardless of storage temperature. Furthermore, exposure to temperature cycles of 25°C and 65°C rapidly resulted in structural changes that led to denaturation (Bhomia et al. (2016)). 12The thermal instability of these two five-coordinate heme or phytoglobin proteins causes the release of heme iron, which plays a role in catalyzing the creation of the characteristic flavors and aromas of meat and meat substitutes. 13 However, as mentioned above, five-coordinate hemoglobin is highly toxic due to its instability and reactivity, and large doses are not advisable for health reasons.

[0174] Example 3: Stability Evaluation - DNA Cutting Free hemoglobin acts as a "Fenton" reagent, making it a biologically harmful molecule. Therefore, other biomolecules 14 ,cell 15 , organization 16 It catalyzes the generation of hydroxyl radicals that directly act on DNA. The rate of radical formation depends on the intrinsic reactivity of the hemoglobin molecule. One way to measure this reactivity is to expose supercoiled DNA (SC) to free hemoglobin and then track its degradation into open-ring DNA (OC) and linear DNA (L). Highly reactive and unstable hemoglobin degrades DNA faster than stable hemoglobin.

[0175] method DNA cleavage experiments were performed in PCR tubes with a final volume of 20 μl. The cleavage reaction system consisted of buffer (20 mM sodium phosphate, pH 7.2), supercoiled plasmid DNA (pUC18, 1.25 ng / μl), and oxidized hemoglobin (Fe(III)). The reaction temperature was 37°C.

[0176] Oxyhemoglobin was obtained by adding potassium ferrocyanide. Excess potassium ferrocyanide was removed by passing the sample through a PD-10 desalting column packed with Sephadex G-25 resin (Cytiva).

[0177] DNA cleavage results were analyzed by agarose electrophoresis. After 4 hours of reaction, the samples were collected, mixed with DNA loading stain 6x (Thermo Fisher Scientific), and loaded onto a 1% agarose gel containing gel red dye (Biotium). Electrophoresis was performed in TAE buffer at 100V for 90 minutes. The gels were imaged using the GelDoc XR system (BioRad), and the relative amounts of complete plasmids (also known as supercoiled (SC)) and partially or completely degraded plasmid DNA (also known as ring-opened (OC) and linear (L)) were estimated using Quantity One software (BioRad). An unpaired, unequal variance t-test (Welch's t-test) was used to compare the amount of residual SC DNA after incubation with hexa-coordinate hemoglobin with the amount of residual SC DNA after incubation with recombinant adult hemoglobin (five-coordinate). A p<0.05 result was considered statistically significant. As a control, incubation of SC DNA without hemoglobin was used.

[0178] Results and Conclusions DNA cleavage was measured by quantifying the remaining SC and OC / L plasmid DNA (pDNA) after incubation at 37°C for 4 hours. Highly reactive hemoglobin would generate high levels of radicals, damaging DNA and reducing the amount of remaining SC pDNA. The cleavage ability of three types of hexacoordinate hemoglobin was compared with recombinant pentacoordinate hemoglobin (adult hemoglobin, rHbA) (Table 3 and Figure 4). The results showed that significantly less SC DNA remained after incubation with recombinant human adult hemoglobin (pentacoordinate) compared with the other three types of hexacoordinate hemoglobin (p<0.05) (Figure 4). No significant differences were observed among the three types of hexacoordinate hemoglobin (p>0.05). DNA cleavage experiments showed that six-coordinate hemoglobin derived from sugar beet, spinach, and quinoa (all belonging to the Amaranthaceae family of the Caryophyllales order) exhibited significantly less DNA degradation than recombinant human adult hemoglobin, which is a five-coordinate protein.

[0179] These results provide new insights into the positive effects of hexa-coordination on hemoglobin stability and demonstrate that hexa-coordinated plant hemoglobin is less reactive than penta-coordinated hemoglobin. [Table 4]

[0180] Example 4: Bioavailability of hexacoordinate phytoglobin from plants of the Caryophyllales (Amaranthaceae) Iron deficiency and anemia (IDA) is a disease affecting 2 billion people worldwide. Currently, IDA is treated with known iron supplements, but the iron used in these products is inorganic iron, or elemental iron. Elemental iron has low absorption rates, and most of it generates reactive oxygen species that damage cells, tissues, and organs as it passes through the digestive system, causing side effects such as digestive problems, abdominal pain, nausea, and constipation. Furthermore, it leaves a metallic aftertaste in the patient's mouth. As a result, fewer than 4 out of 10 patients complete treatment, and the rest remain iron deficient or anemic. Notably, IDA is a recurrent disease prone to relapse even after treatment, making continuous iron supplementation necessary for those affected.

[0181] The primary and best source of iron for humans is found in animal-derived foods, particularly red meat. There, iron exists in its organic form, heme iron, bound to proteins (hemoglobin and myoglobin). However, because heme iron has a loose binding relationship with apolipoproteins (due to its five-coordinate structure), it is easily released, initiating toxic tumorigenic reactions as described in Example 1.

[0182] On the other hand, plant hemoglobin, such as that of the Caryophyllales (Amaranthaceae), is less reactive and more stable because it retains heme iron within the apolipoprotein due to its strong hexa-coordinate structure.

[0183] However, stability alone is not enough to make a plant a sufficiently good iron source. The iron contained in plant hemoglobin must also have bioavailability, that is, the property of being easily absorbed by the body. Therefore, the bioavailability of iron in these plant proteins was tested.

[0184] Next, we describe a bioavailability testing method for heme iron derived from plant hemoglobin using Caco-2 cells. These cells are a standard in vitro model of iron absorption, and this method mimics the entire digestive process, starting from the mouth and ending in the duodenum and proximal jejunum, which are iron absorption sites. This method is robust and produces results that are consistently consistent with human studies.

[0185] method The bioavailability of iron in hemoglobin derived from sugar beet, spinach, and quinoa was measured using a combination of Caco-2 cell lines and in vitro digestion techniques. Specifically, proteins were subjected to simulated gastric digestion followed by enteric digestion in the presence of a Caco-2 cell monolayer. This model not only provides an indicator of absorption via the biological component Caco-2 cell monolayer but also measures iron solubility (Glahn et al. (1998)). 17 This approach utilizes ferritin formation in Caco-2 cells as a marker for iron absorption, avoiding the cost and methodological problems associated with isotopic labeling. Furthermore, the ferritin formation approach is a highly sensitive and cost-effective marker for iron absorption, dramatically improving the system's throughput.

[0186] This procedure consists of four parts, and the details have been previously published (Glahn et al. (2017)). 18The following is an overview. First, the iron content in the sample is measured. For this purpose, a portion of the sample is freeze-dried, and organic matter is decomposed by acid (HNO3 and HClO4) and heat treatment. Iron content is measured using inductively coupled plasma atomic emission spectrometry (ICP-AES). The second stage is in vitro digestion. Starting with the gastric phase, the sample is digested into peptides in an acidic environment of pH 2.0, then the pH is raised to 5.5-6.0, and a pancreatin-bile extract solution is added. At this point, the mixture is called the "digested product". In the third stage, the digested product is added to a monolayer of Caco-2 cells and cultured at 37°C for 22 hours. After that, the cells are aspirated and collected, and subjected to sonication. Cellular proteins were measured using the semi-micro-adapted method of the Bio-Rad DC protein assay kit (Bio-Rad Laboratories). Finally, in the fourth stage, ferritin in Caco-2 cells was measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit. This kit is the same type used for measuring human ferritin levels in clinical settings.

[0187] Data analysis was performed using analysis of variance and Tukey's post-hoc test to determine statistical significance (p<0.05) between samples. Values ​​are shown as mean standard errors (SEM), and the study consisted of three independent replicated experiments.

[0188] Results and Conclusions The results were expressed as ferritin levels (μg) per 1 mg of total protein and per unit of iron, representing the amount of ferritin formed by Caco-2 cells. Differences were observed between samples regarding ferritin / mg protein, with spinach (SO) having the lowest ferritin production relative to total protein and sugar beet (Bv) having the highest (Table 4). This value (ferritin / protein) is calculated based on the amount of intracellular protein during the assay, not the amount of protein added to the cells. Therefore, this value may vary between samples because the amount of ferritin produced depends on the amount of iron in the sample and the amount absorbed. Consequently, if cells receive different amounts of iron, the ferritin level per unit of protein (cell mass) will differ. To directly evaluate the amount of absorbed iron, it is more appropriate to use the amount of ferritin (μg) per unit of iron (μg). This value will be low if the iron is not bioavailable (not absorbed). No significant differences were observed between different samples in the amount of ferritin formed per 1 μg of iron. This indicates that iron contained in plant hemoglobin is absorbed by mammalian cells at a similar rate to iron from beef-derived myoglobin (Figure 5). [Table 5]

[0189] Iron is a nutrient whose absorption is affected by food components. These components can enhance or inhibit the bioavailability of iron. Figure 5B shows the results of the effects of two common foods on iron absorption. The foods tested were a plant-based beverage and sweet potato baby food.

[0190] The results show that hexa-coordinate phytoglobins derived from spinach, quinoa, and sugar beets all exhibit bioavailability equivalent to that of myoglobin in beef, the most common iron source in red meat, and therefore are suitable as iron supplements.

[0191] Corn-derived phytoglobin also showed comparable bioavailability to myoglobin, but as the aforementioned temperature stability test example shows, corn-derived phytoglobin is less suitable as a supplement. The bioavailability of iron in plant hemoglobin is comparable to that of myoglobin in beef, which is the most common iron source in red meat. Taken together, the experimental results show that the phytoglobin according to the present invention possesses both stability and bioavailability.

[0192] Example 5: Taste In Example 4, phytoglobins derived from sugar beets, spinach, and quinoa were demonstrated to be highly bioavailable iron sources. Therefore, these can be used as organic, bio-based iron supplements, and as food ingredients, provided they do not affect the taste of the food additive, for the prevention and treatment of iron deficiency and anemia (IDA).

[0193] Sensory tests were conducted to evaluate whether these proteins have a taste or alter the taste of other foods. Phytoglobin proteins were tested in three food matrices: blueberry juice, rosehip juice, and plum juice, with water also used as a control.

[0194] method Panelists were presented with two sets of opaque cups containing 200ml each of juice. One set had 1ml of 0.3mM phytoglobin added, and the other set had 1ml of water added. This was done to prevent the panelists from perceiving a difference between the phytoglobin-added and unadded cups by diluting the sweetness components in the juice. The same setup was used for the control (water), with one cup containing only water and the other containing 1ml of phytoglobin. A total of four sets of cups were presented to the panelists, and they were asked to evaluate the taste of each set. The objective was to determine whether the panelists could perceive the change in taste caused by the addition of phytoglobin.

[0195] Panel members were asked to rate the taste of the beverages on a scale of 1 to 5 based on the following description: 1 = very bad taste, 3 = neutral taste, and 5 = very good taste. Results were compared pair by pair. This was to determine whether the addition of phytoglobin affected the perception of taste compared to the same juice with only water added. The delta value (Δ value) was determined by calculating the difference in the rating scores for each beverage pair (phytoglobin added or water only).

[0196] Results and conclusions In all cases, for all beverages, including the control water, the panelists assigned the same rating to the presented pair of cups. This means that the Δ value for all beverages was zero (0). These results confidently indicate that the addition of phytoglobin did not alter the taste of the juice, as the panelists could not perceive any change in the juice's flavor. Some commented that they recalled tasting the juice before, but noticed that it was sweeter or had a stronger taste, suggesting that something had been added to both cups. Others stated that the juice tasted diluted. Aside from this detail, they could not perceive any difference in taste even after being told which cup had phytoglobin added. [Table 6]

[0197] None of the panelists could distinguish any difference in taste between cups of juice with phytoglobin added or with water added. Therefore, we concluded that phytoglobin derived from sugar beets, spinach, and quinoa does not affect the taste of the food to which it is added.

[0198] This means that these proteins can be used as food ingredients to enhance the nutritional value of foods. Furthermore, they can be used in their highest concentration form without the need for coatings to mask metallic tastes. This is in contrast to current iron supplements that leave a metallic taste after ingestion. Iron supplements based on these proteins can be supplied in liquid form (e.g., drops or syrup) or tablet form, avoiding the absorption limitations imposed by coatings, as is the case with capsules.

[0199] This lack of flavor is a significant difference compared to five-coordinate hemoglobin (e.g., soy leghemoglobin, bovine myoglobin, hemoglobin) found in meat substitutes and animal-based foods. These catalyze the production of meat-specific flavors and aromas as a result of heme iron loss.

[0200] These proteins according to the present invention are tasteless because iron is released from the proteins only in the duodenum and upper jejunum, which are iron absorption sites, by the iron transport system of the digestive tract. Consequently, the proteins of the present invention have a very low oxidation rate.

[0201] Example 6: Alignment of variable loops (CD region) [Table 7]

[0202] Sequence alignment was performed between the sequences of the variable loop (CD region). This region is highly conserved, and differences within this region are thought to be reflected in variations in molecular stability. [ka]

[0203] Example 7: Further alignment of the CD loop, which is the main structural region. While not bound by theory, it was hypothesized that the insertion of heme groups and their stability within globin proteins depend on the flexibility and componentity provided by the CD loop and its key amino acids. These amino acids play a role in fixing and stabilizing the heme within the pocket. The CD loop controls the opening and closing of helix E during heme insertion during protein synthesis, i.e., during holoprotein formation by heme group insertion into the apolipoprotein (globin protein). The CD loop also controls the rate of heme loss and auto-oxidation throughout the holoprotein's lifespan. Furthermore, the CD loop influences the binding of external ligands (e.g., oxygen, carbon monoxide, carbon dioxide, nitric oxide, sulfates, nitrites, nitrates, cyanides, etc.) to the heme-containing pocket, determining, for example, oxygen transporters, nitrite oxidases, nitrite reductases, peroxidases, sulfide reducers, carbon monoxide carriers, other gas carriers, and other final functions. Given the importance of the CD loop, expanded samples of known and hypothetical CD loops were compared with each other.

[0204] Heme or phytoglobin proteins are classified into three main groups: five-coordinate, six-coordinate, and truncated hemoglobins. Five-coordinate proteins are typically responsible for oxygen transport and scavenging within the cells and tissues in which they express. They are also relatively evolutionarily recent. Six-coordinate proteins, on the other hand, do not necessarily transport oxygen but sense it. These proteins are primarily involved in nitric oxide binding and are evolutionarily older.

[0205] Five-coordinate heme and phytoglobin proteins are found in aerobic animals (e.g., mammals) and leguminous plants, and are collectively called leghemoglobin or symbiotic hemoglobin.

[0206] Hexacoordinate proteins (also called non-symbiotic hemoglobins) are present in all organisms, from archaea to humans, and are classified into two groups: Class 1 and Class 2. This classification is based on differences in protein sequence and oxygen affinity. Class 1 proteins are thought to have a higher oxygen affinity than Class 2 proteins. 2Furthermore, class 2 proteins are phylogenetically closer to leghemoglobin and oxygen-transporting hemoglobin compared to class 1 proteins. To our knowledge, no structural domain or sequence that can distinguish the two classes has been identified. Here, we hypothesize that the CD loop is a crucial region that distinguishes class 1 and class 2 heme and phytoglobin proteins and may contribute significantly to the formation of six-coordinate or five-coordinate heme or phytoglobin proteins. [Table 8]

[0207] method Sequences of known and hypothetical heme-binding proteins were obtained from publicly available databases. The sequences were taken from a wide range of species, from animals to plants and bacteria. Based on the general structure of hemoglobin proteins, the sequences were analyzed to identify the location of the CD loop. After identification, the sequences were extracted and cross-compared using the publicly available online software Clustal Omega. Clustal Omega is a multi-sequence alignment program that generates sequence alignment using seed guide trees and HMM profile-versus-profile techniques.

[0208] Results and Discussion Alignment results showed that the CD loop contains at least two conserved amino acids (P and F at positions 01 and 08) and a semi-conserved amino acid at position 03, compared to a reference CD loop. In addition to these, at least four key positions were identified in regions crucial for the expression and stability of globin proteins containing the heme group. These positions are 08, 13, 16, and 21 (and 08, 13, 18, and 23 in species with longer loops) (Figure 7).

[0209] The identity percentage matrix showed significant variability, with some instances where the similarity between CD loop sequences dropped to as low as 11%. This suggests that, apart from the aforementioned key locations, this region may be a major structural component distinguishing different types and classes of heme or phytoglobin proteins. This result was confirmed by phylogenetic analysis using only CD loops (Figure 8A). This phylogenetic tree shows that even using only CD loops, different types or classes of heme or phytoglobin proteins can be classified according to their classification. To further verify this, the next step involved substituting the CD loops of heme or phytoglobin proteins with the CD loops of the parent sequence (SEQ ID NO: 56) to see if the entire protein with the CD loop of SEQ ID NO: 56 could still be classified into different types and classes. The results in Figure 8B show that the distinction between different classes of heme phytoglobin and iron phytoglobin is not as clear as in Figure 8A. Therefore, it can be concluded that the CD loop may be a key region distinguishing between five-coordinate hemoglobin and six-coordinate hemoglobin. Therefore, this operation can reversibly convert heme or phytoglobin proteins, either partially or completely, from class 1 to class 2, and from five-coordinate to six-coordinate proteins (Figure 8B). [Table 9]

[0210] Sequence alignment was performed between variable loop (CD loop) sequences of globin proteins containing heme groups. [ka] TIFF2026513230000017.tif126159

[0211] Example 8. Modification of the CD loop and its effect on heme-binding protein expression. While not bound by theory, it has been hypothesized that the CD loop, when generated outside of its natural source, is related to the expression and synthesis of heme-binding holoproteins (hemoglobin and phytoglobin, which are formed when globin apoproteins bind to heme iron groups).

[0212] method To test this hypothesis, the CD loop was modified by inserting mutations (i.e., amino acid substitutions) at various positions, including those where sequence matching was confirmed. The modified CD loop was then added by substituting it for the CD loop in the reference sequence (SEQ ID NO: 1). The resulting gene was synthesized, cloned into an expression vector, and then transformed into competent E. coli cells. By culturing the transformed cells in concentrated liquid medium, mutant proteins containing the modified CD loop were generated.

[0213] Results and Discussion Since the heme group bound to globin apolipoprotein contains iron, iron-containing expressed proteins appear red, resulting in a red cell pellet. As expected, the degree of redness of the cell pellet confirmed that modifications to the CD loop affected the expression and production of hemoglobin protein. This confirmed that specific mutations have an enhancing or inhibiting effect on the expression and production of heme-binding proteins. Very poor effects resulted in little to no red pellet, while very good effects resulted in a very dark red pellet. "Poor" and "Good" represent intermediate color categories between "Very Poor" and "Very Good" (Figure 9), with "Good" being redder than "Poor." [Table 10]

[0214] Example 9: CD loop modification and its effect on heme stability The insertion and stabilization of heme groups in globin apolipoproteins affect their expression and synthesis. This effect is thought to be due to improved molecular stability of heme-binding proteins. This beneficial effect can provide resistance to physicochemical events such as high temperatures, as a stable CD loop contributes to the stability of the heme group within the active site of the hemoglobin protein.

[0215] method Three mutants exhibiting very good expression levels and one mutant exhibiting very poor expression results were selected for synthesis in 2 liters of concentrated culture medium. After synthesis, the cells were lysed and the proteins were purified by chromatography. The thermal stability of the proteins (10-20 μL of sample at a concentration of 10-50 μM in 50 mM Tris-HCl, pH 8.5, n>3) was analyzed by nano-differential scanning fluorescence (nanoDSF) using a Prometheus NT.48 instrument (NanoTemper Technologies) equipped with an aggregation optical system. The excitation LED intensity (wavelength 280 nm) was set to 20%. The fluorescence intensity of the samples at 350 nm (F350) and 330 nm (F330) was recorded while the temperature was increased from 20 to 95°C at a rate of 7°C / min. The melting point (Tm) of the proteins was obtained by the first derivative of the F350 / F330 fluorescence ratio plotted against temperature.

[0216] Results and Discussion Very good hemoglobin proteins, together, have an average melting temperature T o The (Tm) temperature exceeds 80°C, indicating high thermal stability compared to other proteins (compared to Table 2 above). On the other hand, the very poor hemoglobin protein had a melting temperature of less than 70°C. This is an expected result because this very poor protein has a mutation (rigid proline amino acids are replaced with small, flexible glycine), which increases the flexibility of the CD loop, resulting in an inability to stabilize the heme group inside the globin apoprotein. This lack of stability reduced the intrinsic stability of this very poor hemoglobin. [Table 11]

[0217] Example 10: Alternative signal peptide In particular, by adding various signal peptides, including heterologous signal peptides, to the N-terminus of recombinant heme or phytoglobin, protein transport to specific sites both intracellularly and extracellularly becomes possible. In cases where molecules already possess signal peptides (e.g., SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 16), these can be substituted with other signal peptides. One application of signal peptide addition is to transport heme proteins outside the producing cell, facilitating downstream processes (i.e., separation from other non-heme molecules). Signal peptides usable to induce the transport of recombinant heme or phytoglobin include SEQ ID NOs: 203-213.

[0218] Example 11: Expression of recombinant heme or phytoglobin in surrogate host strains In addition to Escherichia coli, recombinant heme or phytoglobin has been successfully expressed in the following using the genes disclosed herein and methods known in the art: Arabidopsis thaliana, Sciadophylloides thaliana, tobacco, wheat, barley, rice, quinoa, sugar beet, soybean, P. pastris, Pichia species, budding yeast, Yarrowia species, Y. liporitica, Kluiveromyces lactis, brewer's yeast, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasi, Saccharomyces crivelli, Saccharomyces bensis, Saccharomyces obiformis, Asibia gossipii, H. polymorpha, Hansenula species, C. glutamicum, Bacillus subtilis, Reticulum vulgare, Ptida, and Pseudomonas aeruginosa.

[0219] Example 12: Stabilization of recombinant proteins with salt and / or antioxidants Adding a 500 mM salt selected from NaCl, ammonium sulfate, CaCl2, KCl, and MgCl2, or a 25 mM antioxidant selected from ascorbic acid, 2-mercaptoethanol, dithiothreitol (DTT), superoxide dismutase (SOD), catalase, β-carotene, lycopene, glutathione, melatonin, estrogen, and biquinol-10, N-acetylcysteine, lipoic acid, zinc, selenium, copper, quercetin, catechin, cortisone, estradiol, estriol, and α-tocopherol to a recombinant protein powder or liquid preparation improves its stability and the absorption of iron contained within the heme group.

[0220] References 1.BLASTP online tool(https: / / blast.ncbi.nlm.nih.gov) 2.Leiva Eriksson N, Reeder B, Wilson M, Bulow L, Sugar beet haemoglobin:reactions with nitric oxide and nitrite reveal differential roles for nitrogen metabolism.Biochem J 31 2019;476(14):2111-2125. 3.Sinclair,PR,Gorman,N.and Jacobs,JM(2001)Measurement of haem concentration.Curr.Prot.Toxicol.Chapter 8,Unit 8.3. 4. Sadrzadeh, SM, Graf, E., Panter, SS, Hallaway, PE, and Eaton, JW (1984). Haemglobin. A biologic fenton reagent. J. Biol. Chem. 259, 14354-14356. 5.Fiorito V,Chiabrando D,Petrillo S,Bertino F and Tolosano E (2020)The Multifaceted Role of Haem in Cancer.Front.Oncol.9:1540. 6.FAQ_Impossible Foods webpage;https: / / faq.impossiblefoods.com / hc / en-us 7.M.S Hargrove,J.S Olson.The stability of holomyoglobin is determined by haem affinity.Biochemistry,35(1996),pp.11310-11318. 8.M.S.Hargrove,et al.Characterization of recombinant soybean leghaemglobin a and apolar distal histidine mutants.J.Mol.Biol.,266(1997),pp.1032-1042. 9.M.Renerre,M.Anton,P.Gatellier.Autoxidation of purified myoglobin from two bovine muscles.Meat Science,32(3)(1992),pp.331-342. 10.M.Simons et al.Comparison of the oxidative reactivity of recombinant fetal and adult human haemglobin:implications for the design of haemglobin-based oxygen carriers.Biosci Rep 31 August 2018;38(4):BSR20180370. 11.Rossi-Fanelli,A.,E.Antonini,and A.Caputo.1958.Studies on the structure of haemglobin.I.Physico-chemical properties of human globin.Biochim.Biophys.Acta.30:608. 12.R.Bhomia,V.Trivedi,N.J.Coleman,J.C.Mitchell.The thermal and storage stability of bovine haemglobin by ultraviolet-visible and circular dichroism spectroscopies.J.Pharm.Anal.,6(4)(2016),pp.242-248. 13.https: / / www.fda.gov / media / 124351 / download 14.Chakane S,Matos T,Kettisen K,Bulow L.Fetal haemglobin is much less prone to DNA cleavage compared to the adult protein.Redox Biol.2017,12:114-120. 15.Glei M,Klenow S,Sauer J,Wegewitz U,Richter K,Pool-Zobel BL.Haemglobin and hemin induce DNA damage in human colon tumor cells HT29 clone 19A and in primary human colonocytes.Mutat Res. 2006,22;594(1-2):162-71. 16.Alayash AI,Patel RP,Cashon RE.Redox reactions of haemglobin and myoglobin:biological and toxicological implications.Antioxid Redox Signal.2001.3(2):313-27. 17. Glahn RP, Lee OA, Yeung A, Goldman MI, Miller DD. Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion / Caco-2 cell culture model.J Nutr.1998 128(9):1555-61. 18. Glahn R, Tako E, Hart J, Haas J, Lung'aho M, Beebe S. Iron Bioavailability Studies of the First Generation of Iron-Biofortified Beans Released in Rwanda.Nutrients.2017 21;9(7):787. 19. M. Schultz and JP Burton:Chapter 5 - Escherichia coli Nissle 1917;The Microbiota in Gastrointestinal Pathophysiology;Implications for Human Health,Prebiotics,Probiotics,and Dysbiosis 2017,Pages 59-69.

[0221] item This disclosure further discloses the following first set of itemized embodiments: 1. Recombinant phytoglobin containing a hexa-coordinate heme group, having at least 86% identity with amino acid sequence number 5; or a fragment, variant, or fusion thereof that retains a hexa-coordinate heme group, for use in the treatment or prevention of iron deficiency in a subject. 2. Recombinant phytoglobin according to item 1, having sequence identity of at least 87%, for example, at least 88%, for example, at least 89%, for example, at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99% with the sequence of sequence number 5. 3. Recombinant phytoglobin as described in item 1 or 2, which is a class 1 non-symbiotic hemoglobin. 4. Recombinant phytoglobin as described in any one of items 1 to 3, derived from a carnation substratum, such as spinach or quinoa. 5. Recombinant phytoglobin as described in any one of items 1 to 4, comprising or consisting of amino acid sequence number 5, or a fragment, variant, or fusion thereof that retains a hexacoordinate heme group. 6. Recombinant phytoglobin as described in any one of items 1 to 5, comprising or consisting of amino acid sequence number 4, or a fragment, variant, or fusion thereof that retains a hexacoordinate heme group. 7. Recombinant phytoglobin described in any one of items 1-6, which has improved stability compared to five-coordinate hemoglobin. 8. Auto-oxidation rate less than 0.2, for example less than 0.15, for example in the range of 0.01 to 0.18, or in the range of 0.01 to 0.15, for example about 0.1h -1 Recombinant phytoglobin as described in any one of items 1 to 7. 9. Recombinant phytoglobin as described in any one of items 1 to 8, which does not have a metallic taste or has a neutral taste. Recombinant phytoglobin according to any one of items 1 to 9, having a melting temperature of 10.68°C or higher, for example, in the range of 68 to 75°C, for example, in the range of 70 to 76°C. 11. Recombinant phytoglobin for use in the treatment or prevention of target iron deficiency, further comprising a hexacoordinate heme group and a CD variable loop as described in any one of items 1 to 10, wherein the region or variable region has at least 60% sequence identity with SEQ ID NO: 111. 12. Recombinant phytoglobin for use in the treatment or prevention of target iron deficiency, comprising a hexa-coordinate heme group and further comprising a variable region in which the CD variable loop has at least 90% sequence identity with SEQ ID NO: 111. 13. Recombinant phytoglobin for use in the treatment or prevention of target iron deficiency, comprising a hexacoordinate heme group and further comprising a CD variable loop, wherein the CD variable loop comprises SEQ ID NO: 111 having one substitution or two substitutions. 14. Recombinant phytoglobin having a hexa-coordinate heme group, for use in the treatment or prevention of target iron deficiency, wherein the variable domain contains or comprises SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116. 15. A composition for use in food products comprising at least one recombinant phytoglobin as described in any one of items 1 to 14. 16. A composition comprising at least one recombinant phytoglobin described in any one of items 1 to 14, for use in the treatment or prevention of iron deficiency. 17. The composition according to item 15 or 16, comprising at least 0.002% (by weight / by weight) of recombinant phytoglobin. 18. The composition according to item 15 or 16, comprising at least 0.1 μM, for example, 0.1 μM to 8 M of recombinant phytoglobin. 19. A composition according to any one of items 15 to 18, which does not have a metallic taste. 20. The composition according to any one of items 15 to 19, further comprising one or more reducing agents, such as one or more ascorbic acid (vitamin C), tocopherol, carotenoids, flavonoids, and glutathione. 21. A composition according to any one of items 15 to 20, further comprising at least one vitamin and / or mineral, for example, one or more selected from the group consisting of vitamin C, vitamin D3, vitamin E, vitamin B6, vitamin B12, folate, folic acid, biotin, zinc, copper, magnesium, selenium and combinations thereof. 22. The composition according to any one of items 15 to 21, having a formulation selected from the group consisting of tablets, capsules, liquids, drops, concentrates, powders, granules, and combinations thereof. 23. The composition according to any one of items 15 to 22, which is a feed, food, beverage, food ingredient and / or dietary supplement. 24. The composition according to item 23, which is a dietary supplement. 25. The composition according to item 23, which is a food ingredient. 26. The composition according to any one of items 15 to 21, which is a pharmaceutical composition selected from the group consisting of drugs, vaccines, personal care compositions, and combinations thereof. 27. The composition according to item 26, further comprising a pharmaceutically acceptable carrier. 28. The composition according to any one of items 15 to 23, which is used in a food composition. 29. A recombinant phytoglobin comprising a hexa-coordinated heme group having at least 60% identity with amino acid sequence number 5, or a fragment, variant, or fusion thereof that retains the hexa-coordinated heme group; or a composition comprising the recombinant phytoglobin, for use as a drug. 30. The recombinant phytoglobin and / or the composition comprising the recombinant phytoglobin according to any one of items 15 to 28, for use in the treatment, improvement or prevention of iron deficiency in humans and animals. 31. The recombinant phytoglobin and / or the composition comprising the recombinant phytoglobin for use according to any one of items 15 to 28, wherein the use is for the treatment, improvement or prevention of iron deficiency in women selected from the group consisting of pregnant women, lactating women, and women of reproductive age. 32. The recombinant phytoglobin and / or the composition comprising the recombinant phytoglobin for use according to any one of items 15 to 28, wherein the use is for the treatment, improvement or prevention of iron deficiency in patients having a chronic disease selected from the group consisting of chronic diseases such as endometriosis, dysmenorrhea, menorrhagia, cancer, chronic kidney disease, diabetes, obesity, celiac disease, inflammatory bowel disease, gastrointestinal diseases such as peptic ulcer, hereditary diseases such as hemolytic anemia, autoimmune diseases, and / or diseases associated with aging. 33. The use is for the treatment, improvement or prevention of iron deficiency in a subject having an acute disease such as an acute disease selected from the group consisting of trauma, gastrointestinal surgery, gastrointestinal bleeding, renal insufficiency, and acute toxicity. A recombinant phytoglobin and / or a composition containing recombinant phytoglobin for the use according to any one of items 15 to 28. 34. The use is for the treatment, improvement or prevention of iron deficiency in a human who ingests a plant-based diet selected from the group consisting of vegetarians, vegans and / or flexitarians. A recombinant phytoglobin and / or a composition containing recombinant phytoglobin for the use according to any one of items 15 to 28. 35. The use is for the treatment, improvement or prevention of iron deficiency in animals such as companion animals, poultry or livestock. A recombinant phytoglobin and / or a composition containing recombinant phytoglobin for the use according to any one of items 15 to 28. 36. A composition containing at least 0.002% (weight / weight) of at least one phytoglobin having at least 60% identity with amino acid sequence number 5, or a fragment, variant, or fusion thereof that retains a hexa-coordinate heme group, for use in the treatment or prevention of iron deficiency. 37. A liquid composition containing at least one phytoglobin containing a hexa-coordinate heme group, having at least 86% identity with amino acid sequence number 5, or a fragment, variant, or fusion thereof that retains a hexa-coordinate heme group, for use in the treatment or prevention of iron deficiency in a subject.

[0222] The present disclosure further discloses the following set of itemized second embodiments: 1. A recombinant phytoglobin containing a hexa-coordinate heme group, having at least 86% identity with amino acid sequence number 5, or a fragment, variant, or fusion thereof that retains a hexa-coordinate heme group, for use in food. 2. Recombinant phytoglobin according to item 1, having sequence identity of at least 87%, for example, at least 88%, for example, at least 89%, for example, at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99% with the sequence of sequence number 5. 3. Recombinant phytoglobin as described in item 1 or 2, comprising or consisting of amino acid sequence number 5, or a fragment, variant, or fusion thereof that retains a hexacoordinate heme group. 4. Recombinant phytoglobin as described in any one of items 1 to 3, comprising or consisting of amino acid sequence number 4, or a fragment, variant, or fusion thereof that retains a hexacoordinate heme group. 5. Recombinant phytoglobin for use in the treatment or prevention of target iron deficiency, wherein a region or variable region comprising a hexacoordinate heme group and further comprising the CD variable loop described in the preceding paragraph has at least 60% sequence identity with SEQ ID NO: 111. 6. A composition for use in food products comprising at least one recombinant phytoglobin as described in any one of items 1 to 5. 7. A composition comprising at least one recombinant phytoglobin described in any one of items 1 to 6, for use in the treatment or prevention of iron deficiency. 8. The composition according to item 6 or 7, comprising at least 0.1 μM, for example, 0.1 μM to 8 M of recombinant phytoglobin. 9. A composition according to any one of items 6 to 8, which does not have a metallic taste. 10. A liquid composition for use in the treatment or prevention of iron deficiency in a subject, comprising at least one phytoglobin containing a hexa-coordinate heme group, having at least 86% identity with amino acid sequence number 5; or a fragment, variant, or fusion thereof that retains a hexa-coordinate heme group.

[0223] This disclosure further discloses a set of third embodiments, itemized below: 1. A recombinant heme or phytoglobin protein having at least 50% identical amino acid sequence to the heme or phytoglobin contained in any one of Sequence IDs 1-55, containing C-helices and D-helices covalently linked to the CD loop region, and containing a six-coordinate or five-coordinate prosthetic heme group. 2. The recombinant protein described in item 1, wherein the CD loop comprises one or more modifications compared to the parent CD loop region, and the modified CD loop region binds more strongly to the prosthetic group than the parent's unmodified CD loop region. 3. The parent CD loop region is unique to the protein in question, a recombinant protein as described in item 1 or 2. 4. The recombinant protein described in any one of items 1 to 3, wherein the parent CD loop region is located in the recombinant heme or phytoglobin protein at positions 52 to 78 of SEQ ID NO: 1, and optionally at positions 52 to 72 of SEQ ID NO: 1. 5. The CD loop region has an amino acid sequence that is at least 50% identical to the CD loop region contained in any one of sequence numbers 56-147, and is a recombinant protein as described in any one of items 1-4. 6. A recombinant protein according to any one of items 2 to 5, wherein the modification of the CD loop region is selected from the deletion, substitution, and / or addition of one or more amino acids. 7. Recombinant proteins described in any one of items 2 to 6, wherein the modification of the CD loop region alters the flexibility or rigidity of the CD loop region compared to the parent CD loop region. 8. A recombinant protein according to any one of items 2 to 7, wherein the CD loop region contains two or more substitutions, e.g., three or more, e.g., four or more, compared to the parent CD loop region. 9. A recombinant protein according to any one of items 2 to 8, wherein the amino acid modification in the CD loop corresponds to one or more modifications selected from S13G, S13P, V15G, A25P, and A27G in the CD loop as described in SEQ ID NO: 56. 10. A recombinant protein as described in any one of items 2-8, wherein the amino acids corresponding to the positions P1, A3, F7, S8, S13, P16, P21 and / or P25 of SEQ ID NO: 56 are either conserved or conservedly substituted. 11. A recombinant protein as described in any one of items 2-10, wherein the CD loop contains the amino acid sequence described in any one of sequence numbers 123, 127, 128, 129, 130, 131, 133, 134, 135, 137, 139, 141, 142, or 145. 12. A recombinant protein as described in item 11, wherein the CD loop contains the amino acid sequence described in any one of sequence numbers 127, 128, 130, 131, 135, 139, 141, 142, or 145. 13. A recombinant protein as described in any one of items 1 to 12, further comprising a signal peptide that directs the secretion of a recombinant protein from a cell. 14. Recombinant proteins described in item 13, whose expression is directed by a signal peptide within microbial cells. 15. Recombinant proteins as described in item 13 or 14, in which the signal peptide is heterogeneous to the recombinant protein. 16. Recombinant proteins as described in any one of items 13-15, wherein the microbial cell is a bacterial cell or a fungal cell. 17. A recombinant protein according to any one of items 13 to 16, wherein the signal peptide has at least 50% identical amino acid sequence to the signal peptide contained in any one of sequence numbers 203 to 213. 18. Recombinant proteins, which are non-natural or synthetic, as described in any one of items 1 through 17. 19. Non-natural or synthetic recombinant proteins are fusion proteins, recombinant proteins as described in item 18. 20. A recombinant protein as described in item 19, wherein the fusion protein contains two or more prosthetic molecular groups. 21. A recombinant protein described in any one of items 1-20, which is a class 1 or class 2 non-symbiotic hemoglobin. 22. Recombinant proteins derived from plants of the Caryophyllaceae subclass, as described in any one of items 1 to 21. 23. Recombinant proteins derived from spinach or quinoa, as described in item 22. 24. Recombinant proteins according to any one of items 1 to 23, comprising at least one post-translational modification selected from glycosylation or phosphorylation, compared to the corresponding native protein. 25. A composition comprising a recombinant protein as described in any one of items 1 to 24, and one or more carriers, drugs, additives and / or excipients. 26.1 The composition according to item 25, wherein one or more carriers, agents, additives and / or excipients are selected from salts, antioxidants and / or reducing agents. 27. The composition according to item 26, wherein the salt is selected from NaCl, ammonium sulfate, CaCl2, KCl, and MgCl2. 28. The composition according to item 26 or 27, wherein the amount of salt in the composition is between 10 μM and 2 M. 29. A composition according to any one of items 26 to 28, wherein the antioxidant is selected from ascorbic acid, 2-mercaptoethanol, dithiothreitol (DTT), superoxide dismutase (SOD), catalase, β-carotene, lycopene, glutathione, melatonin, estrogen, and biquinol-10 N-acetylcysteine, lipoic acid, zinc, selenium and / or copper salts, quercetin, catechin, cortisone, estradiol, estriol, and α-tocopherol. 30. A composition according to any one of items 26 to 29, wherein the amount of antioxidant in the composition is 10 μM to 1 M. 31. The composition according to item 26, wherein the reducing agent is selected from ascorbic acid (vitamin C), tocopherol, carotenoids, flavonoids, and glutathione. 32. The composition according to any one of items 25 to 31, further comprising at least one vitamin and / or mineral selected from vitamin C, vitamin D3, vitamin E, vitamin B6, vitamin B12, folate, folic acid, biotin, zinc, copper, magnesium, selenium, and combinations thereof. 33. The composition according to any one of items 25 to 30, containing at least 0.002% (weight / weight) of a recombinant protein. 34. The composition according to any one of items 25 to 30, containing at least 0.1 μM of a recombinant protein, for example, 0.1 μM to 8 M. 35. The composition according to any one of items 25 to 34, selected from food, feed, beverages, food ingredients, health supplements or nutritional supplements and / or pharmaceuticals. 36. The composition according to item 35, which is a nutritional supplement. 37. The composition according to item 35, which is a food ingredient. 38. The composition according to item 35, wherein the composition is suitable for children, the elderly, or subjects undergoing medical or surgical treatment, and is a nutritionally balanced food composition. 39. The composition according to any one of items 25 to 38, having no metallic odor or metallic taste. 40. The composition according to any one of items 25 to 39, formulated as a dry preparation, a liquid preparation, or a slurry or dispersant. 41. The composition according to item 40, having a dosage form selected from the group consisting of tablets, capsules, solutions, drops, concentrates, powders, granules, and combinations thereof. 42. The composition according to any one of items 25 to 41, further comprising a pharmaceutically acceptable carrier. 43. The composition according to any one of items 25 to 42, which is a pharmaceutical composition selected from the group consisting of drugs, vaccines, personal care compositions, and combinations thereof. 44. A non-therapeutic method for improving endurance or hypoxia tolerance in a subject, comprising administering to the subject an amount effective in improving the subject's endurance or hypoxia tolerance, comprising administering to the subject an amount effective in improving the subject's endurance or hypoxia tolerance in any one of items 1 to 24 or any one of items 25 to 43. 45. The method described in item 44, in which the subject is performing physical activity. 46. ​​The method described in item 45, for subjects who are climbing or mountaineering under low-oxygen conditions. 47. A non-therapeutic method for altering the skin color or texture of a subject, comprising administering to the subject skin in an amount effective to alter the skin color or texture of any one of the items described in 48. A method for treating, improving or preventing a disease or deficiency, comprising administering to a subject an amount effective for treating, improving or preventing the disease or deficiency of a recombinant protein described in any one of items 1 to 24 or a composition described in any one of items 25 to 43. 49. The method described in item 48, wherein the disease or deficiency is either an iron deficiency or a disease caused by iron deficiency. 50. Recombinant proteins as described in any one of items 1 to 24 or compositions as described in any one of items 25 to 43, for use in the treatment, improvement or prevention of a disease or deficiency, comprising administering to the subject an amount of recombinant protein or composition effective in improving or preventing the disease or deficiency. 51. Recombinant protein composition as described in item 50, wherein the disease or deficiency is either iron deficiency or a disease caused by iron deficiency. 52. Recombinant protein or composition according to item 50 or 51, wherein the recombinant protein is phytoglobin. 53. Recombinant protein or composition according to any one of items 50 to 52, wherein the recombinant phytoglobin contains a hexa-coordinate heme group and has an amino acid sequence that is at least 86% identical to the phytoglobin contained in SEQ ID NO: 1 or 5. 54. The recombinant protein or composition according to item 53, wherein the recombinant phytoglobin has an amino acid sequence that is at least 87%, for example, at least 88%, for example, at least 89%, for example, at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99% identical to the phytoglobin contained in the sequence of SEQ ID NO: 1 or 5. 55. A recombinant protein or composition according to any one of items 52 to 54, wherein the recombinant phytoglobin is a class 1 non-symbiotic hemoglobin. 56. Recombinant protein or composition according to any one of items 52 to 55, wherein the recombinant phytoglobin is derived from a plant of the Caryophyllaceae substratum, for example, spinach or quinoa. 57. Recombinant protein or composition according to any one of items 52 to 56, wherein recombinant phytoglobin is contained in or derived from SEQ ID NO: 4. 58. A recombinant protein or composition according to any one of items 50 to 57, wherein the recombinant protein contains a hexa-coordinate heme group and exhibits improved stability compared to penta-coordinate hemoglobin. 59. Recombinant protein is less than 0.2, for example less than 0.15, for example in the range of 0.01 to 0.18, or in the range of 0.01 to 0.15, for example about 0.1h. -1 A recombinant protein or composition according to any one of items 50 to 58, having an auto-oxidation rate of 60. A recombinant protein or composition according to any one of items 50 to 59, wherein the recombinant protein does not have a metallic taste or has a neutral taste. 61. The recombinant protein or composition according to any one of items 50 to 60, wherein the melting temperature of the recombinant protein is greater than 68°C, for example, in the range of 68 to 75°C, for example, in the range of 70 to 76°C, for example, greater than 76°C, for example, greater than 77°C, for example, greater than 78°C, for example, greater than 79°C, for example, greater than 80°C, for example, greater than 81°C, for example, greater than 82°C, for example, greater than 83°C, for example, about 84°C. 62. The recombinant protein or composition according to item 52, wherein the recombinant phytoglobin comprises a hexacoordinate heme group and further comprises a CD loop region having an amino acid sequence that is at least 60% sequence-identical to the CD loop region contained in SEQ ID NO: 56, 60, or 111. 63. A recombinant protein or composition according to item 62, wherein the CD loop region has an amino acid sequence that is at least 90% identical to the CD loop region contained in SEQ ID NO: 56, 60, or 111. 64. A recombinant protein or composition according to item 62 or 63, having an amino acid sequence comprising a CD loop region included in SEQ ID NO: 56, 60, or 111, wherein the CD loop region has one or more, two or more, three or more, or four or more amino acid substitutions. 65. A recombinant protein or composition according to any one of items 62 to 64, wherein the CD loop region has an amino acid sequence comprising or consisting of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 116. 66. Recombinant protein or composition as described in any one of items 50 to 65, wherein the subject is a blood donor. 67. Recombinant proteins or compositions according to any one of items 50 to 66, for which the target population is vegetarian, vegan, and / or flexitarian. 68. Recombinant proteins or compositions according to any one of items 50 to 67, wherein the subject is an animal, and in some cases, a pet, poultry, or livestock. 69. Recombinant proteins or compositions according to any one of items 50 to 67, wherein the subjects are women, and optionally selected from the group consisting of pregnant women, lactating women, and women of reproductive age. 70. Recombinant proteins or compositions according to any one of items 50 to 69, wherein the disease is selected from endometriosis, dysmenorrhea, menorrhagia, cancer, kidney disease, diabetes, obesity, metabolic syndromes, gastrointestinal disorders, mental disorders, genetic disorders, age-related disorders, acute diseases, and / or infections. 71. Recombinant protein or composition as described in item 70, wherein the gastrointestinal disorder is selected from celiac disease, inflammatory bowel disease, and / or peptic ulcer. 72. Recombinant proteins or compositions as described in item 70, wherein the hereditary disorder is selected from hemolytic anemia and / or autoimmune diseases. 73. Recombinant proteins or compositions as described in item 70, wherein the mental disorder is selected from bulimia nervosa and / or anorexia nervosa. 74. Recombinant protein or composition as described in item 70, wherein the infectious disease is selected from malaria and / or streptococcal infections. 75. Recombinant proteins or compositions described in item 70, for which the disease is chronic. 76. Recombinant proteins or compositions as described in item 70, wherein the acute disease is selected from trauma, gastrointestinal surgery, gastrointestinal bleeding, renal failure, and / or acute toxicity. 77. A method or recombinant protein or composition according to any one of items 50 to 76, wherein the amount of recombinant protein administered to the subject is 0.1 mg to 350 mg per kg of body weight of the subject, for example, 0.25 mg to 100 mg, for example, 0.5 to 50 mg, for example, 0.75 mg to 10 mg, for example, 0.75 mg to 3 mg per kg of body weight of the subject. 78. The amount of recombinant protein administered to the subject is 0.75 mg to 3 mg per kg of body weight, as described in item 77, or a recombinant protein or composition. 79. The method described in item 78 or recombinant protein or composition, wherein the subject is male. 80. The amount of recombinant protein administered to the subject is 0.75 mg to 1.25 mg per kg of body weight, for example, about 1 mg, according to the method in item 79 or the recombinant protein or composition. 81. The method described in item 78 or recombinant protein or composition, wherein the subject is female. 82. The amount of recombinant protein administered to the subject is 1.3 mg to 1.5 mg / kg body weight, for example, about 1.4 mg / kg body weight, according to the method in item 81 or the recombinant protein or composition. 83. A gene encoding a recombinant protein as described in any one of items 1-24. 84. A gene described in item 83, having a nucleotide sequence that is at least 50% identical to the nucleotide sequence contained in any of the sequence numbers 148-202 that encode the recombinant protein corresponding to sequence numbers 1-55. 85. A polynucleotide construct comprising the nucleotide sequences described in item 83 or 84, operably ligated to a regulatory sequence that directs the transcription and / or translation of a gene within a host cell. 86. A polynucleotide construct as described in item 85, wherein the control sequence is a promoter. 87. A polynucleotide construct as described in item 86, wherein the promoter is an inducible promoter. 88. A polynucleotide construct according to item 87, wherein the promoter is at least 50% identical to the promoters contained in SEQ ID NOs. 225-235. 89. An expression vector, which is a polynucleotide construct as described in any one of items 85-88. 90. Genetically modified host cells expressing a gene or polynucleotide construct described in any one of items 83-89 and a recombinant protein described in any one of items 1-24. 91. The host cell described in item 90, which is a eukaryotic cell, a bacterial cell, or an archaeal cell. 92. A host cell as described in item 91, in which the eukaryotic cell is a fungal cell, plant cell, mammalian cell, or insect cell. 93. Genetically modified host cells as described in item 92, in which plant cells are selected from the genera Arabidopsis, Lepidium, Tobacco, Wheat, Barley, Rice, Chenopodiaceae, Swiss chard, or Soybean. 94. Genetically modified host cells as described in item 93, in which plant cells are selected from seeds of Arabidopsis thaliana, scalycium, tobacco, wheat, barley, rice, quinoa, sugar beet, or soybean. 95. Genetically modified host cells as described in item 92, in which the fungal cell is a yeast or filamentous fungus. 96. Genetically modified host cells as described in item 95, in which the filamentous fungus is selected from the genera Aspergillus, Trichoderma, or Rhizopus. 97. Genetically modified host cells as described in item 96, wherein the filamentous fungus is selected from species of the genera Aspergillus, Aspergillus, Trichoderma, or Rhizopus. 98. Genetically modified host cells as described in item 95, in which yeast is selected from the genera Pichia, Saccharomyces, Yarrowia, Kluiveromyces, Ashbia, or Hansenula. 99. Genetically modified host cells as described in item 98, wherein the yeast is selected from methanol-utilizing yeasts, Pichia species, budding yeasts, Yarrowia species, alkane-utilizing yeasts, Cluyveromyces lactis, brewer's yeast, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasi, Saccharomyces crivelli, Saccharomyces bensis, Saccharomyces obiformis, Asibia gossipii, H. polymorpha, or Hansenula species. 100. Genetically modified host cells as described in item 91, in which bacterial cells are selected from the genera Escherichia coli, Bacillus, Brevibacterium, Burgholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter, or Pseudomonas. 101. Genetically modified host cells as described in item 100, in which bacterial cells are selected from the species of Escherichia coli, glutamate-producing bacteria, Bacillus subtilis, Mycobacterium purpurea, Ptida, Pseudomonas aeruginosa and / or P. mutabilis. 102. An archaeal cell is an algae, and the genetically modified host cell is as described in item 91. 103. A cell culture comprising host cells and culture medium as described in any one of items 90-102. 104. A method for producing a recombinant protein as described in any one of items 1 to 24, including the following: a. Culturing the cell culture described in item 103 under conditions that allow the cells to produce recombinant proteins; and b. Recover and / or isolate the recombinant protein, if necessary. 105. The method described in item 104, further comprising one or more of the following elements: a) Culturing cell cultures in a nutrient medium; b) Culturing cell cultures under aerobic or anaerobic conditions; c) Culture the cell culture under agitation; d) Culturing the cell culture at a temperature of 10-50°C; e) Culture the cell culture at a pH of 3-9; f) Culturing the cell culture for 10 hours to 30 days; and g) Culturing the cell cultures under fed-batch, repeated fed-batch, continuous, or semi-continuous conditions. 106. The method according to item 105, wherein the nutrient medium has the following characteristics: a) pH 5-8, b) Temperature 17~38℃. 107. The recovery and / or separation step comprises separating the liquid phase of cells or cell culture from the solid phase of cells or cell culture to obtain a supernatant containing recombinant protein, and / or subjecting the supernatant to one or more steps selected from below, according to any one of items 104 to 106: a) For example, separating the supernatant from the solid phase of a cell culture by filtration or gravity separation; b) Contact the supernatant with one or more adsorbent resins to obtain at least a portion of the generated recombinant protein; c) Contact the supernatant with one or more ion exchange or reverse-phase chromatography columns, or with a filtration or ultrafiltration apparatus to obtain at least a portion of the recombinant protein; d) Extracting recombinant proteins; and / or e) Precipitating the recombinant protein by crystallization or evaporation of the liquid-phase solvent; and, if necessary, separating the recombinant protein by filtration or gravity separation; This allows for the recovery and / or separation of recombinant proteins. 108. A composition comprising the cell culture described in item 103. 109. The composition described in item 108, wherein the composition is edible and the cell culture is in the form of edible biomass and mycelium.

Claims

1. A recombinant heme or phytoglobin protein comprising C-helices and D-helices covalently linked by a CD loop region, and comprising a hexa-coordinate or penta-coordinate prosthetic molecular heme group, wherein the heme or phytoglobin has an amino acid sequence at least 50% identical to the heme or phytoglobin contained in any one of SEQ ID NOs: 1 to 55, the CD loop region comprises one or more modifications compared to the parent CD loop region, thereby the modified CD loop region is more strongly bound to the prosthetic molecular group than the parent unmodified CD loop region, the parent CD loop region is located at positions 52 to 78 of SEQ ID NO: 1 in the recombinant heme or phytoglobin protein, optionally at positions 52 to 72 of SEQ ID NO: 1, and the CD loop region has an amino acid sequence at least 50% identical to the CD loop region contained in any one of SEQ ID NOs: 56 to 147.

2. The recombinant protein according to claim 1, wherein the parent CD loop region is specific to the protein.

3. The recombinant protein according to claim 1 or 2, wherein the modification in the CD loop region is selected from the deletion, substitution, and / or addition of one or more amino acids.

4. The modification of the CD loop region alters the flexibility or rigidity of the CD loop region compared to the parent CD loop region, according to any one of claims 1 to 3.

5. The recombinant protein according to any one of claims 1 to 4, wherein the CD loop region includes two or more substitutions, for example, three or more, for example, four or more, compared to the parent CD loop region.

6. The recombinant protein according to any one of claims 1 to 5, wherein the amino acid modification in the CD loop corresponds to one or more modifications selected from S13G, S13P, V15G, A25P, and A27G of the CD loop described in SEQ ID NO:

56.

7. The recombinant protein according to any one of claims 1 to 5, wherein the amino acids corresponding to positions P1, A3, F7, S8, S13, P16, P21 and / or P25 in SEQ ID NO: 56 are either conserved or conservatively substituted.

8. The recombinant protein according to any one of claims 1 to 7, wherein the CD loop comprises an amino acid sequence described in any one of sequence numbers 123, 127, 128, 129, 130, 131, 133, 134, 135, 137, 139, 141, 142, or 145.

9. The recombinant protein according to claim 8, wherein the CD loop comprises an amino acid sequence described in any one of sequence numbers 127, 128, 130, 131, 135, 139, 141, 142, or 145.

10. The recombinant protein according to any one of claims 1 to 9, further comprising a signal peptide that instructs the secretion of the recombinant protein from a cell.

11. The recombinant protein according to claim 10, wherein the signal peptide instructs expression within microbial cells.

12. The recombinant protein according to claim 10 or 11, wherein the signal peptide is heterogeneous to the recombinant protein.

13. The recombinant protein according to any one of claims 10 to 12, wherein the microbial cell is a bacterial cell or a fungal cell.

14. The recombinant protein according to any one of claims 10 to 13, wherein the signal peptide has an amino acid sequence that is at least 50% identical to the signal peptide contained in any one of SEQ ID NOs. 203 to 213.

15. The recombinant protein according to any one of claims 1 to 14, wherein the recombinant protein is unnatural or synthetic.

16. The recombinant protein according to claim 15, wherein the non-natural or synthetic recombinant protein is a fusion protein.

17. The recombinant protein according to claim 16, wherein the fusion protein comprises two or more prosthetic molecular groups.

18. The recombinant protein according to any one of claims 1 to 17, wherein the recombinant protein is a class 1 or class 2 non-symbiotic hemoglobin.

19. The recombinant protein is derived from a plant of the Caryophyllaceae family, according to any one of claims 1 to 18.

20. The recombinant protein according to claim 19, wherein the recombinant protein is derived from spinach or quinoa.

21. The recombinant protein according to any one of claims 1 to 20, wherein the recombinant protein comprises at least one post-translational modification selected from glycosylation or phosphorylation, compared to the corresponding native protein.

22. A composition comprising a recombinant protein according to any one of claims 1 to 21, and one or more carriers, drugs, additives and / or excipients.

23. The composition according to claim 22, wherein one or more carriers, agents, additives and / or excipients are selected from salts, antioxidants and / or reducing agents.

24. The aforementioned salts are NaCl, ammonium sulfate, and CaCl ２ , KCl, MgCl ２ A composition according to claim 23, selected from the above.

25. The composition according to claim 23 or 24, wherein the amount of salt in the composition is 10 μM to 2 M.

26. The composition according to any one of claims 23 to 25, wherein the antioxidant is selected from ascorbic acid, 2-mercaptoethanol, dithiothreitol (DTT), superoxide dismutase (SOD), catalase, β-carotene, lycopene, glutathione, melatonin, estrogen, and biquinol-10, N-acetylcysteine, lipoic acid; zinc, selenium, and / or copper salts; quercetin, catechin, cortisone, estradiol, estriol, and α-tocopherol.

27. The composition according to any one of claims 23 to 26, wherein the amount of antioxidant in the composition is 10 μM to 1 M.

28. The composition according to claim 23, wherein the reducing agent is selected from ascorbic acid (vitamin C), tocopherol, carotenoids, flavonoids, and glutathione.

29. The composition according to any one of claims 22 to 28, further comprising at least one vitamin and / or mineral selected from vitamin C, vitamin D3, vitamin E, vitamin B6, vitamin B12, folate, folic acid, biotin, zinc, copper, magnesium, selenium, and combinations thereof.

30. The composition according to any one of claims 22 to 27, comprising at least 0.002% (weight / weight) of the recombinant protein.

31. The composition according to any one of claims 22 to 27, comprising at least 0.1 μM, for example, 0.1 μM to 8 M, of the recombinant protein.

32. The composition according to any one of claims 22 to 31, selected from food, feed, beverage, food ingredient, health supplement or nutritional supplement and / or pharmaceutical.

33. The composition according to claim 32, wherein the composition is a nutritional supplement.

34. The composition is a food ingredient. The composition according to claim 32.

35. The composition according to claim 32, wherein the composition is a nutritionally balanced food composition suitable for children, the elderly, or subjects undergoing medical or surgical treatment.

36. The composition according to any one of claims 22 to 35, wherein the composition does not have a metallic flavor or taste.

37. The composition according to any one of claims 22 to 36, wherein the composition is formulated as a dry preparation, a liquid preparation, or a slurry or dispersion.

38. The composition according to claim 37, comprising a formulation selected from the group consisting of tablets, capsules, liquids, drops, concentrates, powders, granules, and combinations thereof.

39. The composition according to any one of claims 22 to 38, further comprising a pharmaceutically acceptable carrier.

40. The composition according to any one of claims 22 to 39, wherein the composition is a pharmaceutical composition selected from the group consisting of drugs, vaccines, personal care compositions and combinations thereof.

41. A non-therapeutic method for improving the endurance or hypoxia tolerance of a subject, comprising administering to the subject a recombinant protein according to any one of claims 1 to 21 or a composition according to any one of claims 22 to 40 in an amount effective for improving the endurance or hypoxia tolerance of the subject.

42. The method according to claim 41, wherein the subject is performing physical activity.

43. The method according to claim 42, wherein the subject is engaged in mountain climbing or hiking under low-oxygen conditions.

44. A non-therapeutic method for altering the color or texture of a target skin, comprising administering to the target skin an amount effective in altering the color or texture of the recombinant protein described in any one of claims 1 to 21 or a composition described in any one of claims 22 to 40.

45. A recombinant protein or composition according to any one of claims 1 to 21 or any one of claims 22 to 40 for use in the treatment, improvement or prevention of a target disease or deficiency, comprising administering the recombinant protein or composition to the target in an amount effective for improving or preventing the disease or deficiency.

46. The recombinant protein composition according to claim 45, wherein the disease or deficiency is either iron deficiency or a disease caused by iron deficiency.

47. The recombinant protein or composition according to claim 45 or 46, wherein the recombinant protein is phytoglobin.

48. The recombinant protein or composition according to any one of claims 45 to 47, wherein the recombinant phytoglobin comprises a hexa-coordinate heme group and has an amino acid sequence identical to that of the phytoglobin contained in SEQ ID NO: 1 or 5 by at least 86%.

49. The recombinant protein or composition according to claim 48, wherein the recombinant phytoglobin has the same amino acid sequence as the phytoglobin contained in the sequence of SEQ ID NO: 1 or 5 by at least 87%, for example, at least 88%, for example, at least 89%, for example, at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99%.

50. The recombinant protein or composition according to any one of claims 47 to 49, wherein the recombinant phytoglobin is a class 1 non-symbiotic hemoglobin.

51. The recombinant phytoglobin is derived from a plant of the Caryophyllaceae subfamily, for example, spinach or quinoa, according to any one of claims 47 to 50, the recombinant protein or composition.

52. The recombinant protein or composition according to any one of claims 47 to 51, wherein the recombinant phytoglobin has an amino acid sequence contained in or consisting of SEQ ID NO:

4.

53. The recombinant protein or composition according to any one of claims 45 to 52, wherein the recombinant protein contains a hexa-coordinate heme group and has improved stability compared to penta-coordinate hemoglobin.

54. The recombinant protein has a value of less than 0.2, for example, less than 0.15, for example, in the range of 0.01 to 0.18, or in the range of 0.01 to 0.15, for example, about 0.1h －１ A recombinant protein or composition according to any one of claims 45 to 53, having an auto-oxidation rate.

55. The recombinant protein or composition according to any one of claims 45 to 54, wherein the recombinant protein does not have a metallic taste or has a neutral taste.

56. The recombinant protein or composition according to any one of claims 45 to 55, wherein the recombinant protein has a melting temperature above 68°C, for example, in the range of 68 to 75°C, for example, in the range of 70 to 76°C, for example, above 76°C, for example, above 77°C, for example, above 78°C, for example, above 79°C, for example, above 80°C, for example, above 81°C, for example, above 82°C, for example, above 83°C, for example, a melting temperature of about 84°C.

57. The recombinant protein or composition according to claim 47, wherein the recombinant phytoglobin comprises a hexacoordinate heme group and further comprises a CD loop region included in SEQ ID NO: 56, 60, or 111 and the CD loop region having an amino acid sequence with at least 60% sequence identity.

58. The recombinant protein or composition according to claim 57, wherein the CD loop region has an amino acid sequence that is at least 90% identical to the CD loop region contained in SEQ ID NO: 56, 60, or 111.

59. The recombinant protein or composition according to claim 57 or 58, wherein the CD loop region has an amino acid sequence including the CD loop region contained in SEQ ID NO: 56, 60, or 111, which has one or more, two or more, three or more, or four or more amino acid substitutions.

60. The recombinant protein or composition according to any one of claims 57 to 59, wherein the CD loop region has an amino acid sequence comprising or consisting of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO:

116.

61. The subject is a blood donor, according to any one of claims 45 to 60, recombinant protein or composition.

62. The recombinant protein or composition according to any one of claims 45 to 61, wherein the subject is a vegetarian, a vegan, and / or a flexitarian.

63. The recombinant protein or composition according to any one of claims 45 to 62, wherein the subject is an animal, optionally a pet, poultry, or livestock.

64. The recombinant protein or composition according to any one of claims 45 to 62, wherein the subject is, in some cases, a woman selected from the group consisting of pregnant women, lactating women, and women of reproductive age.

65. The recombinant protein or composition according to any one of claims 45 to 64, wherein the disease is selected from endometriosis, dysmenorrhea, menorrhagia, cancer, kidney disease, diabetes, obesity, metabolic syndrome, digestive disorders, mental disorders, genetic disorders, age-related disorders, acute diseases, and / or infections.

66. The recombinant protein or composition according to claim 65, wherein the digestive disorder is selected from celiac disease, inflammatory bowel disease, and / or peptic ulcer.

67. The recombinant protein or composition according to claim 65, wherein the genetic disorder is selected from hemolytic anemia and / or autoimmune diseases.

68. The recombinant protein or composition according to claim 65, wherein the mental disorder is selected from bulimia nervosa and / or anorexia nervosa.

69. The recombinant protein or composition according to claim 65, wherein the infectious disease is selected from malaria and / or streptococcal infections.

70. The recombinant protein or composition according to claim 65, wherein the disease is chronic.

71. The recombinant protein or composition according to claim 65, wherein the acute disease is selected from trauma, gastrointestinal surgery, gastrointestinal bleeding, renal failure, and / or acute toxicity.

72. The amount of recombinant protein administered to the subject is 0.1 mg to 350 mg per kg of the subject's body weight, for example, 0.25 mg to 100 mg, for example, 0.5 to 50 mg, for example, 0.75 mg to 10 mg, for example, 0.75 mg to 3 mg per kg of body weight, a method or a recombinant protein or composition according to any one of claims 45 to 71.

73. The method according to claim 72, or a recombinant protein or composition, wherein the amount of recombinant protein administered to the subject is 0.75 mg to 3 mg per kg of body weight.

74. The method according to claim 73, or a recombinant protein or composition, wherein the subject is male.

75. The method according to claim 74, or the recombinant protein or composition, wherein the amount of recombinant protein administered to the subject is 0.75 mg to 1.25 mg per kg of body weight, for example, about 1 mg.

76. The method according to claim 73, or a recombinant protein or composition, wherein the subject is female.

77. The method according to claim 76, or the recombinant protein or composition, wherein the amount of recombinant protein administered to the subject is 1.3 mg to 1.5 mg per kg of body weight, for example, about 1.4 mg.

78. A gene encoding a recombinant protein according to any one of claims 1 to 21.

79. The gene according to claim 78, having a nucleotide sequence that is at least 50% identical to the nucleotide sequence contained in any of the sequence numbers 148 to 202 that encode recombinant proteins corresponding to sequence numbers 1 to 55.

80. A polynucleotide construct comprising the nucleotide sequence according to claim 78 or 79, operably linked to a control sequence that directs the transcription and / or translation of the gene within a host cell.

81. The polynucleotide construct according to claim 80, wherein the control sequence is a promoter.

82. The polynucleotide construct according to claim 81, wherein the promoter is an inducible promoter.

83. The polynucleotide construct according to claim 82, wherein the promoter is at least 50% identical to the promoters included in SEQ ID NOs. 225 to 235.

84. The aforementioned construct is an expression vector, the polynucleotide construct according to any one of claims 80 to 83.

85. A genetically modified host cell expressing a gene or polynucleotide construct according to any one of claims 78 to 84, and a recombinant protein according to any one of claims 1 to 21.

86. The host cell according to claim 85, wherein the cell is a eukaryotic cell, a bacterial cell, or an archaeal cell.

87. The host cell according to claim 86, wherein the eukaryotic cell is a fungal cell, a plant cell, a mammalian cell, or an insect cell.

88. The genetically modified host cell according to claim 87, wherein the plant cell is selected from the genera Arabidopsis, Lepidium, Tobacco, Wheat, Barley, Rice, Chenopodiaceae, Swiss chard, or Soybean.

89. The genetically modified host cell according to claim 88, wherein the plant cell is selected from seeds of Arabidopsis thaliana, scalycium thaliana, tobacco, wheat, barley, rice, quinoa, sugar beet, or soybean.

90. The genetically modified host cell according to claim 87, wherein the fungal cell is a yeast or a filamentous fungus.

91. The filamentous fungus is selected from the genera Aspergillus, Trichoderma, or Rhizopus, as described in claim 90, for the genetically modified host cell.

92. The genetically modified host cell according to claim 91, wherein the filamentous fungus is selected from species of the genus Aspergillus, Aspergillus, Trichoderma, or Rhizopus.

93. The genetically modified host cell according to claim 90, wherein the yeast is selected from the genera Pichia, Saccharomyces, Yarrowia, Kluiveromyces, Ashvia, or Hansenula.

94. The genetically modified host cell according to claim 93, wherein the yeast is selected from methanol-utilizing yeast, Pichia species, budding yeast, Yarrowia species, alkane-utilizing yeast, Cluyveromyces lactis, brewer's yeast, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasi, Saccharomyces crivelli, Saccharomyces bensis, Saccharomyces obiformis, Asibia gossipii, H. polymorpha, or Hansenula species.

95. The genetically modified host cell according to claim 86, wherein the bacterial cell is selected from the genera Escherichia coli, Bacillus, Brevibacterium, Burgholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter, or Pseudomonas.

96. The genetically modified host cell according to claim 95, wherein the bacterial cell is selected from the species Escherichia coli, Corynebacterium glutamate, Bacillus subtilis, Bacillus marcescens, Bacillus ptycnema, Pseudomonas aeruginosa and / or P. mutabilis.

97. The genetically modified host cell according to claim 86, wherein the archaeal cell is an alga.

98. A cell culture comprising host cells and a growth medium according to any one of claims 85 to 97.

99. A method for producing a recombinant protein according to any one of claims 1 to 21, including the following: c. Culturing the cell culture according to claim 98 under conditions that enable the cells to produce the recombinant protein; and d. If necessary, recover and / or separate the recombinant protein.

100. The method according to claim 99, further comprising one or more elements selected from the following: h) Culturing the cell culture in a nutrient medium; i) Culturing the cell culture under aerobic or anaerobic conditions; j) Culturing the cell culture under agitation; k) Culturing the cell culture at a temperature of 10 to 50°C; l) Culturing the cell culture at a pH of 3 to 9; m) Culturing the cell culture for 10 hours to 30 days; and n) The cell cultures are cultured under fed-batch, repeated fed-batch, continuous, or semi-continuous conditions.

101. The method according to claim 100, wherein the nutrient medium has the following characteristics: c) pH 5-8, d) Temperature 17-38°C.

102. The method according to any one of claims 99 to 101, wherein the recovery and / or separation step comprises separating the liquid phase of the cells or cell culture from the solid phase of the cells or cell culture to obtain a supernatant containing the recombinant protein, and / or subjecting the supernatant to one or more steps selected from the following: a) For example, the step of separating the supernatant from the solid phase of the cell culture by filtration or gravity separation; b) The step of contacting the supernatant with one or more adsorbent resins to obtain at least a portion of the recombinant protein produced; c) The step of contacting the supernatant with one or more ion exchange or reverse-phase chromatography columns, or a filtration or ultrafiltration apparatus to obtain at least a portion of the recombinant protein; d) the step of extracting the recombinant protein; and / or e) Precipitating the recombinant protein by crystallization or evaporation of the solvent in the liquid phase, and further separating the recombinant protein by filtration or gravity separation, if necessary; This step involves recovering and / or separating the recombinant protein.

103. A composition comprising the cell culture described in claim 98.

104. The composition according to claim 103, wherein the composition is edible, and the cell culture is in the form of edible biomass and mycelium.

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