Meat substitute comprising animal myoglobin

Abstract

To provide a gene construct comprising a nucleic acid encoding a protein, in a meat substitute comprising the myoglobin protein, a host cell comprising the gene construct, and a method for producing myoglobin or a meat substitute.SOLUTION: A method for producing a meat substitute comprising bovine myoglobin, the method comprising: a): a step of providing a host cell comprising a gene construct, wherein the gene construct is represented by a specific sequence; b) a step of culturing the host cell in a medium suitable for producing bovine myoglobin represented by a specific sequence different from the foregoing; c) a step of recovering the myoglobin; d) a step of purifying the myoglobin; and e) a step of blending the purified myoglobin into the meat substitute.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Aspects and embodiments described herein include meat substitutes comprising myoglobin protein, A gene construct containing a nucleic acid encoding the protein, the gene construct and methods for producing said myoglobin or said meat substitute. do. [Background technology]

[0002] Several meat substitutes have already been developed and are commercially available. The flavor, color, texture and The nutritional quality is not necessarily optimal in that it does not adequately mimic "meat." Such products also may not have the same nutritional quality as meat.

[0003] Therefore, we are looking for new inventions that do not have all the drawbacks of existing food products, such as meat substitutes. There is still a need for this. Summary of the Invention [Problem to be solved by the invention]

[0004] Accordingly, aspects and embodiments of the present invention as described herein are intended to be illustrative and not restrictive. This invention solves at least some of the problems and needs described above. [Means for solving the problem]

[0005] meat substitute In a first embodiment, animal myoglobin, preferably mammalian myoglobin, more preferably or a mammoth, a pig, a sheep, or a cow, more preferably the mammoth is a woolly mammoth. Mammoth (Mammuthus primigenius) or Steppe Mammoth (Mamm uthus trogontherii), most preferably the mammoth is a steppe mammoth animal myoglobin, or avian myoglobin, preferably chicken myoglobin; Robin or fish myoglobin, preferably tuna fish myoglobin, or A meat substitute or food ingredient is provided comprising myoglobin derived from

[0006] In one embodiment, the meat substitute or food ingredient is derived from mammoth, pig, sheep, cow, or elk. animal myoglobin of tuna or tuna, or animal myoglobin derived therefrom, preferably Or the mammoth in question is a woolly mammoth (Mammuthus primigenius) or the steppe mammoth (Mammuthus trogontherii), Preferably, the mammoth comprises animal myoglobin from a steppe mammoth.

[0007] The animal myoglobin has an amino acid sequence similar to that of a mammoth, preferably a woolly mammoth, or or one of the following: steppe mammoth, pig, sheep, cow, chicken, or tuna Each of these is derived from the addition, deletion, and / or substitution of at least one amino acid. Preferably, woolly mammoths or steppe mammoths, more preferably steppe mammoths. Derived from the myoglobin of mammoths, pigs, sheep, cows, chickens, or tuna It can be said that there are 2, 3, 4, 5, 6, 7, 8, 9, or 10 Amino acid additions, deletions, and / or substitutions are also contemplated by the present invention. Myoglobin A of moth, steppe mammoth, pig, sheep, cow, chicken, or tuna Examples of amino acid sequences are disclosed below by the given SEQ ID NOs. Derived from the myoglobin of mammoth, pig, sheep, cow, chicken, or tuna Such animal myoglobins may also be used in combination with other animal myoglobins, such as those described later in this specification. Mammoths may exert at least detectable levels of activity unless otherwise specifically stated. In the context of this application, the woolly mammoth (Mammuthus primigeniu s) or steppe mammoths (Mammuthus trogontherii) It can be any species in the genus Mammuthus.

[0008] In one embodiment, the myoglobin in the meat substitute or in the food ingredient is from mammoth. It is or is derived from this.

[0009] In a preferred embodiment, the myoglobin in the meat substitute or in the food ingredient is Of or derived from Moss.

[0010] In a preferred embodiment, the myoglobin in the meat substitute or in the food ingredient is step myoglobin. Of or derived from the nummos.

[0011] In one embodiment, the myoglobin in the meat substitute or in the food ingredient has the following amino acid sequence: columns, i.e. a) A sequence that contains at least 70% sequence identity with SEQ ID NO: 3 and contains the following amino acid combinations: Se, that is F at position 30, and / or Q in position 65, and / or H at position 92, and / or H at position 94, and / or F at position 30 and Q at position 65, and / or F at position 30 and H at position 92, and / or F at position 30 and H at position 94, and / or Q at position 65 and H at position 92, and / or Q at position 65 and H at position 94, and / or H at position 92 and H at position 94, and / or F at position 30 and Q at position 65 and H at position 92, and / or F at position 30 and Q at position 65 and H at position 94, and / or F at position 30 and H at position 92 and H at position 94, and / or Q at position 65 and H at position 92 and H at position 94, and / or F at position 30, Q at position 65, H at position 92, and H at position 94 an amino acid sequence having at least one of b) at least 70% sequence identity with SEQ ID NO: 2 or 3, Q or H at position 65 and H at position 94, and optionally at the following positions in SEQ ID NO: 2 or 3: The following amino acids: E at position 9, K at position 13, T at position 14, P at position 27, L at position 31, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88, I at position 102, E at position 123, and / or E at position 143 an amino acid sequence having at least one of the following I: c) at least 70% sequence identity with SEQ ID NO: 1, Q or H at position 65, and 94 H, and optionally the following amino acids at the following positions in SEQ ID NO: 1: E at position 9, K at position 13, T at position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88 Q, I at position 102, and / or E at position 123. amino acid sequence, d) at least 70% identity to SEQ ID NO: 4, 5 or 6, Q or H at position 65 and H at position 94, and optionally the following in SEQ ID NO: 4, 5, or 6: The following amino acids are present: N at position 13, Q at position 27, I at position 31, N at position 7, A at position 128, S at position 133, A at position 145, and / or position 150 an amino acid sequence having at least one of the following Ls: e) at least 70% identity to SEQ ID NO: 7, Q or H at position 65, and Q or H at position 94 and optionally the following amino acid at the following position in SEQ ID NO: 7: , Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M, H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 3, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 111, and I at position 117, A at position 118, A at position 121, S at position 128, and S at position 133 at least one of K at position 145, S at position 145, and / or F at position 150 an amino acid sequence, or f) at least 70% identity to SEQ ID NO: 8, Q or H at position 65, and Q or H at position 94 and an amino acid sequence containing H.

[0012] SEQ ID NO: 1 represents a portion of the amino acid sequence of woolly mammoth myoglobin, and SEQ ID NO: 2 represents the amino acid sequence of woolly mammoth, and SEQ ID NO: 3 represents the amino acid sequence of steppe mammoth myoglobin. SEQ ID NO: 4 represents the amino acid sequence of sheep myoglobin, and the sequence Number 5 represents the amino acid sequence of bovine (cow) myoglobin, and SEQ ID NO: 6 represents the amino acid sequence of porcine myoglobin. SEQ ID NO: 7 represents the amino acid sequence of chicken myoglobin. SEQ ID NO: 8 represents the amino acid sequence of tuna myoglobin. SEQ ID NO: 1, which represents a portion (i.e., a partial sequence) of the amino acid sequence of robin, is It is clear that SEQ ID NO: 2, which represents the (complete) amino acid sequence of smyoglobin, is included. SEQ ID NO:9 is the nucleic acid sequence encoding SEQ ID NO:1. SEQ ID NO:10 is the nucleic acid sequence encoding SEQ ID NO:2. SEQ ID NO: 11 is a nucleic acid sequence that encodes SEQ ID NO: 3 SEQ ID NO: 12 is the nucleic acid sequence encoding SEQ ID NO: 4. SEQ ID NO: 13 is the nucleic acid sequence encoding SEQ ID NO: 4. SEQ ID NO: 14 is a nucleic acid sequence that encodes SEQ ID NO: 6. SEQ ID NO: 15 is the nucleic acid sequence encoding SEQ ID NO: 7. SEQ ID NO: 16 is the nucleic acid sequence encoding SEQ ID NO: nucleotide sequence encoding No. 8.

[0013] Meat substitutes are, by definition, not meat, not natural meat, not real meat. A meat substitute may be considered a non-natural food or edible product. Within the context of the present invention, a meat substitute may be considered a non-natural food or edible product. Not derived from or derived from the meat of cattle, sheep, cattle, chicken, or tuna (Or, if mammoths still existed, which they did not, the diet of these animals (Not meat or derived from meat). Meat substitutes are replicas of meat or meat analogues or It is synonymous with meat-like products.

[0014] Meat substitutes must have the same appearance (pattern, lettering, etc.), shape, structure, and composition (similar to meat) as meat. fat content, protein content and / or heme iron content), texture, color, flavor, aroma and and / or aspects.

[0015] Alternatively, a meat substitute may have a different appearance, shape, structure, texture, color, flavor, or texture from one of the meats. It may have a fragrance and / or appearance.

[0016] Meat substitutes can be prepared as liquid, solid, or semi-solid foods.

[0017] A suitable example of a liquid food product is soup.

[0018] Suitable examples of solid foods include cell-based meat or cultured meat or plant-based meat. Such plant-based meats may include plant proteins such as soy protein. In addition, such plant-based meats contain animal myoglobin as defined herein. In embodiments, the animal myoglobin is the sole source of heme-containing protein.

[0019] Suitable examples of snacks include protein bars or protein shakes.

[0020] Food ingredients can be added to edible products to obtain different edible products. Alternatively, the food ingredient may be consumed as such. The term "food ingredient" may be replaced by the term "food supplement."

[0021] Suitable examples of food ingredients containing myoglobin as identified above are solid forms that can be sold to consumers. (such as powdered spice mixes) or liquid (such as extracts) or semi-liquid or semi-solid (such as peppers) It can be a food ingredient in a variety of foods (such as pasta, toast, gel, sauce, broth, or cream). These food ingredients can be added to food products themselves to provide the myoginseng contained herein. Robin can be replenished and create his own meat substitute.

[0022] Suitable examples of solid food supplements include tablets or tablets containing the animal myoglobins identified above. Tablets, pills or powders contain other compounds such as vitamins or minerals. It can be formulated with

[0023] The present inventors have determined that "sensation for meat" is based on the animal Myoglo as defined herein. We have discovered that this can be mimicked in the meat substitute of the present invention using bottles.

[0024] In one embodiment, the meat substitute of the present invention has the same appearance (pattern, lettering), shape, and texture as meat. , structure, composition (such as similar fat content, protein content and / or heme iron content), wind It may mimic taste, texture, color, aroma, appearance and / or nutritional value. Specifically, the meat substitute may be cell-based meat, cultured meat, or plant-based meat. This is mostly due to the presence of animal myoglobin as described herein. However, meat substitutes cannot have all of the drawbacks of meat. For example, meat substitutes are healthier than meat. The environmental impacts of meat substitutes (long-term direct and indirect effects on climate change) are This is considered to be no more harmful than the known effects of meat.

[0025] In one embodiment, the meat substitute of the present invention has the properties of meat without all of the drawbacks of meat. Aspect (pattern, lettering), shape, structure, composition (similar fat content, protein content) and / or heme iron content), flavor, texture, color, aroma, and / or appearance This is typically the case when meat substitutes are cell-based or cultured or plant-based. This is often the case in the case of animal guinea pigs, as described herein. Due to the existence of bottles, meat substitutes cannot have all the drawbacks of meat. For example, meat substitutes can be Moreover, the environmental impact of meat substitutes (long-term direct impact on climate change) These effects (direct / indirect effects) are likely to be less harmful than the known effects of meat.

[0026] In one embodiment, the meat substitute of the present invention has the properties of meat without all of the drawbacks of meat. Composition (e.g., similar fat content, protein content, and / or heme iron content), flavor, color, and / or fragrances. This is often the case as described herein. This is due to the presence of animal myoglobin.

[0027] In one embodiment, the meat substitute of the present invention has the properties of meat without all of the drawbacks of meat. This is often referred to as simulating a flavor and / or aroma as described herein. This is due to the presence of animal myoglobin, as seen in

[0028] In one embodiment, the meat substitute of the present invention has the properties of meat without all of the drawbacks of meat. This is often the case with behaviors such as those described herein. This is due to the presence of myoglobin.

[0029] In one embodiment, the meat substitute of the present invention has the properties of meat without all of the drawbacks of meat. This is often the case with behaviors such as those described herein. This is due to the presence of myoglobin.

[0030] The inventors furthermore believe that "meat perception" as defined herein is a food component of We discovered that this can be imitated using minutes.

[0031] In one embodiment, the food ingredient of the present invention has the properties of meat without all of the drawbacks of meat. composition (e.g., similar fat content, protein content, and / or heme iron content), flavor, and color This is often referred to herein as mimicking the aroma and / or nutritional value of the food. Due to the presence of animal myoglobin as explained.

[0032] In one embodiment, the food ingredient of the present invention has the properties of meat without all of the drawbacks of meat. composition (e.g., similar fat content, protein content, and / or heme iron content), flavor, and color This is often the case with the imitation of fragrances and / or aromas described herein. This is due to the presence of animal myoglobin, which is thought to be involved in the production of lactic acid bacteria.

[0033] In one embodiment, the food ingredient of the present invention has the properties of meat without all of the drawbacks of meat. This is often the case in the context of the present specification. This is due to the presence of animal myoglobin as explained in the book.

[0034] In one embodiment, the food ingredient of the present invention has the properties of meat without all of the drawbacks of meat. composition (e.g., similar fat content, protein content, and / or heme iron content), flavor, and This is often the case as explained herein. This is due to the presence of animal myoglobin.

[0035] In one embodiment, the food ingredient of the present invention has the properties of meat without all of the drawbacks of meat. composition (e.g., similar fat content, protein content, and / or heme iron content), flavor, and This is likely to be in line with the animal myognomy described herein. Due to Robin's presence.

[0036] In one embodiment, the food ingredient of the present invention has the properties of meat without all of the drawbacks of meat. Composition (similar fat content, protein content and / or heme iron content) and aroma This is most likely due to the animal Myotonia gracilis described herein. Due to the presence of bottles.

[0037] Within the context of this invention, meat perception refers to the perception that a meat substitute and / or food ingredient is perceived by humans. aroma and / or flavor compounds that are recognizable in the food, as well as some that are released when meat is cooked. These odorants and / or flavor compounds may be mimicked to produce the characteristics of these odorants. Some of the formation of ATP can be catalyzed by the heme iron present in myoglobin. However, this formation may be due to lipid oxidation and / or the Maillard reaction. This situation can be mimicked with the myoglobin of the meat substitutes and / or food ingredients of the present invention.

[0038] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the raw meat substitute of the present invention, , believed to mimic the bloody and / or metallic aroma of meat, preferably raw meat. Preferably, the meat substitute or food ingredient of the present invention has the bloody and / or metallic taste of meat. The aroma is imitated to a greater extent than other (i.e., not in accordance with the present invention) meat substitutes or food ingredients. In the context of the present invention, raw refers to meat that has not been subjected to a heat treatment, preferably means uncooked or not grilled.

[0039] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, is The meat is believed to mimic the roasted aroma of meat, especially grilled meat. In other words, the meat substitute or food ingredient of the present invention may be used in combination with other (i.e., non-inventive) meat substitutes or It is capable of imitating the roasted aroma of meat to a greater extent than food ingredients.

[0040] In the context of the present invention and in the embodiments of this section, "other meat substitutes" or "the present invention" The "meat-free substitute" is preferably a plant-based product containing recombinant soybean leghemoglobin (LegH). A base burger, more preferably the commercially available Impossible Burger.

[0041] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, is The meat used in the present invention is superior in taste (grilling) compared to other (i.e., non-inventive) meat substitutes or food ingredients. Higher concentrations of oxidized lipids, lipid acids, and volatile compounds obtained from meat substitutes or food ingredients The higher the better, preferably the higher the fragrance characterized by the oxidative products, pyrazine and / or pyrrole. is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 9 0%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 17 0%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 25 0%, 260%, 270%, 280%, 290%, or 300%. Pyrazines include methylpyrazine, 2,5-dimethylpyrazine, and 2-ethyl-6-methylpyrazine. A preferred pyrrole is pyrrole. A preferred lipid oxidation product is 2-methylpyrrole. butanal and 3-methylbutanal.

[0042] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat of the present invention, The meat substitutes have a low (gly) fat content compared to other (i.e., not in accordance with the present invention) meat substitutes or food ingredients. Higher concentrations of 2,5-dimethylaminobenzoates in volatile compounds obtained from (prepared) meat substitutes or food ingredients The higher preferably means at least 10%, 2% or more of a fragrance characterized by thiamin pyrazine. 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110% , 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% , 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270% , 280%, 290%, or 300%.

[0043] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat of the present invention, The meat substitutes have a low (gly) fat content compared to other (i.e., not in accordance with the present invention) meat substitutes or food ingredients. Higher concentrations of 2-ethyl-6-methyl-2-methylpropional in volatile compounds obtained from (prepared) meat substitutes or food ingredients -methylpyrazine. Higher is preferably at least 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1 10%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 1 90%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 2 This means 70%, 280%, 290%, or 300%.

[0044] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat of the present invention, The meat substitutes have a low (gly) fat content compared to other (i.e., not in accordance with the present invention) meat substitutes or food ingredients. characterized by higher concentrations of pyrrole in volatile compounds obtained from (prepared) meat substitutes or food ingredients. Preferably, higher is at least 10%, 20%, 30%, 40%, or 50% higher. 0%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 13 0%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 21 0%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 29 This means 0% or 300%.

[0045] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat of the present invention, The meat substitutes have a low (gly) fat content compared to other (i.e., not in accordance with the present invention) meat substitutes or food ingredients. Higher concentrations of 2-methylbutadiene in volatile compounds obtained from (prepared) meat substitutes or food ingredients The higher is preferably at least 10%, 20% or more. ,30%,40%,50%,60%,70%,80%,90%,100%,110%,1 20%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 2 00%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 2 This means 80%, 290%, or 300%.

[0046] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat of the present invention, The meat substitutes have a low (gly) fat content compared to other (i.e., not in accordance with the present invention) meat substitutes or food ingredients. Higher concentrations of 3-methylbutadiene in volatile compounds obtained from (prepared) meat substitutes or food ingredients The higher is preferably at least 10%, 20% or more. ,30%,40%,50%,60%,70%,80%,90%,100%,110%,1 20%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 2 00%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 2 This means 80%, 290%, or 300%.

[0047] In Example 2.5, an aroma analysis of meat substitutes according to the present invention is provided. Meat and Meat Substitutes The aroma and concentration of volatile compounds in the tea leaves were determined by the method described in this example. determined by gas chromatography coupled with mass spectrometry of volatile compounds. It can be determined.

[0048] In the present invention, in the context of the present invention, the perception of meat is influenced by the color of the meat substitute, which is similar to the color of the meat. The color of meat is determined by the concentration of heme-containing proteins and / or the amount of heme in the meat. Therefore, as defined herein, The myoglobin will determine the color of the meat substitute. applies to food ingredients.

[0049] In one embodiment, the meat substitute or food ingredient of the present invention, preferably the raw meat substitute of the present invention, It is believed that the color of the colorant mimics the color of meat, preferably raw meat. The color of meat substitutes is often due to the presence of animal myoglobin. The meat or food ingredient has a higher protein content than other (i.e., non-inventive) meat substitutes or food ingredients. To a certain extent, it can mimic the color of meat.

[0050] In a preferred embodiment, the meat substitute or food ingredient of the present invention, preferably the raw The color of the meat substitute preferably remains constant under constant light and at 4°C, more preferably under these conditions. and when measured via the technique of Example 2.4, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or Preferably, the color of the meat substitute or food ingredient of the present invention is essentially stable for 20 days. The color of other (i.e., not according to the invention) meat substitutes or food ingredients is stable, More preferably, a ΔE change of 0% to 10% is defined as stable (see further). Rimo 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, or 20 days longer. Essentially stable for at least X days. Preferably, the difference between 0% and 0.5%, 1%, 1.5%, 2%, or 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5% , 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5% , 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20%, where ΔE is the change in ΔE of a meat substitute or food ingredient between As determined in Example 2.4. Most preferably, the ΔE is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, Not change by more than 10% upon storage for 15, 16, 17, 18, 19, or 20 days.

[0051] Example 2.4 provides a color stability analysis of meat substitutes according to the present invention. Color of Meat and Meat Substitutes and color stability can be determined by the method described in this example.

[0052] Within the context of this invention, meat perception is achieved when the composition of the meat substitute is similar to that of meat. The composition of the meat substitute may be simulated by the presence of the same or similar components, and optionally: When the amount of the constituent present is the same as or similar to the amount present in meat, It may be found that the composition is similar, e.g., similar fat, protein, and / or The iron content may be present in the meat substitute just as it is in meat.

[0053] These animal myoglobins when incorporated into meat substitutes or as food ingredients also contribute to the development of edible myoglobins. It is thought to provide roughly the same amount of available total or heme iron as meat. In this context, "roughly" means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, This means 95%, 96%, 97%, 98%, 99%, 100% or more. The total amount of bioavailable iron in the meat substitute of the present invention is approximately equal to the total amount of available iron in real meat. This means that the myoglobin in these animals contains less than 120% of the total iron or heme iron. This means that it can be considered a unique alternative fortification for improving the iron status of the population.

[0054] These animal myoglobins also have the benefits offered in plant-based meat analog products. Provides an amount of available total or heme iron that is generally greater than the amount of available total or heme iron In this context, "greater" means at least 5%, 6%, 7% ,8%,9%,10%,11%,12%,13%,14%,15%,16%,17%,1 8%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 2 This means 8%, 29%, 30% or more.

[0055] In the preferred definition, the value refers to the amount of available total iron or heme iron. According to the preferred definition, "mimicking meat values" is the same as "meat values" as outlined above. "Provides approximately the same amount of available total or heme iron as a nutrient in a diet containing 200 mg of niacin, 200 mg of niacin, or ...

[0056] Example 2.6 provides a nutritional analysis of a meat substitute according to the present invention, assessing iron bioavailability. Provide.

[0057] Within the context of the present invention, total iron is the total amount of iron that is bioavailable in the meat substitute. Heme iron is the moiety of iron transported by proteins containing a heme domain. The iron is available for absorption by the human body. Such proteins are referred to as amino acids as defined herein. Iron availability is determined by Proulx AK, et al (2006 )., J.Agric.Food.Chem.,(54),1518-1522) and / or can be assessed as described in the method of Example 2.6. Ferritin concentrations were first normalized to cellular protein concentrations. The percentage of lithium was compared to the percentage of FeSO4 as the relative biological value (RBV). represent.

[0058] In one embodiment, the meat substitute is produced by bacteria that live symbiotically in the root nodules of soybean plants. and / or does not contain leghemoglobin produced by the Heme-containing proteins include animal myoglobin.

[0059] In one embodiment, the animal myoglobin disclosed herein is present in the meat substitute of the present invention. This means that the meat substitutes of the present invention are This means that the protein may contain other proteins other than animal myoglobin as disclosed herein. An example of a protein that may be present is soy protein.

[0060] Within the context of the present invention, a heme-containing protein as defined is a protein that contains a covalently attached heme moiety. whole proteins or protein subunits that can be covalently or non-covalently bound to Heme-containing polypeptides can transport or store oxygen. Some examples of heme-containing proteins are globin, hemoglobin, leghemoglobin, There is a bottle.

[0061] In another embodiment, the meat substitute is produced by bacteria that live symbiotically in the root nodules of soybean plants. In one embodiment, the meat substitute does not contain leghemoglobin produced by symbiotic hemoglobin. In one embodiment, the meat substitute does not contain leghemoglobin. In an embodiment, the meat substitute is produced by bacteria that live symbiotically in the root nodules of soybean plants. Contains no raw protein.

[0062] In one embodiment, the meat substitute is produced by bacteria that live constitutively in the root nodules of soybean plants. leghemoglobin produced by the method of the present invention and does not contain ... It contains vin as the only heme-containing protein.

[0063] In one embodiment, the meat substitute contains similar amounts of animal myoglobin as its pure meat counterpart. In one embodiment, the amount is in the range of 0.1 to 5% by weight of animal myoglobin. In one embodiment, the amount is at least 0.1, 0.2, 0.3, 0.4, 0. 5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1. 5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2. 5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3. 5, 4.0, 4.5, 5.0 or more percent by weight animal myoglobin. At least 10%, 20%, 30%, 40%, 50% of the final weight of the composition Those skilled in the art will recognize that the amount may vary depending on the meat substitute. It is.

[0064] In one embodiment, the weight percentage of myoglobin in the meat substitute according to the present invention is 0. 1%-5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3% ,4.2%,4.1%,4%,3.9%,3.8%,3.7%,3.6%,3.5%,3 .4%, 3.3%, 3.2%, 3.1%, 3%, 2.95%, 2.9%, 2.85%, 2 0.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.4 5%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1% ,2.05%,2%,1.95%,1.9%,1.85%,1.8%,1.75%,1. 7%, 1.65%, 1.6%, 1.55%, 1.5%, 1.45%, 1.4%, 1.35 %, 1.3%, 1.25%, 1.2%, 1.15%, 1.1%, 1.05%, 1%, 0. 95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6 %, 0.55%, or 0.5%.

[0065] In one embodiment, the weight percentage of myoglobin in the meat substitute according to the present invention is 0. 5%~5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3% ,4.2%,4.1%,4%,3.9%,3.8%,3.7%,3.6%,3.5%,3 .4%, 3.3%, 3.2%, 3.1%, 3%, 2.95%, 2.9%, 2.85%, 2 0.8%, 2.75%, 2.7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.4 5%, 2.4%, 2.35%, 2.3%, 2.25%, 2.2%, 2.15%, 2.1% ,2.05%,2%,1.95%,1.9%,1.85%,1.8%,1.75%,1. 7%, 1.65%, 1.6%, 1.55%, 1.5%, 1.45%, 1.4%, 1.35 %, 1.3%, 1.25%, 1.2%, 1.15%, 1.1%, 1.05%, 1%, 0. 95%, 0.9%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, or 0 .6.

[0066] In one embodiment, the food ingredient comprises animal myoglobin in the range of 0.1 to 100% by weight. In one embodiment, the amount of animal myoglobin present in the product is determined based on the final weight of the product. Amount of at least 0.1%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 4.0 %, 4.5%, 5.0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%. Those skilled in the art will recognize that these amounts vary depending on the food ingredients. I know I'll get it.

[0067] In one embodiment, the meat substitute comprises 10% to 40% protein source, 5% to 30% fat. source, 0.1% to 5% NaCl, 0.1% to 5% fiber source, and 0.1% to 5% of the present invention or prepared by mixing them. In one embodiment, the meat substitute comprises 20% to 30% protein and 10% to 20% fat. source, 0.5% to 2% NaCl, 0.5% to 1.5% fiber source, and 0.1% to 3% It contains or is prepared by mixing the myoglobin according to the invention.

[0068] Preferably, the protein source is textured soy protein, pea protein, or Protein isolate, mung bean protein, textured wheat protein, rice protein or potato protein. In this context, textured soy protein The texture may be textured vegetable protein (TVP), as will be apparent to those skilled in the art. ) or soy meat.

[0069] Preferably, the liquid source is sunflower oil, coconut oil, safflower oil or cocoa butter. do.

[0070] Preferably, the fiber source is methylcellulose or potato starch.

[0071] In a more preferred embodiment, the meat substitute comprises 20% to 30% textured soybeans. Protein, 10%-20% sunflower oil, 0.5%-2% NaCl, 0.5%-1. 5% methylcellulose and 0.1% to 10% of the myoglobin according to the present invention; is prepared by mixing these. Even more preferably, The weight percentage of myoglobin in meat substitutes varies depending on the form: 0.1% to 5%, 4.9%, 4. .8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4 %, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2 %, 3.1%, 3%, 2.95%, 2.9%, 2.85%, 2.8%, 2.75%, 2. 7%, 2.65%, 2.6%, 2.55%, 2.5%, 2.45%, 2.4%, 2.35 %, 2.3%, 2.25%, 2.2%, 2.15%, 2.1%, 2.05%, 2%, 1. 95%, 1.9%, 1.85%, 1.8%, 1.75%, 1.7%, 1.65%, 1.6 %, 1.55%, 1.5%, 1.45%, 1.4%, 1.35%, 1.3%, 1.25% ,1.2%,1.15%,1.1%,1.05%,1%,0.95%,0.9%,0.8 5%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55%, or 0. It is 5%.

[0072] In one embodiment, the meat substitute comprises 20% to 30% textured soy protein. , 10%~20% sunflower oil, 1%~2% NaCl, 0.5%~1.5% methyl selenite cellulose, 50% to 70% water, and 0.2% to 0.8% myoglobin, or It is prepared by mixing these.

[0073] In a preferred embodiment, the meat substitute is 23% to 27% textured soy protein. Protein, 13%-17% sunflower oil, 1.25%-1.75% NaCl, 0.75% ~1.25% methylcellulose, 55%~60% water, and 0.45%~0.55% It contains myoglobin or is prepared by mixing them.

[0074] In one embodiment, the meat substitute comprises 25% textured soy protein, 15% of sunflower oil, 1.5% NaCl, 1.0% methylcellulose, 57% water, and 0 0.5% myoglobin, or prepared by mixing these, These weight percentages are rounded to two significant digits.

[0075] In one embodiment, the meat substitute comprises 15% to 35% textured soy protein. , 10%~20% sunflower oil, 0.5%~2.5% NaCl, 0.5%~1.5% Contains methylcellulose, 50% to 70% water, and 0.5% to 1.5% myoglobin. or by mixing these.

[0076] In a preferred embodiment, the meat substitute is 22% to 27% textured soy protein. Protein, 13%-17% sunflower oil, 1.25%-1.75% NaCl, 0.75% ~1.25% methylcellulose, 50%~60% water, and 0.75%~1.25% It contains myoglobin or is prepared by mixing them.

[0077] In one embodiment, the meat substitute comprises 25% textured soy protein, 15% of sunflower oil, 1.5% NaCl, 1.0% methylcellulose, 57% water, and 1 0.0% myoglobin, or prepared by mixing these, These weight percentages are rounded to two significant digits.

[0078] In the above meat substitutes, % refers to weight percentage. The weight percentages that describe the composition of the meat substitute are , unless adding up to 100%, the addition of water to the corresponding percentage is assumed.

[0079] Example 1.4 provides an example of the preparation of a meat substitute according to the present invention.

[0080] In one embodiment, the meat substitute has a similar amount of bioavailability to its real meat counterpart. In one embodiment, the amount is per 100 grams of meat substitute. In one embodiment, the range is 0.3 to 20 mg of bioavailable iron or heme iron. The amount ranges from 0.1mg to 16.5mg, 16.1mg, per 100g of meat substitute. 7mg, 15.84mg, 15.51mg, 15.18mg, 14.85mg, 14.5 2mg, 14.19mg, 13.86mg, 13.53mg, 13.2mg, 12.87 mg, 12.54mg, 12.21mg, 11.88mg, 11.55mg, 11.22 mg, 10.89mg, 10.56mg, 10.23mg, 9.9mg, 9.735mg , 9.57mg, 9.405mg, 9.24mg, 9.075mg, 8.91mg, 8. 745mg, 8.58mg, 8.415mg, 8.25mg, 8.085mg, 7.92 mg, 7.755mg, 7.59mg, 7.425mg, 7.26mg, 7.095mg , 6.93mg, 6.765mg, 6.6mg, 6.435mg, 6.27mg, 6.1 05mg, 5.94mg, 5.775mg, 5.61mg, 5.445mg, 5.28m g, 5.115mg, 4.95mg, 4.785mg, 4.62mg, 4.455mg, 4.29mg, 4.125mg, 3.96mg, 3.795mg, 3.63mg, 3.4 65mg, 3.3mg, 3.135mg, 2.97mg, 2.805mg, 2.64mg , 2.475mg, 2.31mg, 2.145mg, 1.98mg, 1.815mg, or is in the range of 1.65 mg of bioavailable iron or heme iron. The amounts required are 0.3, 0.4, 0.5, 0.6, and 0.7 per 100 grams of meat substitute. 7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2. 8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4. 9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8. 1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11 .8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6 , 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 1 3.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14. 3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16 .8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6 ,17.7,17.8,17.9,18,18.1,18.2,18.3,18.4,1 8.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19. 3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, or 20 mg of raw It is biologically available iron or heme iron.

[0081] In one embodiment, the amino acid sequence of the animal myoglobin identified herein above is: The following amino acid combinations F in position 30, and / or Q in position 65, and / or H at position 92, and / or H at position 94, and / or F in position 30 and Q in position 65, and / or F at position 30 and H at position 92, and / or F at position 30 and H at position 94, and / or Q at position 65 and H at position 92, and / or Q at position 65 and H at position 94, and / or H at position 92 and H at position 94, and / or F at position 30 and Q at position 65 and H at position 92, and / or F at position 30 and Q at position 65 and H at position 94, and / or F at position 30 and H at position 92 and H at position 94, and / or Q at position 65 and H at position 92 and H at position 94, and / or F at position 30, Q at position 65, H at position 92, and H at position 94 and at least seven of the sequences identified herein above in combination with at least one of 0%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, At least 84%, at least 85%, at least 86%, at least 87%, at least At least 88%, at least 89%, at least 90%, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least have 97%, at least 98%, at least 99% or 100% identity or similarity Contains an array that

[0082] In one embodiment, the amino acid sequence of the animal myoglobin identified herein above is For each of the identified sequences, the combination of Q or H at position 65 and H at position 94 and preferably further in combination with I at position 143, At least 70%, at least 71%, at least 72%, at least 73%, At least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82% , at least 83%, at least 84%, at least 85%, at least 86%, less At least 87%, at least 88%, at least 89%, at least 90%, at least 91% %, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99% or 100% This includes sequences that have identity or similarity.

[0083] In one embodiment, the amino acid sequence of the animal myoglobin identified herein above is For each of the identified sequences, Q or H at position 65, H at position 92, and In combination with H, and preferably also in combination with I in position 143, at least 70%, at least 71%, at least 72%, at least at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least At least 86%, at least 87%, at least 88%, at least 89%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least At least 95%, at least 96%, at least 97%, at least 98%, at least 99% % or 100% identity or similarity.

[0084] In one embodiment, the amino acid sequence of the animal myoglobin identified herein above is For each of the identified sequences, Q or H at position 65, H at position 92, and In combination with H, and preferably also in combination with I at position 143, at least 70%, at least 71%, at least 72%, at least at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least At least 86%, at least 87%, at least 88%, at least 89%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least At least 95%, at least 96%, at least 97%, at least 98%, at least 99% % or 100% identity or similarity.

[0085] In one embodiment, the amino acid sequence of the animal myoglobin identified herein above is For each of the identified sequences, in combination with H at position 92, preferably further , in combination with I at position 143, and at least 70 %, at least 71%, at least 72%, at least 73%, at least 74%, at least at least 75%, at least 76%, at least 77%, at least 78%, at least 7 9%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least have 97%, at least 98%, at least 99% or 100% identity or similarity Contains an array of

[0086] In one embodiment, the meat substitute or food ingredient comprises a Q or H at position 65 and an H at position 94. and optionally the following amino acids at the following positions within SEQ ID NO: 1: , E at position 9, K at position 13, T as position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88, SEQ ID NO: 1, having at least one of I at position 102 and / or E at position 123 1 and a myoglobin represented by an amino acid sequence having at least 70% sequence identity with In one embodiment, the meat substitute or food ingredient comprises a Q or H at position 65 and a Q or H at position 94. in combination with H, and optionally at the following positions within SEQ ID NO: 2 or 3: , i.e., E at position 9, K at position 13, T at position 14, P at position 23, and L at position 31, V at position 54, Q at position 65, V at position 67, Q at position 84, at least one of Q at position 88, I at position 102, E at position 123, and / or I at position 143 an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2 or 3; Preferably, the animal myoglobin to be identified is represented by the sequence The amino acid sequence is SEQ ID NO: 1, 2, or 3 in combination with Q or H at position 65 and H at position 94. or 3 and at least 70%, at least 71%, at least 72%, at least 73%, At least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82% , at least 83%, at least 84%, at least 85%, at least 86%, less At least 87%, at least 88%, at least 89%, at least 90%, at least 91% %, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99% or 100% SEQ ID NO: 1 is a sequence of a portion of myoglobin derived from mammoth. SEQ ID NOs: 2 and 3 are myoglobins from mammoths. In one embodiment, In one embodiment, the meat substitute comprises myoglobin comprising the amino acid sequence of SEQ ID NO: 1. In this case, the meat substitute is mammoth myocardium, which is represented by the amino acid sequence of SEQ ID NO: 2 or 3. Since such myoglobin is stable to oxidation, especially at low pH, This myoglobin is believed to have attractive properties. It is believed that smyoglobin is stable because it resists oxidation. It is also likely to be less likely to be converted into carcinogenic products than real meat. , providing additional health properties to the meat substitutes of the present invention.

[0087] In one embodiment, the meat substitute or food ingredient has a Q or H at position 65, a Q at position 92 and In combination with H at position 94, and optionally at the following locations within SEQ ID NO:2: acid, i.e., E at position 9, K at position 13, T at position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, At least one of Q at position 88, I at position 102, and / or E at position 123 and is represented by an amino acid sequence having at least 70% sequence identity with SEQ ID NO:2. Preferably, the amino acid sequence of the identified animal myoglobin comprises the amino acid sequence at the position Q or H at position 65, Q at position 92 and H at position 94 in combination with SEQ ID NO: 2 At least 70%, at least 71%, at least 72%, at least 73%, at least 74% , at least 75%, at least 76%, at least 77%, at least 78%, less At least 79%, at least 80%, at least 81%, at least 82%, at least 83% %, at least 84%, at least 85%, at least 86%, at least 87%, at least at least 88%, at least 89%, at least 90%, at least 91%, at least 9 2%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity or similarity SEQ ID NO: 2 is myoglobin from woolly mammoth. In one embodiment, the meat substitute comprises woolly mammoth myoglobin represented by SEQ ID NO: 2. This myoglobin is stable to oxidation, especially at low pH. The color of the meat substitute of the present invention is believed to be similar to that of woolly mammoth meat. Robin is considered stable because it resists oxidation. This stability against oxidation also This invention is likely to be less likely to be converted into carcinogenic products than real meat. provides additional health properties to meat substitutes.

[0088] In one embodiment, the meat substitute or food ingredient has a Q or H at position 65, an H at position 92 and in combination with H at position 94, and optionally at the following locations within SEQ ID NO:3: acid, i.e., E at position 9, K at position 13, T at position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, At least one of Q at position 88, I at position 102, and / or E at position 123 and is represented by an amino acid sequence having at least 70% sequence identity with SEQ ID NO:3. Preferably, the amino acid sequence of the identified animal myoglobin comprises the amino acid sequence at the position Q or H at position 65, H at position 92 and H at position 94 in combination with SEQ ID NO: 3 At least 70%, at least 71%, at least 72%, at least 73%, at least 74% , at least 75%, at least 76%, at least 77%, at least 78%, less At least 79%, at least 80%, at least 81%, at least 82%, at least 83% %, at least 84%, at least 85%, at least 86%, at least 87%, at least at least 88%, at least 89%, at least 90%, at least 91%, at least 9 2%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity or similarity SEQ ID NO: 3 is myoglobin from the steppe mammoth. In an embodiment, the meat substitute is a stepma represented by the amino acid sequence of SEQ ID NO:3. This myoglobin is stable to oxidation, especially at low pH. Therefore, this myoglobin is believed to have attractive properties. It is believed that step mammoth myoglobin is stable because it resists oxidation. Its resistance to oxidation also makes it less likely to convert into carcinogenic products than real meat. This would provide additional health benefits to the meat substitutes of the present invention.

[0089] In one embodiment, the meat substitute or food ingredient has SEQ ID NO: 1 in combination with F at position 30. No. 3 and at least 70%, at least 71%, 72%, 73%, 74%, 75%, 76% ,77%,78%,79%,80%,81%,82%,83%,84%,85%,86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% , 97%, 98%, or 99% identity or similarity. Contains myoglobin.

[0090] In one embodiment, the meat substitute or food ingredient has SEQ ID NO: 5 in combination with Q at position 65. No. 3 and at least 70%, at least 71%, 72%, 73%, 74%, 75%, 76% ,77%,78%,79%,80%,81%,82%,83%,84%,85%,86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% , 97%, 98%, or 99% identity or similarity. Contains myoglobin.

[0091] In one embodiment, the meat substitute or food ingredient has SEQ ID NO: 1 in combination with H at position 92. No. 3 and at least 70%, at least 71%, 72%, 73%, 74%, 75%, 76% ,77%,78%,79%,80%,81%,82%,83%,84%,85%,86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% , 97%, 98%, or 99% identity or similarity. Contains myoglobin.

[0092] In one embodiment, the meat substitute or food ingredient has SEQ ID NO: 1 in combination with H at position 94. No. 3 and at least 70%, at least 71%, 72%, 73%, 74%, 75%, 76% ,77%,78%,79%,80%,81%,82%,83%,84%,85%,86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% , 97%, 98%, or 99% identity or similarity. Contains myoglobin.

[0093] In one embodiment, the meat substitute or food ingredient comprises a combination of F at position 30 and Q at position 65. Combined, at least 70%, at least 71%, 72%, 73%, 74% with SEQ ID NO: 3 ,75%,76%,77%,78%,79%,80%,81%,82%,83%,84% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98%, or 99% identity or similarity This includes myoglobin, which is represented by:

[0094] In one embodiment, the meat substitute or food ingredient has a combination of F at position 30 and H at position 92. Combined, at least 70%, at least 71%, 72%, 73%, 74% with SEQ ID NO: 3 ,75%,76%,77%,78%,79%,80%,81%,82%,83%,84% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98%, or 99% identity or similarity This includes myoglobin, which is represented by:

[0095] In one embodiment, the meat substitute or food ingredient has a combination of F at position 30 and H at position 94. Combined, at least 70%, at least 71%, 72%, 73%, 74% with SEQ ID NO: 3 ,75%,76%,77%,78%,79%,80%,81%,82%,83%,84% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98%, or 99% identity or similarity This includes myoglobin, which is represented by:

[0096] In one embodiment, the meat substitute or food ingredient has a combination of Q at position 65 and H at position 92. Combined, at least 70%, at least 71%, 72%, 73%, 74% with SEQ ID NO: 3 ,75%,76%,77%,78%,79%,80%,81%,82%,83%,84% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98%, or 99% identity or similarity This includes myoglobin, which is represented by:

[0097] In one embodiment, the meat substitute or food ingredient has a combination of Q at position 65 and H at position 94. Combined, at least 70%, at least 71%, 72%, 73%, 74% with SEQ ID NO: 3 ,75%,76%,77%,78%,79%,80%,81%,82%,83%,84% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98%, or 99% identity or similarity This includes myoglobin, which is represented by:

[0098] In one embodiment, the meat substitute or food ingredient has a combination of H at position 92 and H at position 94. Combined, at least 70%, at least 71%, 72%, 73%, 74% with SEQ ID NO: 3 ,75%,76%,77%,78%,79%,80%,81%,82%,83%,84% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98%, or 99% identity or similarity This includes myoglobin, which is represented by:

[0099] In one embodiment, the meat substitute or food ingredient has an F at position 30 and a Q at position 65 and a In combination with H at 92, at least 70%, at least 71%, 72% with SEQ ID NO: 3 ,73%,74%,75%,76%,77%,78%,79%,80%,81%,82% ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity and myoglobin represented by an amino acid sequence comprising:

[0100] In one embodiment, the meat substitute or food ingredient has an F at position 30 and a Q at position 65 and a In combination with H of 94, at least 70%, at least 71%, 72% with SEQ ID NO: 3 ,73%,74%,75%,76%,77%,78%,79%,80%,81%,82% ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity and myoglobin represented by an amino acid sequence comprising:

[0101] In one embodiment, the meat substitute or food ingredient has an F at position 30 and an H at position 92 and an H at position In combination with H of 94, at least 70%, at least 71%, 72% with SEQ ID NO: 3 ,73%,74%,75%,76%,77%,78%,79%,80%,81%,82% ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity and myoglobin represented by an amino acid sequence comprising:

[0102] In one embodiment, the meat substitute or food ingredient has a Q at position 65 and an H at position 92 and an H at position In combination with H of 94, at least 70%, at least 71%, 72% with SEQ ID NO: 3 ,73%,74%,75%,76%,77%,78%,79%,80%,81%,82% ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity and myoglobin represented by an amino acid sequence comprising:

[0103] In one embodiment, the meat substitute or food ingredient has an F at position 30 and a Q at position 65 and a In combination with H at position 92 and H at position 94, it has at least 70% similarity to SEQ ID NO: 3, Also 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% ,81%,82%,83%,84%,85%,86%,87%,88%,89%,90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% This includes myoglobin, which is represented by an amino acid sequence containing identity or similarity.

[0104] In one embodiment, the meat substitute comprises a steroid hormone represented by the amino acid sequence of SEQ ID NO:3. Such myoglobins are particularly resistant to oxidation at low pH. Because it is stable, this myoglobin is believed to have attractive properties. The color of the meat is thought to be stable because mammoth myoglobin resists oxidation. The stability to oxidation is also higher than that of real meat and / or the amino acid sequence SEQ ID NO: 3 than meat substitutes that do not contain mammoth myoglobin, a carcinogenic product The meat substitutes of the present invention may have additional health benefits because they are less likely to be converted into lactic acid bacteria. Example 2.4 provides a method for the preparation of step-wise mammoth myoglobin according to the present invention. The present invention provides a color analysis of meat substitutes containing Tep Mammoth Myoglobin. Example 2.5 provides an aroma analysis of the meat substitutes, while Example 2.6 provides an analysis of the iron bioavailability. Provides nutritional analysis of myoglobin to assess use.

[0105] In one embodiment, the meat substitute is made from β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin and and transferrin, and the dairy protein is derived from woolly mammoth. In another embodiment, the meat substitute is , β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lacto Albumin, β-lactoglobulin, lactoferrin and transferrin does not contain one or more milk proteins selected from the group consisting of mammary It is derived from the Japanese name.

[0106] In one embodiment, the meat substitute is made from β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin and and transferrin, and the milk protein is not produced by a mammal or mammalian cells.

[0107] In a more preferred embodiment, the meat substitute is a mixture of β-casein, κ-casein, α-S1- Casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lacto It does not contain milk proteins such as ferrin or transferrin, and among these, In another more preferred embodiment, the meat substitute is β- Casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin Milk proteins such as lactoferrin, β-lactoglobulin, lactoferrin, or transferrin It does not contain any of the above, and the milk protein is derived from mammoths.

[0108] In another more preferred embodiment, β-casein, κ-casein, α-S1-casein α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin does not contain milk proteins such as lactic acid bacteria or transferrin, and wherein the milk proteins are: It is not produced by a mammal or mammalian cells.

[0109] In one embodiment, the meat substitute does not contain κ-casein from woolly mammoths. In some embodiments, the meat substitute is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 1 Sequences with 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity In a further embodiment, the meat substitute does not contain a protein represented by SEQ ID NO: 1. It does not contain the protein represented by 7.

[0110] In one embodiment, the meat substitute does not contain beta-casein from woolly mammoths. In some embodiments, the meat substitute is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 1 Sequences with 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity In a further embodiment, the meat substitute does not contain a protein represented by SEQ ID NO: 1. It does not contain the protein represented by 8.

[0111] meat In a second embodiment, animal myoglobin, preferably mammalian myoglobin, more preferably or mammalian myoglobin derived from mammoth, pig, sheep, or cow, preferably More preferably, the mammoth is a woolly mammoth (Mammuthus primigeni us) or the steppe mammoth (Mammuthus trogontherii) mammalian myoglobin or avian myoglobin, preferably a steppe mammoth bin, preferably avian myoglobin from chicken, or fish myoglobin, preferably Myoglobin derived from tuna or meat products containing myoglobin derived from these. Therefore, animal myoglobin is not produced by or contained in the animal. The meat product is provided by the method of claim 1, which is not a cell derived from the cell from which the meat product is obtained. For example, meat products contain myoglobin that originates from an animal but was not produced there.

[0112] In one embodiment, exogenous myoglobin, preferably exogenous animal myoglobin, more preferably Preferably derived from mammoth, pig, sheep or cow, more preferably from mammoth It is a woolly mammoth (Mammuthus primigenius) or a steppe mammoth. Mammuthus trogontherii, preferably steppe mammoth exogenous animal myoglobin or avian myoglobin, preferably from chicken; Alternatively, a meat product containing fish myoglobin, preferably derived from tuna, is provided. So, exogenous myoglobin is defined as myoglobin that is not endogenously present in the meat. will be done.

[0113] In one embodiment, the myoglobin in the meat product of the above embodiment is Acid sequence, i.e. a) A sequence having at least 70% sequence identity with SEQ ID NO: 3 and the following amino acid combination: That is, F at position 30, and / or Q in position 65, and / or H at position 92, and / or H at position 94, and / or F at position 30 and Q at position 65, and / or F at position 30 and H at position 92, and / or F at position 30 and H at position 94, and / or Q at position 65 and H at position 92, and / or Q at position 65 and H at position 94, and / or H at position 92 and H at position 94, and / or F at position 30 and Q at position 65 and H at position 92, and / or F at position 30 and Q at position 65 and H at position 94, and / or F at position 30 and H at position 92 and H at position 94, and / or Q at position 65 and H at position 92 and H at position 94, and / or F at position 30, Q at position 65, H at position 92, and H at position 94 an amino acid sequence having at least one of b) at least 70% sequence identity with SEQ ID NO: 2 or 3, Q or H at position 65 and H at position 94, and optionally at the following positions in SEQ ID NO: 2 or 3: The following amino acids: E at position 9, K at position 13, T at position 14, P at position 27, L at position 31, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88, I at position 102, E at position 123, and / or E at position 143 an amino acid sequence having at least one of the following I: c) at least 70% sequence identity with SEQ ID NO: 1, Q or H at position 65, and 94 H, and optionally the following amino acids at the following positions in SEQ ID NO: 1: E at position 9, K at position 13, T at position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88 Q, I at position 102, and / or E at position 123. amino acid sequence, d) at least 70% identity to SEQ ID NO: 4, 5 or 6, Q or H at position 65 and H at position 94, and optionally the following in SEQ ID NO: 4, 5, or 6: The following amino acids are present: N at position 13, Q at position 27, I at position 31, N at position 7, A at position 128, S at position 133, A at position 145, and / or position 150 an amino acid sequence having at least one of the following Ls: e) at least 70% identity to SEQ ID NO: 7, Q or H at position 65, and Q or H at position 94 and optionally the following amino acid at the following position in SEQ ID NO: 7: , Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M, H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 3, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 111, and I at position 117, A at position 118, A at position 121, S at position 128, and S at position 133 at least one of K at position 145, S at position 145, and / or F at position 150 an amino acid sequence, or f) at least 70% identity to SEQ ID NO: 8, Q or H at position 65, and Q or H at position 94 Amino acid sequence containing H is represented by one of

[0114] In one embodiment, a meat product comprising myoglobin is provided, wherein the myoglobin in the meat product is The myoglobin concentration is higher than the average myoglobin concentration in the meat from which the meat product is derived. As used herein, the concentration of myoglobin is determined by the percentage of all myoglobin variants present in the meat product. of myoglobin in the meat from which the meat product is derived. The average concentration of myoglobin in the meat is determined over several corresponding animals. This refers to the average concentration of myoglobin added after harvest.

[0115] myoglobin In a third aspect, the present invention provides a protein that can be represented by a sequence comprising SEQ ID NO:3. to provide.

[0116] In a preferred embodiment, the protein can be represented by a sequence comprising SEQ ID NO:3. The sequence is 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, or 300 Proteins are provided having a length of amino acid residues.

[0117] In a preferred embodiment, a sequence consisting of SEQ ID NO: 3 and the N-terminal end sequence of SEQ ID NO: 3 is Preferably, the terminal sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 8 5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99, or 100 amino acid residues in length.

[0118] In an alternative embodiment, a sequence consisting of SEQ ID NO: 3 and the C-terminal end sequence of SEQ ID NO: 3 is Preferably, the terminal sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 8 5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99, or 100 amino acid residues in length.

[0119] In a preferred embodiment, the sequence comprises or consists of SEQ ID NO: 3 and a tag. and preferably, the tag is a poly-histidine tag. This provides protein.

[0120] In a preferred embodiment, the present invention relates to a myoglobin, wherein the myoglobin is a s Preferably, the myoglobin is derived from the Steppe Mammoth, and the myoglobin is represented by SEQ ID NO: 3. The myoglobin can be substituted as described above. The composition may be for use in meat, food ingredients or meat products.

[0121] In Example 2.2, the structural features of myoglobin represented by SEQ ID NO: 3 are studied. Specifically, SEQ ID NO: 3 surprisingly exhibits a higher activity compared to other myoglobins. It has been shown that this corresponds to a positive surface charge. Without being bound by this theory, The myoglobin represented by No. 3 is therefore characterized by high stability and low tendency to aggregate. This means that these ingredients in meat substitutes, food ingredients, or meat products as described above can be It would be advantageous to use myoglobin of

[0122] The present invention further relates to a composition comprising a protein according to this third aspect, as described above. to provide.

[0123] nucleic acid In a fourth aspect, the present invention provides a nucleic acid, the nucleic acid being represented by SEQ ID NO:3. A nucleic acid that can be represented by a sequence containing a subsequence that encodes a protein that can be expressed as a protein, preferably Preferably, the nucleic acid is provided, the partial sequence of which can be represented by SEQ ID NO:11.

[0124] In a preferred embodiment, the portion encoding the protein can be represented by SEQ ID NO:3. A partial sequence, preferably a partial sequence that can be represented by SEQ ID NO: 11. A nucleic acid that can be represented by a sequence comprising 462, 463, 464, 46 5, 466, 467, 468, 469, 470, 471, 472, 473, 474, 47 5, 476, 477, 478, 479, 480, 481, 482, 483, 484, 48 5, 486, 487, 488, 489, 490, 491, 492, 493, 494, 49 5, 496, 497, 498, 499, 500, 501, 502, 503, 504, 50 5, 506, 507, 508, 509, 510, 511, 512, 513, 514, 51 5, 516, 517, 518, 519, 520, 521, 522, 523, 524, 52 5, 526, 527, 528, 529, 530, 531, 532, 533, 534, 53 5, 536, 537, 538, 539, 540, 541, 542, 543, 544, 54 5, 546, 547, 548, 549, 550, 551, 552, 553, 554, 55 5, 556, 557, 558, 559, 560, 561, 562, 563, 564, 56 5, 566, 567, 568, 569, 570, 571, 572, 573, 574, 57 5, 576, 577, 578, 579, 580, 581, 582, 583, 584, 58 5, 586, 587, 588, 589, 590, 591, 592, 593, 594, 59 Nucleic acids having a length of 5, 596, 597, 598, 599, or 600 bases are provided. will be done.

[0125] In a preferred embodiment, the portion encoding the protein can be represented by SEQ ID NO:3. a partial sequence, preferably a partial sequence that can be represented by SEQ ID NO: 11; and a terminal sequence at the 5' end of the partial sequence. Preferably, the subsequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 6 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, having a length of 92, 93, 94, 95, 96, 97, 98, 99, or 100 bases .

[0126] In a preferred embodiment, the portion encoding the protein can be represented by SEQ ID NO:3. a partial sequence, preferably a partial sequence that can be represented by SEQ ID NO: 11; and a terminal sequence at the 3' end of the partial sequence. Preferably, the subsequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 6 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, having a length of 92, 93, 94, 95, 96, 97, 98, 99, or 100 bases .

[0127] In a preferred embodiment, the nucleic acid is a myogog from a steppe mammoth. Preferably, the myoglobin is represented by SEQ ID NO:3. and most preferably, the nucleic acid can be represented by SEQ ID NO: 11. It is served.

[0128] The present invention further provides a composition comprising a nucleic acid according to this fourth aspect, as described above. do.

[0129] Gene constructs In a fifth embodiment, a nucleic acid encoding a myoglobin as defined herein above A genetic construct comprising:

[0130] A "genetic construct" as described herein would be understood by one of skill in the art in light of this disclosure. "Genetic construct" has its ordinary and usual meaning as understood in the art. It can also be called an "expression cassette" or "expression construct," and is used to express a protein of interest. a gene encoding the protein of interest operably linked to a promoter that controls expression of the gene This application, entitled "General Information," Some of these contain more details about "gene constructs." The term "operably linked" as used herein is further defined in the section of this application entitled "General Information." This is explained in that part.

[0131] The nucleotide sequence encoding animal myoglobin can be found in any animal myoglobin gene. A human or animal myoglobin coding sequence, preferably mammoth, porcine, ovine, bovine, or guinea pig myoglobin coding sequence. Animal myoglobin gene or animal myoglobin code derived from chicken or tuna sequences, or preferably derived from mammoth, pig, sheep, cow, chicken, or tuna May be derived from a mutated animal myoglobin gene or animal myoglobin coding sequence .

[0132] Thus, in some embodiments, preferred nucleotides encoding animal myoglobins are The octide sequence has at least 70% identical identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, At least 71%, at least 72%, at least 73%, at least 74%, at least 75% %, at least 76%, at least 77%, at least 78%, at least 79%, at least at least 80%, at least 81%, at least 82%, at least 83%, at least 8 4%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, Contains sequences with at least 98%, at least 99%, or 100% identity or similarity and at the positions identified herein above (i.e., Q or H at position 65 and Q or H at position 9 A polypeptide represented by an amino acid sequence with individual amino acids (in combination with H in 4) The position of an amino acid may also be taken into consideration.

[0133] In some embodiments, preferred nucleotide sequences encoding animal myoglobins are The sequence is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 9 73%, at least 74%, at least 75%, at least 76%, at least 77% , at least 78%, at least 79%, at least 80%, at least 81%, less At least 82%, at least 83%, at least 84%, at least 85%, at least 86% %, at least 87%, at least 88%, at least 89%, at least 90%, at least at least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99% or The following amino acid combinations have 100% identity or similarity: F at position 30, and / or Q in position 65, and / or H at position 92, and / or H at position 94, and / or F at position 30 and Q at position 65, and / or F at position 30 and H at position 92, and / or F at position 30 and H at position 94, and / or Q at position 65 and H at position 92, and / or Q at position 65 and H at position 94, and / or H at position 92 and H at position 94, and / or F at position 30 and Q at position 65 and H at position 92, and / or F at position 30 and Q at position 65 and H at position 94, and / or F at position 30 and H at position 92 and H at position 94, and / or Q at position 65 and H at position 92 and H at position 94, and / or F at position 30, Q at position 65, H at position 92, and H at position 94 A polypeptide represented by an amino acid sequence comprising a sequence having at least one of Code the following.

[0134] In some embodiments, the animal mitochondria present in the genetic construct according to the present invention The nucleotide sequences encoding globin are SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 4, 15, or 16. at least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, At least 79%, at least 80%, at least 81%, at least 82%, at least At least 83%, at least 84%, at least 85%, at least 86%, at least 87% , at least 88%, at least 89%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, at least 99% or 100% identity do.

[0135] The descriptions of "identity" or "sequence identity" and "similarity" or "sequence similarity" are used in "general The information is provided under the section entitled "Reference Information."

[0136] In some embodiments, the genetic construct is as described herein. Therefore, the nucleotide sequence encoding animal myoglobin is (a) an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8 and at least 75 %, at least 76%, at least 77%, at least 78%, at least 79%, at least at least 80%, at least 81%, at least 82%, at least 83%, at least 8 4%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, have at least 98%, at least 99%, or 100% sequence identity or similarity The sequence is determined by an amino acid sequence containing Q or H at position 65 in combination with H at position 94. A nucleotide sequence encoding a polypeptide represented by the formula (I) and optionally taking into account the amino acid positions. This can be taken into consideration. (b) the nucleotide sequence of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15 or 16; At least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68% , at least 69%, at least 70%, at least 71%, at least 72%, less At least 73%, at least 74%, at least 75%, at least 76%, at least 77% %, at least 78%, at least 79%, at least 80%, at least 81%, at least at least 82%, at least 83%, at least 84%, at least 85%, at least 8 6%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or indicates a nucleotide sequence with 100% sequence identity. (c) A nucleotide sequence that differs from the sequence of nucleotides in (b) due to the degeneracy of the genetic code. column A genetic construct is provided, wherein the genetic construct is selected from the group consisting of:

[0137] In some embodiments, the genetic constructs described herein are The nucleotide sequence encoding the myoglobin is (a) at least 75%, at least 76%, at least 78%, or at least 79% of the amino acid sequence of SEQ ID NO: 3; 7%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least At least 95%, at least 96%, at least 97%, at least 98%, at least 99% or a sequence having 100% sequence identity or similarity and comprising the following amino acid combinations: combination F at position 30, and / or Q in position 65, and / or H at position 92, and / or H at position 94, and / or F at position 30 and Q at position 65, and / or F at position 30 and H at position 92, and / or F at position 30 and H at position 94, and / or Q at position 65 and H at position 92, and / or Q at position 65 and H at position 94, and / or H at position 92 and H at position 94, and / or F at position 30 and Q at position 65 and H at position 92, and / or F at position 30 and Q at position 65 and H at position 94, and / or F at position 30 and H at position 92 and H at position 94, and / or Q at position 65 and H at position 92 and H at position 94, and / or F at position 30, Q at position 65, H at position 92, and H at position 94 A polypeptide represented by an amino acid sequence having at least one of the nucleotide sequence (b) a nucleotide sequence that is at least 60%, at least 61%, or at least At least 62%, at least 63%, at least 64%, at least 65%, at least 66 %, at least 67%, at least 68%, at least 69%, at least 70%, at least at least 71%, at least 72%, at least 73%, at least 74%, at least 7 5%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, At least 89%, at least 90%, at least 91%, at least 92%, at least At least 93%, at least 94%, at least 95%, at least 96%, at least 97% , nucleotides having at least 98%, at least 99%, or 100% sequence identity array. (c) A nucleotide sequence that differs from the sequence of the nucleotide sequence in (b) due to the degeneracy of the genetic code. column A genetic construct is provided, wherein the genetic construct is selected from the group consisting of:

[0138] The nucleotide sequences described herein or the animal myoglobins described herein The nucleotide sequences derived from the animal myoglobins are known to those skilled in the art. Such animal myoglobins exhibit at least a detectable level of activity.

[0139] The activity of animal myoglobin may be to transport iron through its heme domain. This activity can be assessed by methods known to those skilled in the art, for example as defined herein above. It can be done.

[0140] In some embodiments, the nucleotide sequence encoding the animal myoglobin is A gene described herein selected from numbers 19, 20, 21, 22, 23 and 24. A child construct is provided.

[0141] As used herein, SEQ ID NO: 19 refers to the steppe mammoth, represented by SEQ ID NO: 3. SEQ ID NO: 20 represents the nucleotide sequence encoding the nucleotide sequence represented by SEQ ID NO: 4. SEQ ID NO: 21 represents the nucleotide sequence encoding sheep myoglobin, 5, and SEQ ID NO: 2. 2 represents the nucleotide sequence encoding the porcine myoglobin represented by SEQ ID NO:6. SEQ ID NO: 23 is a sequence encoding chicken myoglobin represented by SEQ ID NO: 7. SEQ ID NO: 24 represents the nucleotide sequence of the tuna myoglobin represented by SEQ ID NO: 8. These nucleotide sequences represent nucleotide sequences encoding Pichia pastoris. Squirrel (Pichia pastoris) (Komagata The codons have been optimized for expression in Saccharomyces cerevisiae. The optimization of is detailed in Example 2.1.

[0142] In some embodiments, the genetic construct further comprises a regulatory region. In one embodiment, the genetic construct further comprises a promoter. In this case, the gene construct comprises a promoter, a terminator, and optionally a signal peptide. The gene construct may also include a marker. Such a signal peptide preferably facilitates export of the expressed myoglobin. The vendor must optimize the identity of the regulatory region and marker depending on the host cell used. I know that I will have to.

[0143] In some embodiments, the genetic construct is as described herein. Therefore, the gene constructs represented by SEQ ID NOs: 25, 26, 27, 28, 29 or 30 Lactic acid is provided.

[0144] As used herein, SEQ ID NOs: 25 to 30 are represented by SEQ ID NOs: 19 to 24, respectively. Steppe mammoth myoglobin and Saccharomyces cerevisiae It encodes the signal peptide of mating factor alpha of Yeast cerevisiae. Pichia pastoris (Co) containing the nucleotide sequence Expression in Komagataella phaffii In other words, SEQ ID NO: 25 represents a codon-optimized gene construct for ~30 are the numbers of steppe mammoth, sheep, cow, pig, chicken, and tuna minnows, respectively. Figure 1 corresponds to the gene construct for expression of globin. Codon optimization is shown in Example 1. More details are provided in 2.1.

[0145] For example, when bacteria (preferably E. coli) are used as host cells, In this case, the following regulatory regions can be used: Suitable promoters for use in bacteria include , lac, trp, tac, T7 (used in the examples), phoA, ara, xapA, ca d, P1 and P2 from the recA, spc, bla, and rrnB ribosomal RNA operons , P derived from phage λ L A promoter. A terminator suitable for use in bacteria. The genes are lac, trp, tac, T7 (used in the examples), phoA, ara, xapA, P1 and P2 from the cad, recA, spc, bla, and rrnB ribosomal RNA operons P2 is the PL terminator from phage λ. The preferred promoter used is the T7 promoter. A preferred promoter and / or terminator is the T7 terminator. The signal peptide is the E. coli Sec recognition peptide (SecA), (E. coli) Tet recognition peptide, E. coli (E. coli) dsbA, E. coli (E. coli) phoA, E. coli (E. coli) pelB, E. coli (E. coli) MBP (maltose binding protein). Suitable markers for E. coli are or the proBA operon derived from E. coli K12 strain. The nucleotide sequence of the ... do.

[0146] In another example, when yeast is used as a host cell, the following regulatory regions can be used: A promoter suitable for use in yeast may be a constitutive promoter. Examples of suitable constitutive promoters include FBA1, TPI1, PGK1, PYK1, and TDH3. , ENO2, HXK2, PGI1, PFK1, PFK2, GPM1 genes There is a glycolytic promoter or a non-glycolytic promoter of the TEF2 gene.

[0147] Suitable promoters for use in yeast may be inducible. In the case of Pichia, the methanol-inducible promoter AOX1 is preferred. Otherwise, the yeast is Saccharomyces cerevisiae (S. cerevisiae) When the promoter is in the host cell, the GAL1 promoter (galactose inducible) can be used. The genes described as being derived from yeast as the host cell are also It can also be used to induce release factors. Preferred signal peptides for export for Saccharomyces include: Saccharomyces cerevisiae (S. cerevisiae) alpha mating factor prepro Secretory signal peptide, Saccharomyces cerevisiae (S. cerevisiae) O st1 signal peptide, Saccharomyces cerevisiae (S. cerevisiae) Aga2 signal peptide, and fusions thereof.

[0148] Example 2.1 shows a gene construct encoding myoglobin as defined hereinabove. AOX1 is a cyclin-dependent enzyme that regulates the synthesis of lactate. ris) used for expression in strain GS115.

[0149] In another example, when a filamentous fungus is used as the host cell, the following regulatory region may be used: The following promoters may be used: Aspergillus niger (As pergillus niger glucoamylase promoter (glaA), Aspergillus niger Aspergillus nidulans alcohol dehydrate The enzyme promoter (alcA) or Aspergillus oryzae oryzae) Taka-amylase A promoter (amyB), Aspergillus niga -(Aspergillus niger) alcohol dehydrogenase promoter (adh A) Trichoderma reesei pyruvate pki promoter or Aspergillus nidulans s nidulas) glyceraldehyde-3-phosphate dehydrogenase promoter (gpd A) The promoter has been described as being derived from a filamentous fungus as a host cell. The gene can also be used to induce termination factors for the same filamentous fungi. Suitable for excretion of filamentous fungi, preferably Aspergillus. A preferred signal peptide is the Aspergillus niger ger) glucoamylase signal peptide (glaA), Aspergillus niger (A Spergillus niger α-galactosidase signal peptide (AglB) ) and Trichoderma reesei cellobiohide Lorase I (CbhI) is available.

[0150] For Aspergillus (more preferably Aspergillus Preferred promoter for Aspergillus niger The release factor and release factor of Aspergillus niger The glucoamylase promoter and the glucoamylase terminator.

[0151] Expression vector The genetic constructs described herein can be placed into expression vectors. Thus, in another aspect, any of the components described in any of the preceding embodiments may be used. An expression vector containing such a gene construct is provided.

[0152] A description of "Expression Vectors" is provided under the section entitled "General Information."

[0153] composition In a further embodiment, the genetic construct as described above and / or the Compositions are provided that include such expression vectors and, optionally, further include one or more components. It is served.

[0154] host cell In a further embodiment, a host comprising a genetic construct as defined herein. A host cell is provided. The host cell can be prokaryotic or eukaryotic.

[0155] The prokaryotic organism may be a bacterium. The bacterium may be any of the following: bsidia), Achromobacter, Acinetobacter - genus (Acinetobacter), genus (Aeribacillus) , Aneurinibacillus spp., Agrobacterium spp. (Agrobacterium), Aeromonas, Alkalige Alcaligenes, Arthrobacter , Arzoarcus, Azomonas, Azo Azospirillum, Azotobacter r), Bacillus, Beijerincki a), Bradyrhizobium spp., Brevibacillus spp. (B revibacillus), Burkholderia, Bayso Byssochlamys, Citrobacter ), Clostridium genus, Comamonas genus s), Cupriavidus, Corynebacterium nebacterium), Deinococcus, Escherichia Escherichia, Enterobacter, Flavobacterium, Fusobacterium obacterium, Gossypium, Klebsiella levsiella), Lactobacillus spp., Listeria spp. isteria), Megasphaera, Micrococcus Micrococcus, Mycobacterium, Norca Norcadia, Porphyromonas, Propionibacterium, Pseudomonas Pseudomonas, Ralstonia, Rhizobium zobium, Rhodopseudomonas, Rhodos Rhodospirillum, Rhodococcus , Roseburia, Shewanella, Streptomycetes, Xanthomonas onas, Xylella, Yersinia, Treponema ulcerata Treponema, Vibrio, Streptococcus eptococcus, Lactococcus, Zymomonas Zymomonas, Staphylococcus, Salmonella, Sphingomonas s), Sphingobium, Novosphingobium vosphingobium), Brucella and Microscilla The bacterium may be a gram-positive / gram-negative bacterium selected from the group consisting of: Bacillus subtilis, Bacillus angustifolium, Bacillus subtilis ...

[0156] Preferred bacteria include Aeribacillus pallidus idus), Aneurinibacillus terranovensis (Aneurinibacillus terranovensis, Bacillus subtilis, Bacillus amyloliquefaciens ens), Bacillus coagulans, Bacillus Bacillus licheniformis, Bacillus Bacillus megaterium, Bacillus halodurans (Bacillus halodurans), Bacillus pumilus (Bacillus pumilus), Brevibacillus thermolvar (Brevibacillus t hermoruber), Brevibacillus pantyfumi (Brevibacillus panacihumi), Cupriavidus basilensis (Cupriavidus basilensis), Geobacillus lraustopsis (G.lraustopsis hilus), Gluconobacter oxydans dans), Caulobacter crescens tus) CB 15, Methylobacterium extorquens (Methylobact erium extorquens, Rhodobacter sphaeroides (Rhodob acter sphaeroides), Pelotomaculum thermopropionicum (Pe lotomaculum thermopropionicum), Pseudomonas Pseudomonas zeaxanthinifaciens ens), Pseudomonas putida, Paracoccus Paracoccus denitrificans, Escherichia coli, Corynebacterium glutanicum (C orynebacterium glutamicum), Staphylococcus carnosus Staphylococcus carnosus, Streptomyces lycopersici Streptomyces lividans, Sinorhizobium melioides Sinorhizobium melioti, Sphingobium ngobium sp.), Novosphingobium sp.), Sphingomonas henshuiensis (Sphingomonas henshuiensis) nshuiensis, and Rhizobium radiobacter adiobacter. The preferred bacteria is Escherichia coli. oli).

[0157] Preferred Escherichia coli strains include 58, 679, and WG1 , DH5α, TG1, TOP10, K12 (used in the examples), BL21, BL21 DE3, XL1-Blue, XL10-Gold, TB1, REG-12, W945, H B101, DH1, DP50, AB284, JC9387, AG1, C600, Cavalli (Cavalli)Hfr, Y10 available.

[0158] The eukaryote may be a yeast or a filamentous fungus.

[0159] Preferred yeasts include those of the genera Saccharomyces, Kluyveromyces, Kluyveromyces, Candida, Pichia chia), Schizosaccharomyces, Han Hansenula, Kloeckera, Schwannio Schwanniomyces, Yarrowia, Chestnut Cryptococcus, Debaromyces s), Saccharomycecopsis, Saccharomycecopsis Saccharomycodes, Wickerhamia a), Debayomyces, Hanseniaspora iaspora), Ogataea, Kuraishi a), Komagataella, Metschn ikowia), Williopsis, Nakazawaea azawaea, Torulaspora, Bulle ra), Rhodotorula, Sporobolomyces Among yeasts, there is Kluyveromyces lactis. myces lactis, Saccharomyces cerevisiae cerevisiae), Hansenula polymorpha ymorpha) (Ogataea henricii) also known as Yarrowia lipolytica, Candida tropicalis and Pichia pastoris Squirrel (Pichia pastoris) (Komagata The species P. ella phaffii (also known as P. ella phaffii) is preferred.

[0160] Preferred Pichia strains are those in the following list: Bg09, Bg10 , Bg11, Bg12 (exemplified), Bg20, Bg21, Bg22, Bg23, Bg 24, Bg25, Bg26, Bg40, Bg43, Bg44, Bg45, Y-11430 , X-33, GS115, KM71, SMD1168, SMD1165, MC100-3 Most preferably, Bg10 and derivatives.

[0161] In Example 2.3, Pichia pastoris G The S115(his4) strain is used as the host cell.

[0162] Preferred Saccharomyces strains are those listed below: Chi, S288C, CEN.PK family, CBS 2354, ATCC 2360, A TCC 4098, ATCC 4124, ATCC 4126, ATCC 4127, A TCC 4921, ATCC 7754, ATCC 9763, ATCC 20598, ATCC 24855, ATCC 24858, ATCC 24860, ATCC 26 422, ATCC 46523, ATCC 56069, ATCC 60222, ATC C 60223, ATCC 60493, ATCC 66348, ATCC 66349 ATCC 96581. Preferred yeasts are Pichia strains. More preferably, it is Pichia pastoris.

[0163] The filamentous fungus may be selected from the following list, including the genus Acremonium onium, Agaricus, Aspergillus lus), Aureobasidium, Chrysosporium Genus (Chrysosporium), Genus (Coprinus), Cryptococcus Cryptococcus, Filibasidium ), Fusarium, Humicola, Magnapol Magnaporthe, Mucor, Myceliophthora eliophthora, Neocallinastix , Neurospora spp., Paecilomyces spp. s), Penicillium, Piromyces ), Panerochaete, Pleurotus , Schizophyllum, Talaromyces ces), Thermoascus, Thielavia avia, Tolypocladium, Ustilago tilago and Trichoderma.

[0164] Preferred filamentous fungi are those listed below: Aspergillus niger (Aspe Aspergillus niger, Aspergillus nidulans s nidulans), Aspergillus fumigatus (Aspergillus f umigatus), Aspergillus oryzae e), Aspergillus vadensis, Pe Penicillium chrysogenum, Penicillium chrysogenum Psyllium citrinum (Penicillium citrinum), Penicillium Rubens (Penicillium rubens), Penicillium oxalicum ( Penicillium oxalicum, Penicillium sublubescens (Pe nicillium subrubescens), Rasamsonia emersonii (Ra samsonia emersonii), Talaromyces emersonii (Talaro myces emersonii, Acremonium chrysogenum (Acremoni um chrysogenum), Trichoderma reesei (Trichoderma reesei, Aspergillus sojae, and and Chrysosporium lucknowe nse).

[0165] Preferred strains of filamentous fungi are listed below: Aspergillus niger (As pergillus niger)CBS 513.88, N593, CBS 120. 49 (as used in the examples), N402, ATCC 1015 Aspergillus Aspergillus oryzae ATCC 20423, IF O 4177, ATCC 1011, ATCC 9576, ATCC 14488-14 491, ATCC 11601, ATCC12892, Aspergillus badensis (A spergillus vadensis)CBS 113365, CBS 10278 7, IMI 142717, IBT 24658, CBS 113226, Penicillium · Chrysogenum (Penicillium chrysogenum) CBS 455. 95. Penicillium citrinum ATC C 38065, Penicillium chrysogenum genum) P2, Wisconsin 54-1255, Penicillium Penicillium subrubescens CBS 132785, FBCC 1632, Talaromyces emersonii (Talaromyc es emersonii) CBS 393.64, Acremonium chrysogenum (A cremonium chrysogenum) ATCC 36225 or ATCC 4 8272, Trichoderma reesei ATCC 26921 or ATCC56765 or ATCC 26921, Aspergillus sojae Aspergillus sojae ATCC11906, Chrysosporium la Chrysosporium lucknowense ATCC440 06 is selected.

[0166] In a preferred embodiment, the Aspergillus is a filamentous As a fungus, Aspergillus niger is preferably used. us niger) strain is used.

[0167] method The host cells of the invention are useful for producing myoglobin. The myoglobin is derived from a mammoth, preferably a woolly mammoth or a steppe mammoth. In some embodiments, the myoglobin is of porcine origin. In some embodiments, the myoglobin is derived from sheep. The globin is of bovine origin. In some embodiments, the myoglobin is of chicken origin. In some embodiments, the myoglobin is derived from tuna. In one embodiment, the present invention provides a method for producing a cell culture medium comprising culturing a host cell of the present invention in a suitable medium, and optionally and recovering the host cell and / or myoglobin by The present invention provides a method for producing such myoglobin. The bin does not include a signal peptide as defined elsewhere herein.

[0168] In one embodiment, the present invention relates to mammoth-derived myogol as defined hereinabove. Robin, preferably myoglobin comprising at least 70% sequence identity with SEQ ID NO: 1; More preferably, a Q or H at position 65 in combination with an H at position 94 is at least as good as SEQ ID NO: 1. 1. amino acids, namely, E at position 9, K at position 13, T as position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, at least one of Q, Q at position 88, I at position 102, and / or E at position 123 1. A method for producing myoglobin having the formula: and optionally recovering the host cells and / or myoglobin. A method is provided.

[0169] In one embodiment, the present invention relates to a method for producing a mammalian cell derived from a woolly mammoth as defined hereinabove. Myoglobin, preferably a myoglobin having at least 70% sequence identity with SEQ ID NO: 2. more preferably, in combination with Q or H at position 65, Q at position 92, and H at position 94. 2, and optionally a sequence identity of at least 70% with SEQ ID NO: 2. The following amino acids are present in the following positions: E at position 9, K at position 13, T at position 1, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88, I at position 102, E at position 123, and / or is a method for producing myoglobin having at least one I at position 143 Thus, the method comprises culturing the host cells of the present invention in an appropriate medium and, optionally, culturing the host cells and / or and recovering the myoglobin.

[0170] In one embodiment, the present invention relates to a steppe mammoth derived mammoth mammoth as defined herein above. myoglobin, preferably a myoglobin comprising at least 70% sequence identity with SEQ ID NO: 3. Robin, more preferably in combination with Q or H at position 65, H at position 92, and H at position 94 collectively have at least 70% sequence identity with SEQ ID NO: 3, and optionally SEQ ID NO: 3 The following amino acids are present in the following locations: E at position 9, K at position 13, and T at position 23, P at position 27, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88, I at position 102, E at position 123, and / or or a method for producing myoglobin having at least one I at position 143 by culturing the host cells of the invention in a suitable medium and optionally or recovering the myoglobin.

[0171] In one embodiment, the present invention relates to a steppe mammoth derived mammoth mammoth as defined herein above. myoglobin, preferably a myoglobin comprising at least 70% sequence identity with SEQ ID NO: 3. Robin, more preferably the following amino acid combination: F in position 30, and / or Q in position 65, and / or H at position 92, and / or H at position 94, and / or F in position 30 and Q in position 65, and / or F at position 30 and H at position 92, and / or F at position 30 and H at position 94, and / or Q at position 65 and H at position 92, and / or Q at position 65 and H at position 94, and / or H at position 92 and H at position 94, and / or F at position 30 and Q at position 65 and H at position 92, and / or F at position 30 and Q at position 65 and H at position 94, and / or F at position 30 and H at position 92 and H at position 94, and / or Q at position 65 and H at position 92 and H at position 94, and / or F at position 30, Q at position 65, H at position 92, and H at position 94 and (iii) a sequence identity of at least 70% with SEQ ID NO: 3 in combination with at least one of 1. A method for the production of myoglobin comprising culturing a host cell of the invention in a suitable medium. and optionally recovering the host cells and / or myoglobin. to provide.

[0172] In one embodiment, the present invention relates to a myoglo of ovine origin as defined herein above. globin, preferably myoglobin, which contains at least 70% sequence identity with SEQ ID NO: 4, More preferably, in combination with a Q or H at position 65 and an H at position 94, at least SEQ ID NO: 4 and optionally the following sequences at the following locations in SEQ ID NO: 4: amino acids, i.e., N at position 13, Q at position 27, I at position 31, N at position 67, at least one of A at position 28, S at position 133, A at position 145, and / or L at position 150 A method for producing myoglobin having both the host cell of the present invention and the host cell of the present invention in a suitable medium is also provided. Culturing the host cells and optionally recovering the host cells and / or myoglobin. The present invention provides a method including:

[0173] In one embodiment, the present invention relates to myoglobin of bovine origin as defined herein above. myoglobin, preferably a myoglobin comprising at least 70% sequence identity with SEQ ID NO: 5, more preferably Preferably, at least SEQ ID NO: 5 in combination with Q or H at position 65 and H at position 94 70% sequence identity, and optionally the following amino acid at the following positions in SEQ ID NO:5: acid, i.e., N at position 13, Q at position 27, I at position 31, N at position 67, at least one of A at position 8, S at position 133, A at position 145, and / or L at position 150; a method for producing myoglobin having one of the following structures: Culturing the cells and optionally harvesting the host cells and / or myoglobin; The present invention provides a method comprising:

[0174] In one embodiment, the present invention relates to a myoglobin derived from pigs as defined herein above. myoglobin, preferably a myoglobin comprising at least 70% sequence identity with SEQ ID NO: 6, more preferably a myoglobin comprising at least 70% sequence identity with SEQ ID NO: 6 Preferably, at least SEQ ID NO: 6 in combination with Q or H at position 65 and H at position 94 70% sequence identity, and optionally the following amino acid at the following positions in SEQ ID NO: 6: acid, i.e., N at position 13, Q at position 27, I at position 31, N at position 67, at least one of A at position 8, S at position 133, A at position 145, and / or L at position 150; a method for producing myoglobin having one of the following structures: Culturing the cells and optionally harvesting the host cells and / or myoglobin; The present invention provides a method comprising:

[0175] In one embodiment, the present invention relates to myoginseng derived from chickens as defined hereinabove. Robin, preferably myoglobin comprising at least 70% sequence identity with SEQ ID NO: 7; More preferably, a Q or H at position 65 in combination with an H at position 94 is at least SEQ ID NO: 7. 7, and optionally at the following positions in SEQ ID NO: 7: Amino acids, namely, Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 7, M at position 31, H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72 , K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 1, I at position 116, A at position 117, E at position 118, A at position 121, at least one of S at position 8, K at position 133, S at position 145, and / or F at position 150; a method for producing myoglobin having one of the following structures: Culturing the cells and optionally harvesting the host cells and / or myoglobin; The present invention provides a method comprising:

[0176] In one embodiment, the present invention relates to a fish called bluefin tuna, derived from tuna as defined herein above. globin, preferably myoglobin, which contains at least 70% sequence identity with SEQ ID NO: 8, More preferably, in combination with Q or H at position 65 and H at position 94, at least SEQ ID NO: 8 The present invention provides a method for the production of myoglobin containing 70% sequence identity with the nucleotide sequence of ...

[0177] The features of this embodiment are preferably as described elsewhere herein. Alternatively, it may be referred to as the process according to the invention. Suitable cell culture methods for use in the present invention are known to those skilled in the art and are described, for example, in the article by van't Rie t, K. and Tramper, J., 1st edition, Basic Bio Discussed in Reactor Design, CRC Press, NY, 1991 Such methods include submerged fermentation in a liquid medium, surface fermentation on a liquid medium, and solid fermentation. Cell cultures include, but are not limited to, fermentation in a laboratory setting. in microtiter plates, shake flasks, and small-scale benchtop bioreactors in industrial settings. The method is carried out by culturing in a microbioreactor, a medium-scale bioreactor, and / or a large-scale bioreactor. Suitable cell culture modes include continuous fermentation, batch fermentation and / or fed-batch fermentation, and In one embodiment, the cell culture is In a preferred embodiment, the cell culture is carried out using batch fermentation. In a more preferred embodiment, the cell culture is carried out using fed-batch fermentation. do.

[0178] In the context of the present invention, the term "culture medium", hereinafter alternatively referred to as "growth medium", is used. "Ground" refers to the case where there are no cultured cells and the case where cultured cells are present in a culture medium. "Culture broth" refers to the culture medium in which cultured cells reside. "Culture supernatant" refers to the culture medium without the cultured cells. "Cell-free extract" refers to the culture medium without the cultured cells. refers to a cell lysate free of cell debris. The cell culture as part of the process of the present invention may The method can be carried out under conditions conducive to the production of the introduced myoglobin, and the conditions are Such conditions include the duration of incubation, temperature, as well as the chemical composition of the culture medium. , O2 levels in the culture broth and / or headspace, Other parameters include CO2 levels in the headspace, pH, ionic strength, agitation speed, hydrostatic pressure, etc. Depends on process parameters. Cell culture is maintained with adequate nutrients such as carbon and nitrogen sources, and inorganic nutrients. and culturing media containing salts and additional compounds such as vitamins, as known in the art. This can be done using procedures (e.g., Bennett, W. and Lasure, L.,1 st edition,More Gene Manipulations i (See, for example, "Fungi," Academic Press, CA, 1991). Suitable growth media are available from commercial sources or may be adapted for individual hosts (e.g., For example, Centraalbureau Voor Schimmelcultures collection (CBS) catalogue or American Type Collection The compositions published in the American Type Culture Collection (ATCC) catalogue are It can be prepared using

[0179] The actual composition of the growth medium and the values ​​of the culture process parameters are not a critical feature of the present invention. Any growth medium composition is not sufficient to support the growth of the host cells and the expression of the introduced myoglobin. The growth medium is typically suitable for the growth of the cells being cultured. Those skilled in the art will understand that suitable carbon sources may be added exogenously to the growth medium. It is understood that carbon sources may be added individually or in combination or may already be present in the medium. The carbon source may be present in or added to a mixture of several carbon sources. Examples include simple sugars such as glucose, maltose, sucrose, xylose, and arabinose. Pure sugar, maltodextrin, hydrolyzed starch, starch, complex sugars such as molasses, and Second-generation feedstocks can be particularly attractive because they Second generation feedstocks are typically lignocellulosic because they have a lower carbon footprint. Such materials may include any lignocellulosic and / or hemicellulose-based materials. Such materials are suitable for agricultural, industrial or municipal use, preferably agricultural Examples of suitable materials include (agricultural) biomass, commercial waste streams, Virgin biomass such as organic matter, municipal solid waste, waste paper and yard waste, Common forms of biomass include trees, shrubs and Pasture, wheat, straw, sugarcane bagasse, switchgrass, miscanthus, corn, Corn stover, corn cob, canola stalk, soybean stalk, sweet corn, corn Koshi kernel, often called "bran or fiber," corn, wheat, and barley Products and by-products from grain milling (including wet and dry milling), such as Biomass can also be found in grassy materials, agricultural residues, forestry residues, and municipal solid waste. It can be agricultural solid waste, waste paper, and pulp and paper mill residues. Includes branches, shrubs, cane, corn and corn straw, energy crops, forests, and fruits. , flowers, cereals, pastures, herbaceous crops, leaves, bark, needles, logs, roots, young trees, short-term rotational tree crops, low Trees, switch herbs, trees, vegetables, fruits, wine, sugar beet pulp, wheat milling by-products, eng Baku rice husks, and hard and soft trees (excluding poisonous trees), and agricultural and forestry activities There is organic waste material, particularly forestry wood waste, resulting from agricultural processes including: Biomass may be any of the above alone or any combination or mixture thereof. Carbon sources such as organic acids, aldehydes, ketones, esters, and alcohols can also be used. The use of growth media containing a combination of multiple different carbon sources is also contemplated in accordance with the present invention. Such media may contain more acids, such as, but not limited to, organic acids. The reduced carbon source may be combined with a more reduced carbon source, such as an alcohol. Examples of suitable nitrogen sources known in the art include soybean meal, corn steep liquor, yeast extract, Whey protein, egg protein, casein hydrolysate, urea, ammonia, ammonium Examples of additional suitable compounds known in the art include phosphates, , sulfates, magnesium and other metals, trace elements and vitamins. Requirements will vary based on the host cell, e.g., between yeast, bacteria, and filamentous fungi. The growth medium may be a complete (rich) medium or a minimally oxidized medium, the requirements of which will be known to those skilled in the art. A small medium, i.e., a medium containing the components absolutely necessary for growth depending on the host cell being cultured. The medium may contain only the lactic acid bacteria.

[0180] The process parameters, as well as the composition of the growth medium, affect the growth of the host cells and the amount of myoglobin introduced. Any value can be assigned as long as it allows for the production of globin. Typically, The value will vary based on the host cell being cultured and will be known to those skilled in the art. Preferably, the process according to the invention is an oxygen-limited or aerobic process, Preferably, the cell culture is carried out under oxygen-limited or aerobic conditions, more preferably under process conditions. Oxygen-limited conditions, also known as microaerobic conditions, mean that the water is oxygen-limited. is a culture condition in which oxygen consumption is limited by oxygen availability. The amount and composition of the incoming gas stream and the actual mixing / mass transfer characteristics of the fermentation equipment used will determine the Preferably, under oxygen-limited conditions in liquid culture, the oxygen consumption is low. at least about 5.5 mmol / L / hr, more preferably at least about 6 mmol / L / hr; And even more preferably at least about 7 mmol / L / hr. Culture conditions in which oxygen consumption is not limited by oxygen availability.

[0181] Cell culture is performed at the temperature that is optimal for cells, typically in the range of 16-42°C. In some embodiments, the temperature is preferably between 20 and 40°C. The temperature range is preferably between 25 and 38°C, and most preferably between 28 and 36°C. In the most preferred embodiment, a temperature value of about 30-36°C is used.

[0182] Cell culture can be performed at a pH value that is optimal for the cells. The culture pH was about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, and about pH 4. .5, about pH5, about pH5.5, about pH6, about pH6.5, about pH7, about pH7.5, about pH 8.0, about pH 8.5, about pH 9. In a preferred embodiment, the pH is about pH The pH ranges from about pH 3.0 to about pH 9, and more preferably from about pH 3.5 to pH 7. In a most preferred embodiment, a pH value of about 6 is used.

[0183] Cell culture is performed at the ionic strength value of the culture medium that is optimal for the cells, typically 50 mM In some embodiments, the ionic strength of the culture medium can range from 0.1 to 2 M. The concentration is preferably in the range of 75 mM to 1 M, more preferably 100 mM to 750 mM. In some most preferred embodiments, an ionic strength value of about 100 mM is used. .

[0184] Cell culture was performed at 14, 13.5, 13, 12.5, 12, 11.5, 11, 10.5, 1 0, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3. It can be performed for a duration of 5, 3, 2.5, 2, 1.5 or 1 day, and the duration is 20% Preferably, the cell culture is 1.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5 Duration of 0.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 day The duration may deviate by as much as 10%. More preferably, the cell culture is carried out for 5 days, The duration may deviate by as much as 20%, most preferably by as much as 10%.

[0185] The process according to the invention typically involves the production of at least 100 mg / L, 200 mg / L, 3 00mg / L, 400mg / L, 500mg / L, 600mg / L, 700mg / L, 8 00mg / L, 900mg / L, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 11g / L, 12g / L, 13 g / L, 14g / L, 15g / L, 16g / L, 17g / L, 18g / L, 19g / L, 20g / L, 21g / L, 22g / L, 23g / L, 24g / L, 25g / L, 50g / 100g / L, 75g / L, 100g / L, 200g / L or 300g / L of myoglobin will result.

[0186] In the process according to the invention, typically at least 10% of the carbon source in the growth medium, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, preferably at least At least 20%, more preferably at least 40%, and most preferably at least 60% of the myocardium It will be converted to globin.

[0187] Cell culture can also be achieved by implementing a multi-step, preferably two-step, culture method. For example, the step of producing myoglobin can be carried out after the step of growing the cell biomass. Therefore, it can be preempted and only limited production occurs or no production occurs at all. Steps may require different culture formats and / or methods depending on the goal of each step and / or the cells being cultured. Alternatively, it may be performed using a different culture medium and / or different culture process parameter values. The biomass during the production step may or may not be actively growing.

[0188] In the context of the process of the present invention, the host cell and / or the myoglobin may optionally be: When present intracellularly, myoglobin can optionally be recovered from the culture medium. The myoglobin can be recovered from the cell biomass. Optionally, the recovered myoglobin can be purified. Preferably, the purification of myoglobin results in at least 70%, more preferably at least 70%. At least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and most preferably will result in substantially pure myoglobin.

[0189] In one embodiment, the host cell expresses myoglobin extracellularly in the process according to the invention. In the process according to this embodiment, myoglobin is produced in the host cell. The process according to this embodiment is carried out according to the present invention. In this context, both secretory fermentation and extracellular fermentation are , considered to be extracellularly produced.

[0190] Without being bound by this theory, it is believed that the extracellular process according to the present invention is responsible for the production of myoglobin. The advantage is that downstream processing to recover the carbon is simpler, more efficient, and / or more effective. Furthermore, the process by which this product is produced results in myoglobin being synthesized. at least 60% with minimal downstream processing compared to intracellular methods that are not transported outside of the host cell , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% This can result in compositions containing highly pure myoglobin, such as 99% or higher.

[0191] Without being bound by this theory, the extracellular process according to the present invention is The optional step of lysing the host cells has the advantage that it does not involve lysing the host cells. Thus, the extracellular process according to the present invention results in the host cell being able to express the host cell as described in the following embodiment. This can result in a composition having a low concentration of nucleic acids originating from the host cells.

[0192] Thus, in a further aspect, the present invention provides a myoglomerate as defined herein above. A method for the production of bottles, comprising culturing a host cell of the invention in a suitable medium; and optionally recovering the host cells and / or myoglobin. .

[0193] In one embodiment, the extracellular process according to the present invention results in at least 60%, 6 5%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or The composition preferably has a purity of 99% myoglobin, and most preferably has a purity of substantially pure myoglobin. Produces globin.

[0194] Preferably, the purity is the amount of free radicals obtained at the end of the process or extracellular process according to the invention. It is measured as a weight percentage of the total protein fraction in the cell supernatant.

[0195] In one embodiment, the extracellular process according to the present invention results in the nuclear transfer of the host cell. Acid 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3. 4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6% ,2.5%,2.4%,2.3%,2.2%,2.1%,2%,1.9%,1.8%,1 0.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9 %, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0 Resulting in a composition containing less than 0.1%.

[0196] In one embodiment, the present invention relates to a method for producing a mammalian cell derived from a woolly mammoth as defined hereinabove. Myoglobin, preferably a myoglobin having at least 70% sequence identity with SEQ ID NO: 2. bin, more preferably a combination of Q or H at position 65, Q at position 92, and H at position 94. 2. The sequence of SEQ ID NO: 2 is at least 70% identical to SEQ ID NO: 2. the following amino acids in position 9: E, K, T as position 14; P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V, Q at position 84, Q at position 88, I at position 102, E at position 123, and / or The present invention relates to a method for producing myoglobin, which may optionally have at least one of the I groups of 43. an extracellular process according to the invention, comprising culturing a host cell of the invention in an appropriate medium; and optionally recovering the host cell and / or myoglobin. Provide.

[0197] In one embodiment, the present invention relates to a steppe mammoth derived mammoth mammoth as defined herein above. myoglobin, preferably a myoglobin comprising at least 70% sequence identity with SEQ ID NO: 3. Robin, more preferably the following amino acid combination: F at position 30, and / or Q in position 65, and / or H at position 92, and / or H at position 94, and / or F at position 30 and Q at position 65, and / or F at position 30 and H at position 92, and / or F at position 30 and H at position 94, and / or Q at position 65 and H at position 92, and / or Q at position 65 and H at position 94, and / or H at position 92 and H at position 94, and / or F at position 30 and Q at position 65 and H at position 92, and / or F at position 30 and Q at position 65 and H at position 94, and / or F at position 30 and H at position 92 and H at position 94, and / or Q at position 65 and H at position 92 and H at position 94, and / or F at position 30, Q at position 65, H at position 92, and H at position 94 and (iii) a sequence identity of at least 70% with SEQ ID NO: 3 in combination with at least one of An extracellular process according to the invention for the production of myoglobin, comprising: Cultivating the cells in an appropriate medium and optionally harvesting the host cells and / or myoglobin. and extracellular processes including:

[0198] Example 2.3 provides the extracellular production of step mammoth myoglobin.

[0199] In one embodiment, the present invention relates to a myoglo of ovine origin as defined herein above. bin, preferably myoglobin comprising at least 70% sequence identity with SEQ ID NO: 4, Preferably, the combination of Q or H at position 65 and H at position 94 is at least SEQ ID NO: 4 and optionally the following at the following positions in SEQ ID NO: 4: The amino acids are: N at position 13, Q at position 27, I at position 31, N at position 67, At least one of A at position 128, S at position 133, A at position 145, and / or L at position 150 1. An extracellular process according to the invention for the production of myoglobin having at least one Cultivating the host cells of the present invention in an appropriate medium and, optionally, culturing the host cells and / or myoglobin and recovering globin.

[0200] In one embodiment, myoglobin of bovine origin, as defined herein above, preferably or a myoglobin comprising at least 70% sequence identity with SEQ ID NO: 5, more preferably Q or H at position 65 in combination with H at position 94 has at least 70% affinity to SEQ ID NO: 5 The following amino acids, including sequence identity, and optionally at the following positions in SEQ ID NO: 5: That is, N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, at least one of S at position 133, A at position 145, and / or L at position 150 An extracellular process according to the invention for the production of myoglobin comprising a host cell of the invention. Cultivating the cells in an appropriate medium and optionally recovering the host cells and / or myoglobin The extracellular process involves the collection of ATP.

[0201] In one embodiment, the present invention relates to a myoglobin derived from pigs as defined herein above. myoglobin, preferably comprising at least 70% sequence identity with SEQ ID NO: 6; more preferably Preferably, the amino acid sequence of SEQ ID NO: 6 in combination with Q or H at position 65 and H at position 94 is at least 70% sequence identity, and optionally the following amino acid at the following location in SEQ ID NO: 6: amino acids, i.e., N at position 13, Q at position 27, I at position 31, N at position 67, at least one of A at position 28, S at position 133, A at position 145, and / or L at position 150 An extracellular process according to the invention for the production of myoglobin having one of the following: Cultivating the host cells of the invention in a suitable medium and optionally culturing the host cells and / or myoglobin. and recovering the robin.

[0202] In one embodiment, the present invention relates to myoginseng derived from chickens as defined hereinabove. globin, preferably myoglobin comprising at least 70% sequence identity with SEQ ID NO: 7, More preferably, in combination with Q or H at position 65 and H at position 94, SEQ ID NO: 7 or less 7, and optionally, the following positions in SEQ ID NO: 7: amino acids, namely, Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M at position 31, H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, and V at position 11, I at position 116, A at position 117, E at position 118, A at position 121, at least one of S at position 28, K at position 133, S at position 145, and / or F at position 150 an extracellular process according to the invention for the production of myoglobin having at least one The host cells of the present invention are cultured in an appropriate medium, and optionally the host cells and / or mitochondria are cultured in an appropriate medium. and recovering the globin.

[0203] In one embodiment, the present invention relates to a fish called bluefin tuna, derived from tuna as defined herein above. bin, preferably myoglobin comprising at least 70% sequence identity with SEQ ID NO: 8, Preferably, at least SEQ ID NO: 8 in combination with Q or H at position 65 and H at position 94 The extracellular process according to the present invention for the production of myoglobin containing 70% sequence identity to provide.

[0204] The associated downstream processing techniques that may be suitable for recovery and / or purification are not a critical feature of the present invention. It depends on whether myoglobin is accumulated in the cultured cells or excreted. Such processing techniques and related options will be known to those skilled in the art and are described, for example, in Wesse Lingh, JA and Krijgsman, J., 1st edition, D ownstream Processing in Biotechnology,De This is discussed in lft Academic Press, NL, 2013. In a non-limiting example of a process, the biomass is separated from the culture medium using, for example, centrifugation or filtration. If the produced myoglobin is accumulated in cells, it is recovered from the soil. If excreted, they can be recovered and / or purified from the cell-free medium. or directly from the culture broth if the biomass separation step is skipped. Recovery and / or purification can be carried out by any conventional recovery method known in the art. The methods for recovery and / or purification of proteins may be performed by the methods described herein. As known to those skilled in the art, Sambrook and Russell, Molecular C loning:A Laboratory Manual,3rd edition,C old Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, NY, 2001, or Ausub el F.et al,eds.,Current protocols in mol ecular biology, Green Publishing and Wile Considered in standard handbooks such as University Interscience, NY, 2003 Examples of widely used recovery and / or purification methods include gel filtration chromatography. chromatography, ion exchange chromatography, immunoaffinity chromatography, metal chromatographic methods such as affinity chromatography and gel filtration chromatography; Fractionation using ammonium sulfate and polyethylene glycol precipitates, gel electrophoresis The methods include salting out and dialysis. Preferably, metal affinity chromatography or size exclusion chromatography is used. Recovery and / or purification may optionally be performed using well-known molecular level techniques. Using the toolbox technology, enzyme polypeptides, such as those containing a GST domain, This may be enhanced by linking to sequences that facilitate purification. The facilitating sequence and / or signal peptide that facilitates export of myoglobin are described in the art. Techniques known in the art, such as the addition of sequences and / or signal peptides to facilitate purification, may be used. Using proteolysis by endopeptidases targeting the linker between robin and In some embodiments, the enzyme polypeptide is, inter alia, Many of these vectors are commercially available as pET23a(+) vectors (Genescript Bio Tags provided by otech, Leiden, The Netherlands or the like, linked (fused) to a hexa-histidine peptide. z et al.,Proc.Natl.Acad.Sci.USA 86:821-8 24 (1989), the hexa-histidine peptide is a fusion protein. A simple purification of the protein is provided.

[0205] In a preferred embodiment, the produced myoglobin is recovered from the culture medium and / or This can be achieved either continuously with the production process or after the production process. .

[0206] In a preferred embodiment, the produced myoglobin is recovered from the cultured cells and This can be achieved by harvesting a fraction of growing cells continuously throughout the production process. This may be achieved by or after such.

[0207] In a preferred embodiment, the cultured host cells used in the methods of the present invention are Fixation of cells is described in Guisan, JM, Bolivar, JM, Lo pez-Gallego, F., Rocha-Martin, J. (Eds.), Imm. obilization of Enzymes and Cells:Methods and Protocols, Springer US, USA, 2020. By any means known to those skilled in the art, such as those discussed in standard handbooks. Typically, host cells are supported on semi-solid or solid supports by three different methods: The first method involves polymerizing the spore- or cell-containing solution or Examples of polymerizable or coagulable solutions include alginate. Salt, λ-carrageenan, chitosan, polyacrylamide, polyacrylamide-hydrazide , agarose, polypropylene, polyethylene glycol, dimethyl acrylate, poly Styrene divinylbenzene, polyvinylbenzene, polyvinyl alcohol, epoxy carrier , cellulose, cellulose acetate, photocrosslinkable resins, prepolymers, urethane, and gelatin The second method involves cell adsorption onto a support. Examples of such supports include: Bone charcoal, cork, clay, resin, sand-like porous alumina beads, porous brick, porous silica The support can be a substrate, celite, or wood chips. The host cells can colonize the support and The third method involves the use of glutaraldehyde, O-dianisidine, (U.S. Pat. No. 3,983,000), isocyanate polymers (U.S. Pat. No. 4,000,000), ,071,409), silanes (U.S. Pat. Nos. 3,519,538 and 3,519,539), No. 3,652,761), hydroxyethyl acrylate, transition metal activated support , cyanuric chloride, sodium periodate, toluene, etc. to induce cell proliferation in the host cells. The cultured host cells include, for example, covalently attaching the cells to a support. The host cells may be fixed in any phase of growth after reaching a desired cell density in the culture medium. Suitable culture formats and / or different culture process parameter values ​​can be determined by those skilled in the art. Colin R. Phillips CR, Poon YC ,Immobilization of Cells:In Biotechnolog y Monographs book series(Biotechnology,v olume 5), Springer, Berlin, Germany, 1988, Ta mpion J.,Tampion MD,Immobilized Cells: Principles and Applications, Cambridge Un University Press, UK, 1987. Preferably, immobilized cells are cultured in a manner also known as a plug-flow bioreactor. The cells are cultured in a conventional packed bed bioreactor or an expanded (fluidized) bed bioreactor. Appropriate growth media and recovery and / or purification methods are further discussed elsewhere herein.

[0208] In a further aspect, a myoglobin obtainable from the method defined herein above. a method for producing a meat substitute as defined above, including blending the meat substitute into a meat substitute; Depending on the meat substitute and / or food ingredient envisaged, one skilled in the art will be able to determine which formulation is You will know which is best for you.

[0209] General information Unless otherwise defined, all technical and scientific terms used herein are intended to be understood as meaning the principles of the present invention. as commonly and ordinarily understood by those of ordinary skill in the art who belong to this class and who read this disclosure. has the same meaning as

[0210] As used herein, the term "promoter" or "regulatory sequence" refers to one or more refers to a nucleic acid fragment that functions to control the transcription of a coding sequence of a Located upstream in the direction of transcription and a binding site for DNA-dependent RNA polymerase , transcription initiation sites, and transcription factor binding sites, repressor and activator protein binding any other DNA sequence, including but not limited to, a promoter Nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription of A "constitutive promoter" is a promoter that is structurally identified by the presence of any other sequence in the promoter. Inducible promoters are promoters that are active under most physiological and developmental conditions. " and / or "repressible" promoters are promoters that are not sensitive to, for example, chemical inducers or repression signals. Physiologically or developmentally regulated processes that are induced and / or suppressed by application It is a motor.

[0211] As used herein, the term "operably linked" refers to a functional relationship. refers to the linkage of polynucleotide elements in a nucleic acid sequence. For example, a transcriptional regulatory sequence such as a promoter is "operably linked" when An amino acid sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Linked means that the DNA sequences being linked are typically contiguous and are easily linked by the two proteins. If it is necessary to splice together the coding regions, they must be contiguous and in reading frame. do.

[0212] As used herein, a "regulatory element" or "transcriptional regulator" refers to a specific DNA sequence By binding to It is a regulatory protein.

[0213] The terms "protein" and "polypeptide" are used interchangeably and are not intended to be limiting unless otherwise specified. Consisting of a chain of several amino acids, without regard to mode of action, size, three-dimensional structure or origin Refers to the molecule.

[0214] The term "gene" refers to a gene that is operably linked to appropriate regulatory regions (e.g., promoter). It contains a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in the cell. A gene is a DNA fragment that contains a promoter, a 5' leader sequence, a coding region, and a , and 3' untranslated sequences, including, for example, polyadenylation sites and / or transcription termination sites ( 3'-end).

[0215] "Expression of a gene" refers to the expression of a gene that is operably linked to an appropriate regulatory region, particularly a promoter. The NA region is transcribed into RNA, and the RNA is biologically active, i.e., The term refers to the process by which a protein can be translated into an environmentally active protein or peptide.

[0216] In amino acid sequences as described herein, amino acids or "residues" are represented by the three letter code These three-letter symbols and their corresponding one-letter symbols are well known to those skilled in the art. A (Ala) is alanine, and C (Cys) is cysteine. D (Asp) is aspartic acid, and E (Glu) is glutamic acid. where F (Phe) is phenylalanine, G (Gly) is glycine, and H (H is histidine, I (Ile) is isoleucine, and K (Lys) is lysine. where L (Leu) is leucine, M (Met) is methionine, and N (Asn ) is asparagine, P (Pro) is proline, and Q (Gln) is glutamine. where R (Arg) is arginine, S (Ser) is serine, and T (Thr) is Threonine, V (Val) is valine, and W (Trp) is tryptophan. , Y (Tyr) is tyrosine. The residue can be any proteinogenic amino acid. formed by any non-proteinogenic amino acid, such as D-amino acids, and post-translational modifications modified amino acids, as well as any unnatural amino acids, as described herein. It is also possible.

[0217] In the context of this application, all percentages in the context of concentrations or compositions are used unless otherwise defined. Unless otherwise specified, percentages refer to weight percentages.

[0218] In the context of this application, a table such as "a parameter having a value of at least X, Y or Z" Currently, the parameter has at least X, at least Y, or at least Z values. shall be construed as

[0219] Sequence identity In the context of the present invention, nucleic acid molecules, such as nucleic acid molecules encoding animal myoglobins, are Protein fragments or nucleic acids encoding polypeptides or peptides or resulting peptides is represented by a sequence of nucleotides.

[0220] Each nucleic acid molecule as identified herein by a given sequence identification number (SEQ ID NO:) or Protein fragment or polypeptide or peptide or resulting peptide or construct It is to be understood that the present invention is not limited to this particular sequence as disclosed. Each coding sequence as identified herein encodes a given protein fragment or polypeptide. A protein encoding a peptide or peptides or a resulting peptide, or Protein fragment or polypeptide or construct or peptide or obtained It is a peptide.

[0221] Throughout this application, a given protein fragment or polypeptide or peptide or product may be referred to as a Each time the SEQ ID NO of a specific nucleotide sequence encoding a peptide is referenced (e.g., X), it may be replaced by: i. A nucleic acid sequence containing a nucleotide sequence having at least 60% sequence identity to SEQ ID NO:X. leotide sequence, ii. Nucleotides whose sequence differs from the sequence of the nucleic acid molecule in (i) due to the degeneracy of the genetic code. an array, or iii. The amino acid sequence encoded by the nucleotide sequence SEQ ID NO: X is at least Nucleotide sequences encoding amino acid sequences with at least 60% amino acid identity or similarity Column.

[0222] Another preferred level of sequence identity or similarity is 70%. The sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is Another preferred level of sequence identity or similarity is 95%. A preferred level of sequence identity or similarity is 99%.

[0223] Throughout this application, whenever a specific amino acid sequence SEQ ID NO is referenced (e.g., Y), it may be replaced by: an amino acid sequence having at least 60% sequence identity or similarity with the amino acid sequence Another preferred level of sequence identity or similarity is a polypeptide represented by the amino acid sequence Another preferred level of sequence identity or similarity is 80%. A preferred level of sequence identity or similarity is 90%. The identity or similarity is 95%. Another preferred level of sequence identity or similarity is 9 It is 9%.

[0224] Percent identity or similarity to a given nucleotide or amino acid sequence, respectively Each nucleotide or amino acid sequence described herein is further preferred in accordance with the present invention. In the form of a given nucleotide or amino acid sequence, at least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, At least 79%, at least 80%, at least 81%, at least 82%, at least At least 83%, at least 84%, at least 85%, at least 86%, at least 87% , at least 88%, at least 89%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, at least 99% or 100% identity or There are similarities.

[0225] The terms "homology," "sequence identity," and the like are used interchangeably herein. Sequence identity, as used herein, refers to the degree to which two sequences are identical, as determined by comparing the sequences. between two or more amino acid (polypeptide or protein) sequences or between two or more nucleic acids (polynucleotides) In a preferred embodiment, sequence identity is described as the relatedness between sequences of a given nucleotide. It is calculated based on the full length of the two given SEQ ID NOs or on a portion thereof. Preferably, at least 50%, 60%, 70%, 80%, 90%, or 100%. In the art, "identity" is also sometimes used to refer to amino acid sequence. Sequence relationships between or among nucleic acid sequences as determined by matches between such sequences. The "similarity" between two amino acid sequences refers to the degree of similarity between the amino acid sequences of a single polypeptide and and its conserved amino acid substitutions are determined by comparing the sequence of the second polypeptide. "Identity" and "similarity" are defined in accordance with the principles of Biology, each of which is incorporated herein by reference. oinformatics and the Cell:Modern Computa tional Approaches in Genomics,Proteomics and transcriptomics, Xia X., Springer Int. ernational Publishing, New York, 2018, and Bi oinformatics: Sequence and Genome Analysis s,Mount D.,Cold Spring Harbor Laboratory Press, New York, 2004. It can be easily calculated by known methods without any restrictions.

[0226] "Sequence identity" and "sequence similarity" refer to the degree of similarity between two peptide sequences or two nucleotide sequences. Depending on the length of the sequence, a global or local alignment algorithm is used to align the two sequences. Sequences of similar length can be determined by sequence alignment. Using a global alignment algorithm (e.g., Needleman-Wunsch) This allows sequences of substantially different lengths to be aligned using local alignment algorithms ( (e.g., Smith-Waterman) along its length, while maintaining a preferred alignment. The sequences are then optimally aligned across the Optimally aligned by the MBOSS needle or EMBOSS water program. If the sequences share at least a certain minimum percentage of sequence identity (as explained later), When two or more people share the same characteristics (such as the original or the original text), they may be referred to as "substantially identical" or "essentially similar."

[0227] Global alignment determines sequence identity when two sequences have similar lengths. When sequences have substantially different overall lengths, the Smith-W Local alignments, such as those using the Terman algorithm, are preferred. The needle is then aligned with the needleman to align the two sequences over its entire length. The Wunsch global alignment algorithm is used to maximize the number of matches and eliminate gaps. EMBOSS water is a Smith-Waterman topical anti-aging treatment. Generally, EMBOSS needle and EMBOSS wave alignment algorithms are used. The default parameters of water were used, with gap opening penalty = 10(nucleotide sequence) / 10 (protein) and gap extension penalty = 0.5 (nucleotide sequence) / 0.5 (protein For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is ​​Blosu m62 (Henikoff & Henikoff, incorporated herein by reference). ff,1992,PNAS 89,915-919).

[0228] Alternatively, the percentage of similarity or identity may be calculated using algorithms such as FASTA, BLAST, etc. The present invention can be determined by searching against public databases using the The nucleic acid and protein sequences of some embodiments of the present invention may also be searched for in public databases. Searches can be performed to identify, for example, other family members or related sequences. Such searches can be used as "reference sequences." Altschul, et al. (1990) J. Mol. Biol. 215: This was performed using the BLASTn and BLASTx programs (version 2.0) in 403-10. BLAST nucleotide searches can be performed to identify nucleotides homologous to the oxidoreductase nucleic acid molecules of the present invention. To obtain the nucleotide sequence, use the NBLAST program, score=100, word length=1. BLAST protein searches can be performed using the protein molecules of the present invention. To obtain homologous amino acid sequences, use the BLASTx program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, For this purpose, Gapped BLAST was used as described in Altschul's l et al., (1997) Nucleic Acids Res.25(17): BLAST programs can be used as described in 3389-3402. When using the Gapped BLAST and Gapped BLAST programs, For example, the default parameters of BLASTx and BLASTn can be used. Available on the web at www.ncbi.nlm.nih.gov / National Center for Biotechnology Inform Please refer to the ation homepage.

[0229] In some cases, in determining the degree of amino acid similarity, the skilled artisan may also consider so-called conservative amino acid similarities. As used herein, "conservative" amino acid substitutions are those that are It refers to the interchangeability of residues with similar side chains. Examples of such cases are given in the table below.

[0230] [Table 1]

[0231] Alternative conservative amino acid residue substitution classes:

[0232] [Table 2]

[0233] Alternative physical and functional classifications of amino acid residues:

[0234] [Table 3]

[0235] For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine and isoleucine, and the group of amino acids with aliphatic hydroxyl side chains is serine and and threonine, and the group of amino acids with amide-containing side chains is asparagine and glutamic acid. The group of amino acids with aromatic side chains is phenylalanine, tyrosine, and thiamin The group of amino acids with basic side chains is tryptophan, lysine, arginine, and hyaluronic acid. The group of amino acids that are stigmine and have sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine -Tyrosine, Lysine-Arginine, Alanine-Valine, and Asparagine-Glutamine Substitution variants of the amino acid sequences disclosed herein, including at least one of the amino acid sequences disclosed, At the very least, one residue has been removed and a different residue inserted in its place. For each of the naturally occurring amino acids, the amino acid changes are conservative. Preferred conservative substitutions are The amino acid sequence is as follows: Ala → Ser, Arg → Lys, Asn → Gln or His, Asp → Glu, Cys → Ser or Ala, Gln → Asn, Glu → As p, Gly → Pro, His → Asn or Gln, Ile → Leu or Val, Leu → Ile or Val, Lys→Arg, Gln or Glu, Met→Leu or Ile, P he → Met, Leu or Tyr, Ser → Thr, Thr → Ser, Trp → Tyr, Tyr→Trp or Phe, and Val→Ile or Leu.

[0236] Gene or coding sequence The term "gene" refers to a gene that acts within a cell on an appropriate regulatory region (e.g., a promoter). It contains a region that is transcribed into an RNA molecule (e.g., mRNA) operably linked to the A gene is a DNA fragment that contains a promoter, a 5' leader sequence, a coding region, and a and 3' untranslated sequences (3' "Expression of a gene" may include several operably linked fragments, such as the nucleotide sequence of the gene (terminal fragment). A DNA region operably linked to an appropriate regulatory region, particularly a promoter, is biologically Active, i.e., capable of being translated into a biologically active protein or peptide This refers to the process by which DNA is transcribed into RNA.

[0237] promoter As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to one A nucleic acid fragment that functions to control the transcription of the coding sequence, including the initiation of transcription of the coding sequence. Located upstream of the transcriptional direction of the site, binding to DNA-dependent RNA polymerase sites, transcription initiation sites, and transcription factor binding sites, repressor and activator proteins Any other DNA sequences, including but not limited to binding sites, as well as promoters Nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from A "constitutive promoter" is a promoter that is structurally identified by the presence of any other sequence in the promoter. A promoter is one that is active under most physiological and developmental conditions. An "inducible" promoter is one that can be activated physiologically or developmentally, for example, by application of a chemical inducer. It is an up-regulated promoter.

[0238] operably linked As used herein, the term "operably linked" refers to a functional relationship. refers to the linkage of polynucleotide elements in a nucleic acid sequence. For example, a transcriptional regulatory sequence is "operably linked" when it controls the transcription of a coding sequence. A coding sequence is operably linked if it has an effect on the coding sequence. The DNA sequences that are linked together are typically contiguous and join two protein-coding regions. When necessary, contiguous and in reading frame are meant. Ligation at suitable restriction sites or with adapters or linkers inserted instead This can be achieved by gene synthesis or by gene synthesis.

[0239] Proteins and amino acids The terms "protein" or "polypeptide" or "amino acid sequence" are used interchangeably. and are used in a variety of ways, without regard to their specific mode of action, size, three-dimensional structure or origin. A molecule consisting of a single chain of amino acids. The amino acid sequence as described herein In the present specification, amino acids or "residues" are designated by three letter symbols. and the corresponding one-letter symbols are well known to those skilled in the art and have the following meanings: A( Ala) is alanine, C (Cys) is cysteine, and D (Asp) is asparagus. E (Glu) is glutamic acid, and F (Phe) is phenylalanine. G (Gly) is glycine, H (His) is histidine, and I (Ile) is isoleucine, K(Lys) is lysine, and L(Leu) is leucine; M (Met) is methionine, N (Asn) is asparagine, and P (Pro) is is proline, Q (Gln) is glutamine, and R (Arg) is arginine; S (Ser) is serine, T (Thr) is threonine, and V (Val) is valine. where W (Trp) is tryptophan and Y (Tyr) is tyrosine. The residues are , can be any proteinogenic amino acid, or any non-proteinogenic amino acid, such as a D amino acid. De novo amino acids and modified amino acids formed by post-translational modifications, as well as any non- It can also be a naturally occurring amino acid.

[0240] Gene constructs Genetic constructs as described herein may be used in any manner known to those skilled in the art. may be prepared using cloning and / or recombinant DNA techniques, in which the Nucleotide sequences encoding globin are incorporated herein by reference in their entirety. This document also incorporates Ausubel et al., “Current Protocol s in Molecular Biology”,Greene Publishing g and Wiley Interscience, New York (1987) and Sambrook and Russell (2001, supra) The method is carried out in a suitable cell, e.g., a cultured cell or a cell of a multicellular organism, as described herein. Also, Kunkel (1985) Proc. Natl. Acad. Sci. 82 :488 (describing site-directed mutagenesis), and Roberts et al. 87) Nature 328:731 734 or Wells, JA, et al. (1985) Gene 34:315 (describing cassette mutagenesis).

[0241] Expression vector The phrase "expression vector" or "vector" generally refers to a vector that contains, for example, a gene sequence or coding sequence. Nucleotides capable of effecting expression of such sequences in host cells compatible with such sequences. A gene expression vector used to obtain gene expression in cells by introducing a sequence into the vector. An expression vector is a tool in molecular biology that can be stabilized in cells and Within the context of the present invention, cells possess a genome that remains ubiquitous. This includes cells used to generate or to which the construct will be administered. Alternatively, the vector may be inserted into the cell genome, for example, by homologous recombination or other It can be incorporated through other means.

[0242] These expression vectors typically contain at least a suitable promoter sequence and, optionally, Additional factors necessary or helpful in effecting expression may also include transcription termination signals. It may also be used as described herein. The DNA sequence or nucleotide sequence is capable of being introduced into an in vitro cell culture and reacting therewith. Specifically, the DNA construct is incorporated into a DNA construct that allows expression in the target cell. The construct may be used for replication in a prokaryotic host, such as a bacterium, e.g., E. coli. Suitable for or cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungus The vector can be introduced into bacterial or other eukaryotic cell lines.

[0243] DNA constructs prepared for introduction into a particular host contain a sequence that is recognized by the host. The replication system to be used, the intended DNA segment encoding the desired polypeptide, and the polypeptide transcriptional and translational initiation regulatory sequences operably linked to the peptide-encoding segment; and termination regulatory sequences. The term "operably linked" is used herein to refer to As already explained, for example, a promoter or enhancer may be used to direct the transcription of a coding sequence. If it stimulates the transcription of a gene, it is operably linked to the sequence. A polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide Generally, the operably linked D The NA sequences are contiguous and, in the case of a signal sequence, contiguous and in reading frame. However, enhancers do not have to be contiguous with the coding sequence whose transcription they control. Ligation can be at convenient restriction sites or with adapters or linkers inserted instead. This can be achieved by ligation of a suitable promoter to a target gene or by gene synthesis. The selection of sequence will generally depend on the host cell selected for expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters well known in the art. There are also promoters (e.g., Sambrook and Russell, 2001 , supra). Transcriptional regulatory sequences typically include a heterologous sequence that is recognized by the host. The selection of an appropriate promoter depends on the host. rp, lac and phage promoters, tRNA promoters, and glycolytic enzyme promoters Promoters such as ribonucleotides are known and available (see, e.g., Sambrook and Russell, 2001, supra). A replication system and transcription and translation systems, together with an insertion site for a segment encoding a peptide. In most cases, the replication system is determined by the cell type used to generate the vector. It only functions in bacteria (such as E. coli). Plasmids and vectors do not replicate in cells that are infected with the vector. Examples of operable combinations of expression vectors are given in Sambrook and Russell, (2001, supra) and Metzger et al. (1988) Nat ure 334:31-36. For example, suitable expression vectors include: Yeasts, such as Saccharomyces cerevisiae (S. cerevisiae), e.g. Insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells, and bacterial cells, e.g., For example, it can be expressed in E. coli. The cell is therefore a prokaryotic The host cells may be bacterial or eukaryotic. The cells may be grown in liquid medium or on solid medium. The cells may be suitable for the

[0244] Alternatively, the host cell may be a cell that is part of a multicellular organism, such as a transgenic plant or animal. It is a cell.

[0245] Selection of an appropriate promoter sequence is generally selected for expression of the DNA segment. Depending on the host cell, examples of suitable promoter sequences include those known in the art for prokaryotes and and eukaryotic promoters (e.g., Sambrook and Russell , 2001, supra). Transcriptional regulatory sequences typically include a sequence that is recognized by the host. There are heterologous enhancers or promoters that can be used. The selection of an appropriate promoter depends on the host. Depending on the promoter, trp, lac, and phage promoters, tRNA promoters, and Glycoenzyme promoters are known and available (see, for example, Sambrook and (See Russell, 2001, supra). The expression vector contains a polypeptide. Replication systems and transcriptional and translational controls, along with insertion sites for the coding segments. In most cases, the replication system is determined by the cell (large cell) used to produce the vector. It only functions in bacterial cells such as E. coli. The smids and vectors do not replicate in cells infected with the vectors. Cell lines and expression Examples of workable combinations of vectors are described in Sambrook and Russell ( 2001, supra) and Metzger et al. (1988) Nature e 334:31-36. For example, suitable expression vectors are , e.g., Saccharomyces cerevisiae (S. cerevisiae), and bacterial cells For example, the gene can be expressed in E. coli. The host cells may be karyotic or eukaryotic. The cells may be grown in liquid medium or on solid medium. The cells may be suitable for culture.

[0246] Expression Expression can be assessed by any method known to those of skill in the art. For example, expression can be assessed by measuring mRNA expression. Measure the level of transgene expression in the transfected tissue in terms of A or protein levels. or by quantitative PCR, RNA sequencing, Northern blot analysis, Those skilled in the art can perform various methods, such as stain blot analysis, mass spectrometry of protein-derived peptides, or ELISA. The activity of the antibody can be assessed by standard assays known to those skilled in the art.

[0247] Expression can be achieved by the expression of a genetic construct, expression vector, or composition as described herein. In some embodiments herein, expression may be assessed at any time after administration. is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 The patient may be evaluated after 28 weeks, 32 weeks, 36 weeks, 40 weeks, or longer. do.

[0248] In this document and in the claims of this document, the verb "comprises" and its conjugations are used in an open-ended sense to mean the following item in which the word appears. The term "consisting of" is used in this context, but does not exclude items not specifically mentioned. The verb "to produce" refers to a meat substitute, genetic construct, host cell (or or a method) includes additional components (or additional steps) other than those specifically identified. The meaning of "consisting essentially of" can be replaced by "consisting essentially of" and the additional constituent element ... The elements do not change the unique features of the present invention. The methods as described herein may include additional steps other than those specifically identified. may be replaced by "consisting essentially of" which means that the additional step The use of a different type of filter may not alter the unique characteristics of the present invention.

[0249] A reference to an element by the indefinite article "a" or "an" refers to one and only one of the elements. Unless the context clearly requires that there be more than one of the elements The indefinite article "a" or "an" therefore usually means "at least" It means "at least one."

[0250] As used herein, "at least" means a particular value is greater than or equal to the particular value. For example, "at least 2" is the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. It is understood.

[0251] Furthermore, the use of first, second, third, etc. in this specification and in the claims The term is used to distinguish between similar elements and to describe sequential or chronological order. Terms so used are interchangeable under appropriate circumstances. and that the embodiments of the invention described herein are not intended to be limiting unless otherwise specified. It should be understood that sequences other than those shown may be operable.

[0252] The words "about" or "approximately" when used in connection with a numerical value (e.g., about 10 ), preferably the value can be 0.1% (of 10) more or less than the given value As used herein, the term "and / or" means that the One or more of the cases may occur alone or in combination with at least one of the cases listed. Therefore, it indicates that up to a maximum of all combinations of the listed cases may occur.

[0253] Various embodiments are described herein. Each embodiment as identified herein may be combined together unless otherwise stated.

[0254] In any case, the language in this disclosure will govern any definitions, disclaimers, or except as expressly permitted by law, and to the extent that the incorporated material is inconsistent with the express disclosure of this specification. Except as provided herein, all patent applications, patents, and printed publications cited herein are hereby incorporated by reference in their entirety. is incorporated herein by reference in its entirety.

[0255] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein. Such methods and materials may be used in the practice of the present invention. The invention is in no way limited to the methods and materials described.

[0256] The present invention further provides the following disclosures, which should not be construed as limiting the scope of the present invention. This will be explained by way of example. [Brief explanation of the drawings]

[0257] [Figure 1] The three-dimensional structure of bovine bioglobin. The structure of wild-type porcine Mb, resolved by X-ray diffraction at 1.8 Å resolution (Krzywda et al. 1998), shows the heme, proximal histidine (His94), and distal histidine (His65). [Figure 2] Comparison of myoglobin protein sequences from six animal species. Sequence alignment shows sequences from bluefin tuna (Thunnus orientalis), red jungle fowl (Gallus gallus), steppe mammoth (Mammuthus trogontherii), wild boar (Sus scrofa), cattle (Bos taurus), and sheep (Ovis aries). The level of amino acid conversion is indicated by blue shading using BLOSUM62 scores. The proximal histidine (His94, boxed) is present in all six species. The steppe mammoth possesses a glutamine (Gln65, boxed) at the position normally occupied by the distal histidine (His65, boxed), a phenylalanine at position 30 (Phe30, boxed), and a histidine at position 92 (His92, boxed). [Figure 3] Models of the 3D structure of myoglobin from Asian elephant (left) and steppe mammoth (right). Ribbon (top) and surface (bottom) views are shown. The heme molecule is depicted, so is the residue at position 92 (glutamine in elephant, histidine in mammoth). In the surface view, the positive charge is shown in dark gray. [Figure 4] Production of extracellular animal Mycobacterium bacterium in Pichia pastoris. Cell-free supernatants were collected after fermentation with (+) or without (-) methanol induction and concentrated 10-fold by ultrafiltration. The concentrated samples obtained after methanol induction exhibited a deep red color (A) and, after analysis by SDS-PAGE, revealed a protein with the molecular weight expected for Mycobacterium b ... [Figure 5] Absence of recombinant DNA in myoglobin preparations obtained by fermentation. The same gel is shown with two different exposure times (top and bottom panels). The asterisks indicate the primers remaining after the PCR reaction. [Figure 6] Sequence coverage of mammoth myoglobin identification by mass spectrometry. The bar graph (bottom panel) shows the intensity of different peptides on a logarithmic scale. The sequences of the peptides at the bottom of the graph correspond to SEQ ID NOs: 31-80. [Figure 7] Absorption spectra of mammoth and bovine myoglobin solutions. (A) Absorption spectra of recombinant steppe mammoth myoglobin (black circles), bovine myoglobin (white circles), and commercially available myoglobin purified from horse muscle (triangles) were recorded at room temperature. (B) Absorption spectrum of horse myoglobin during 24 h of incubation at pH 5.6 and 25 °C. The inset shows the ratio between absorbance at 580 nm (MbO2) and 505 nm (MetMb) for recombinant mammoth myoglobin (black bars), bovine myoglobin (gray bars), and commercially available horse myoglobin (white bars). Asterisks indicate statistically significant differences between recombinant and horse myoglobin. (*) = p < 0.05, (**) = p < 0.01. [Figure 8] Color of laboratory-made meat analogs containing myoglobin. Photograph (A) and quantification (B) show the effect of myoglobin addition on the color of plant-based burgers. [Figure 9] Color stability of meat analogs containing myoglobin. Photographs (A) and quantification (B) show the color changes observed over time in plant-based burgers containing various concentrations of purified equine myoglobin exposed to constant light at 4° C. A commercial burger containing soy leghemoglobin was included for comparison. [Figure 10]Volatile compounds extracted from laboratory-made meat analogs containing recombinant hemoproteins. (A) Quantification of the number of volatile compounds interrogated from plant-based burgers by HS-SPME GC-MS. Values ​​not sharing the same subscript were found to be significantly different from each other at p<0.05 by Tukey's HSD test. (B) Principal component analysis (PCA) of volatiles from laboratory-made plant-based burgers, either raw (shown in green) or gillnet-caught (shown in blue). A commercially available plant-based burger containing recombinant soybean leghemoglobin (LegH) was used as a reference. [Figure 11] Volatile compounds associated with the addition of recombinant myoglobin to laboratory-made meat analogs. (A) Relative amounts of volatile flavor-active compounds detected in grilled plant-based burgers normalized to levels observed in plant-based burgers without Mb. Asterisks indicate statistically significant differences between burgers without and with Mb. (*)=p<0.5, (**)=p<0.01, (***)=p<0.001. $ indicates statistically significant differences between mammoth and bovine Mb. ($)=p<0.05, ($$)=p<0.05. (B) Description of the aroma of volatile compounds also found in cooked meat (van Ba ​​et al. 2021, The Good Scent Company information system). [Figure 12] Iron bioavailability from myoglobin. Iron uptake (ng ferritin content normalized to mg total protein content) by human Caco-2 intestinal cells exposed to 0.5 mg / ml, 1.0 mg / ml, or 2.0 mg / ml purified bovine myoglobin compared to control medium alone (CM). Asterisks indicate statistically significant differences between CM and treatment. (****)=p<0.0001. [Figure 13] Effect of recombinant myoglobin on differentiated Caco-2 monolayers. Cytotoxicity was measured after 24 hours of incubation in the presence of recombinant mammoth or bovine myoglobin, or complete medium (CM) as a control. Asterisks indicate statistically significant differences between CM and treatment. (*)=p<0.05, (**)=p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0258] Example Example 1: Production Method A Example 1.1: General Construction of Gene Constructs and Vectors In some cases, Pichia pastoris, Escherichia coli and Aspergillus niger Complete vectors and / or genomic integration suitable for expression in Gillus niger The cassette is synthesized by a commercial supplier. and / or construct a genome integration cassette using commercially available polynucleotides and fragments according to the manufacturer's protocol. P using primers designed for the introduction of compatible overhangs between the nucleotides. CR was performed, followed by in vitro analysis using a commercially available kit according to the manufacturer's protocol. Complete vector and / or genome integration cassette assembly was performed in some cases. In this case, the complete vector assembly is transformed into yeast cells as previously described in the art. This was done in vivo by transformation of the vector fragment into cells (Kuijpers et al. al. 2013 ), followed by isolation of the complete vector using a commercially available kit.

[0259] The complete vector comprises a known sequence of replication for maintenance of the vector in an individual organism. The necessary origin, the nucleotide sequence to be expressed (SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16), regulatory elements suitable for expression in individual organisms (at least one promoter promoter and one termination factor), and screening and characterization of correct transformants. It contains a selectable marker that allows maintenance of the vector in transformed strains. The expressed nucleotide sequences (SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16), regulatory elements suitable for expression in the respective organism, and optionally, homologous 30-3000 bp of homology with individual genomic integration sites to facilitate recombination In some cases, the genomic integration cassette contains 5' and 3' regions of appropriate length. The kit may be fused to an appropriate selectable marker to facilitate screening and / or In some cases, the genomic integration cassette and the selectable marker are co-transformed. The fused appropriate selectable marker is then transfected by counterselection in the appropriate medium, followed by the Cre-Lox recombinase. Use of CRISPR / Cas systems, or to create marker-free strains and removing it from the final production strain after transformation using methods commonly known in the art, such as using In some cases, expression from a vector and / or genomic integration cassette The nucleotide sequence may be modified to include appropriate signals to facilitate export of the expressed fusion protein. The peptide-encoding sequence and / or its subsequent excretion facilitates purification of the expressed fusion protein. In some cases, the sequence is operably linked to a sequence tag for ease of identification. The linked sequences result in the expressed peptide fused to the N-terminus of the fusion protein, etc. In some cases, the fused sequence may be used to enhance the expression of the expressed fusion protein by the cell. During or after excretion, the signal peptide and / or tag from the fusion protein is removed. It contains recognition sites for natural or synthetic peptidases to facilitate cleavage.

[0260] Example 1.2: General strain construction Pichia pastoris, Escherichia coli hia coli and Aspergillus niger ) strain is transformed using molecular toolbox techniques known in the art. Selection of transformants is determined by whether a selection marker is used and the type of selection marker ( Depending on the dominant / auxotrophic nature of the colony, sufficient time was allowed for colony growth to be observed. Thereafter, the cells are cultured in a suitable, known selective growth medium containing antibiotics or the like, as known for each organism. Correct transformants in each case were identified by diagnostic PCR or Southern blot analysis. The genome integration is confirmed by other suitable known molecular toolbox methods, such as the genomic integration method. In some cases used for transformation, the strain to be transformed contains a nucleotide sequence that facilitates homologous recombination. The non-homologous end joining repair machinery is defective to allow for the transformation. In some cases, selection markers are used to generate marker-free strains, etc. The correctly transformed strains are then removed as described in Example 1. Store in glycerol stocks at -80°C.

[0261] Example 1.3: Myoglobin production by transformed strains The transformed strains obtained in Example 2 are tested for the production of extracellular myoglobin. Cell cultures are controlled according to commonly known parameters such as temperature, pH and ionic strength of the growth medium, and agitation rate. Cultivation in shake flasks under conditions conducive to the production of proteins appropriate for each known host. In some cases, the temperature ranges from 16 to 40°C, and the pH value The value of the ionic strength is in the range of 100 to 1000 mM, and the stirring The rotation speed is in the range of 100-300 rpm. The growth medium suitable for culturing each host is generally It contains sources of carbon and nitrogen, as well as additional nutrients such as inorganic salts and vitamins. The culture is inoculated from a frozen stock of the transformed strain. After 12-24 hours of incubation, the culture broth The culture supernatant is then separated into 1000 ml of the culture medium and centrifuged to remove the biomass. SDS-PA by band identification or Western blotting using commercially available antibodies The presence of produced myoglobin is tested using known techniques such as GE. The production of extracellular myoglobin was confirmed. The produced myoglobin was extracted from the culture supernatant. The product is then purified by chromatographic techniques according to standard protocols. The absence of an excretion peptide (cleaved during excretion) was confirmed by standard protein sequencing protocols. The purified myoglobin was stored at -20°C for further use. Store at °C.

[0262] Example 1.4: Production of meat substitutes The purified myoglobin produced in Example 3 was extracted from muscle tissue and Used in the production of adipose (fatty) tissue. Purified myoglo to produce muscle tissue substitutes. The bottle is cross-linked with endobicillin protein via transglutaminase. Muscle tissue can also be heated to form a gel of endobicillin protein. The purified myoglobin was added to the heated gel, and the heated gel was cooled to room temperature. The muscle tissue substitute was also mixed with purified myoglobin and endobicillin protein. The fat substitute is also produced by co-extrusion with the protein. By forming a heated gel consisting of albumin protein, oil, and lecithin. The purified myoglobin was added to the gel, and the heated gel was cooled to room temperature. The connective tissue substitute is extruded into the zein by known procedures. The meat is made using a protein source. Muscle substitutes, fat substitutes and connective tissue substitutes are ground in a meat grinder. In some cases, the meat substitute is then prepared by combining the ingredients in the desired ratio. Cook the meat.

[0263] Example 1.5: Purified myoglobin from Escherichia coli Production of Complete set of woolly mammoths, steppe mammoths, sheep, cows, pigs, chickens, and tuna The full-length myoglobin gene (SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16) , a modified pET-23 containing a T7 promoter and terminator and a C-terminal hexaHis tag a(+) vector (Genscript Biotech, Leiden, the Netherlands) The sequences were synthesized and cloned by the International Center for Microbiology and Oncology (ICH) at the University of Illinois (Illinois). Although 9 encodes only a portion of myoglobin, the vector It is understood that the am naturally present in the vector may be synthesized and cloned. The pR marker gene was cloned into the E. coli strain K12 containing the original transcriptional regulatory element. The proBA operon is replaced by the proBA operon to facilitate selection without antibiotics. The constructed plasmid was confirmed by PCR and transformed into proline-auxotrophic Escherichia coli (E. coli). i) Transform the protein-producing strain (E. coli K12 ΔproBA) The transformed strain was grown in minimal medium (10.5 g / L K2HPO4, 4.5 g / L KH2PO4, 1.0 g / L (NH4)2SO4, 0.12 g / L MgSO 4, 0.5 g / L sodium citrate, 2 g / L glucose, and 5.0 mg / L thiamin The incubation was continued overnight in a shake flask containing ethanol HCl at 37°C and 150 rpm (pH 6). Incubate 500 μl of the overnight culture in 1 L of PBS containing 500 mL of minimal medium. Transfer to a shake flask and incubate at 37°C and 150 rpm until an OD600 of 0.4-1 is reached. Incubate in 100 μM of IPTG (isopropyl-β-D-thiogalactose) The culture was then incubated at 16°C and 150 rpm for 24 hours. The culture is harvested and centrifuged at 3500 x g (4°C) for 15 minutes. Discard the supernatant and resuspend the pellet in 1 KU of lysozyme / ml (Sigma-Aldrich), 2 5 U of Benzonase® Nuclease and cOmplete™ , EDTA-free Protease Inhibitor Cocktail (Roch e) 50mL of BugBuster Protein Extraction The pellet was dissolved in Reagent (Novagen). The dissolved pellet was stirred for 4 min in a shaker. Incubate at 4°C for 30 minutes. Repeat the centrifugation step to obtain the cell-free extract (supernatant). The cells are harvested and assayed by SDS-PAGE to confirm the production of myoglobin. Transformed strains of woolly mammoths, steppe mammoths, sheep, cattle, pigs, chickens, The production of myoglobin in chicken and tuna was confirmed by the presence of protein bands of the correct size. Check that.

[0264] For purification, the cell-free extract containing the soluble fraction of the protein was purified using the AKTA starting system. Load onto a coupled HisTrap FF 1 mL column (Cytiva, MA, USA). The column was washed with 20 mM HEPES, 0.4 M NaCl, and 20 mM imidazole. Equilibrate the protein in 20 mM HEPES, 0.5% CO₂, pH 7.5, at a flow rate of 1 mL / min. Elute with 0.4 M NaCl and 400 mM imidazole, pH 7.5. Fractions containing ATP were pooled, concentrated, and analyzed by SDS-PAGE and anti-histidine tag antibody ( The total myoglobin was confirmed by Western blotting using a ELISA kit (Bio-Rad) Verify the success of the preparation. Store the purified myoglobin at -20°C for further use. .

[0265] Example 1.6: Aspergillus niger Production of purified myoglobin From woolly mammoths, steppe mammoths, sheep, cattle, pigs, chickens, and tuna The genes (SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16) were used to identify myoglobin Pectin methylesters of Aspergillus niger (A. niger) to facilitate excretion Aspergillus niger (An) operably linked to the efflux signal sequence of sterase A full-length myoglobin expression vector containing the glaA promoter and release factor In this specification, SEQ ID NO: 9 encodes only a portion of myoglobin. However, it is understood that they may nevertheless be included in the expression cassette. The genomic integration cassette was transfected with the myoglobin expression cassette into Aspergillus oryzae (Asp Orotidine 5'-phosphate decarboxylase gene from Sterilus oryzae Gene sequence (pyrG) and Aspergillus niger (A. niger) pyrG gene 1000 bp at the 5' and 3' ends that are homologous to the native upstream and downstream sequences of the clone. Assemble by fusion PCR.

[0266] The genome integration cassette was transfected into Aspergillus niger (A. niger) CBS 120. 49 Transform into protoplasts of ΔkusA ΔpyrG. The cells were incubated at 30°C and 250 rpm in complete medium (2% (wt / vol) glucose, 6 g / L NaNO3, 1.5g / L KH2PO4, 0.5g / L KCl, 0.5g / L MgSO4·7H2O, 0.2% (wt / vol) tryptone, 0.1% (wt / vol) yeast extract, 0.1% (wt / vol) casamino acids, 0.05% (wt / vol) yeast RNA, and trace elements according to Vishniac (1957)) overnight in shake flasks. The mycelium is harvested by filtration and diluted in PS buffer (0.2 M sodium phosphate buffer). Dissolve 1 g of mycelium in 100 ml of thorium buffer (0.8 M L-sorbitol, pH 6). 0.5g of VinoTaste® Pro lytic enzyme was added per body, followed by 3 Incubate at 0°C and 100 rpm. Remove undigested mycelium via filtration. Protoplasts were harvested by gentle centrifugation (1500 × g, 3°C) and placed in SC solution (182. 2g L -1 of sorbitol, 7.35g L -1 CaCl2·2H2O) and 10 in the same solution 8 Resuspend fresh protoplasts to a concentration of 0.5 protoplasts / mL. 20 μL of 4 M ATA (aurintricarboxylic acid ammonium salt) and 20% PEG- 200 μL of protoplast suspension was diluted to 5 μL in a mixture containing an additional 100 μL of 4000 The resulting mixture was mixed with the recombinant DNA cassette of 100 kDa and incubated for 10 minutes. Then, 5 mL of 1.2 M sorbitol solution was added and the mixture was incubated for another 10 minutes. Transformed protoplasts were harvested by gentle centrifugation and aliquoted into 1 mL The transformed protoplasts were plated on minimal medium agar plates. (1.5% (mass / volume) agar, 2% (mass / volume) glucose, 6 g / L NaNO3 , 1.5g / L KH2PO4, 0.5g / L KCl, 0.5g / L MgSO4·7 HO and trace elements according to Vishniac (1957) and incubated at 30°C for 4 days. The DNA from the selected transformants was then incubated for 1 hour (pH 5). Extracted using alcohol / chloroform extraction. Woolly mammoth, steppe mammoth, sheep Genomic integration cassettes containing myoglobin genes from bovine, porcine, chicken, and tuna Correct integration of the vector is confirmed by Southern blotting.

[0267] Spores of correct transformants were cultured by inoculation on complete medium agar plates at 30°C for 4 days. 10 mL of N-(2-acetamido)-2-aminoethanesulfonic acid (ACE S) buffer. 2 × 10 8 Using spores, 400 mL of minimal medium containing Inoculate shake flask cultures and incubate overnight at 30°C and 250 rpm. Add 400 mL of the medium to a fresh shake flask culture at 30°C for 250 min. The cells were transferred to PBS for 24 hours. The culture was harvested and then centrifuged at 3,200 x g for 10 minutes at 4 °C. The supernatant was assayed by SDS-PAGE to determine the amount of myoglobin. The production of the gene was confirmed in individual transformants of woolly mammoths, steppe mammoths, sheep, and Expression of bovine, porcine, chicken, and tuna myoglobin was analyzed to identify proteins of the correct size. Confirmed by the presence of a band.

[0268] For purification, the culture supernatant containing the soluble fraction of the protein was loaded onto HiLoad 16 / 600 Superdex 75pg column (10mm x 300mm) (Cytiva, The column was loaded onto a column of 0.15 M ammonium acetate, pH 6.0, 0. Equilibrate at a flow rate of 75 mL / min. The myoglobin-containing fractions are pooled, concentrated, and standardized. The results are confirmed by SDS-PAGE and LC-MS according to standard protocols. The presence of purified myoglobin without the signal peptide is confirmed.

[0269] Example 1.7: Purified by Pichia pastoris Myoglobin production From woolly mammoths, steppe mammoths, sheep, cattle, pigs, chickens, and tuna The genes (SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16) were transfected into Pichia passivata. Contains P. pastoris native pgk1 promoter and release factor under control Synthesize a full-length myoglobin expression cassette. Native Pichia pastoris (P. pastoris) under the control of motor and release factors The gene encoding the histidine biosynthetic trifunctional protein gene (his4), and Native upstream and downstream sequences of the P. pastoris his4 locus The genome was then cloned by PCR with 1000 bp of homology at the 5' and 3' ends. Assemble the integration cassette. The genomic integration cassette is inserted into a vector containing no antibiotic resistance marker. Histidine auxotrophic Pichia pastoris (P. pastoris) strain Bg12 (B ioGrammatics Inc. Carlsbad, CA), Gietz and Woods(2002)(Gietz RD et al.,(2002),Meth Acetic acid as previously described in [Illegible Text] (Obsidian Enzymol., 350:87-96). Transform using the lithium transformation protocol. Correct transformants were identified using Verdyu n et al.(1992)(Verduyn C.,et al(1992),Ye As previously described in [Dr.] Ast, 8:501-517, a synthetic medium (to facilitate selection) was used. Histidine-free (to prevent oxidative stress), i.e., 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 0.5g / L MgSO 4· 7H2O, 4.5 mg / L ZnSO 4· 7 H20, 0.3 mg / L CoCl 2· 6H2O, 1 mg / L MnCl 2· 4H2O, 0.3 mg / L CuSO 4· 5H2O, 4.5 mg / L CaCl 2· H2O, 3mg / L FeSO 4· 7H2O, 0.4 mg / L NaMoO 4· 2H2O, 1mg / L H3BO3, 0.1 mg / L KI, 0.05 g / L biotin, 1 mg / L pantothenate calcium phosphate, 1 mg / L nicotinic acid, 25 mg / L inositol, 1 mg / L Thiamine.HCl, 1 mg / L pyridoxine.HCl, 0.2 mg / L para-amino 2% w / v containing benzoic acid and 2% w / v glucose (pH 5) as a carbon source The plates are incubated at 30°C for 4 days. Incubate: woolly mammoths, steppe mammoths, sheep, cows, pigs, chickens The precise integration of the genome integration cassette containing tuna myoglobin was confirmed by the manufacturer. The Yeast Protein Kit (ZymoResearch) was used according to the protocol. Confirm by colony PCR after DNA preparation using the ELISA kit (Irvine, CA). Glycerol stocks of confirmed transformants are prepared and stored at -80°C.

[0270] For myoglobin production, use frozen glycerol stocks to make a 500 mL total volume. A 1 L pre-culture shake flask containing the fermented medium was inoculated and incubated overnight at 30°C and 250 rpm. Incubate the pre-culture and subsequent shake flask cultures at a starting O of 0.2. Inoculate to D660. Incubate the culture at 30°C and 250 rpm for 24 hours. Harvest the culture and centrifuge it at 3500 x g (4°C) for 15 minutes. Discard the supernatant. The cell pellet is resuspended in ice-cold demineralized water and the centrifugation step is repeated. Discard the contents and resuspend the cells in Yeast Pro along with mechanical disruption according to the manufacturer's protocol. Lyse the cells using a staining kit (ZymoResearch, Irvine, CA). The mixture containing cell debris and soluble proteins was centrifuged at 3500 x g (4°C) for 15 minutes. The supernatant (cell-free extract) containing soluble proteins was collected and analyzed by SDS-PAG. Assay by E. coli to confirm myoglobin production. Woolly mammoths, steppe mammoths, sheep, cattle, pigs, chickens, and tuna mio Globin production is confirmed by the presence of a protein band of the correct size.

[0271] For purification, the culture supernatant containing the soluble fraction of the protein was loaded onto HiLoad 16 / 600 Superdex 75pg column (10mm x 300mm) (Cytiva, The column was loaded onto a column of 0.15 M ammonium acetate, pH 6.0, 0. Equilibrate at a flow rate of 75 mL / min. The myoglobin-containing fractions are pooled, concentrated, and standardized. The results are confirmed by SDS-PAGE and LC-MS according to standard protocols. Check for the presence of purified myoglobin.

[0272] Example 2: Production Method B Unless expressly stated otherwise, "mammoth" and "mammoth myoglobin" are used throughout the examples. 2 refers to the "step mammoth" and "step mammoth myoglobin."

[0273] Example 2.1. Materials and Methods Sequence analysis Cattle (Bos taurus), Red jungle fowl (Gallus gallus), Bluefin tuna (Thunnus orientalis), sheepshead (Ovis aries The sequences encoding myoglobin from wild boar (Sus scrofa) were iprot (Accession numbers: P02192, P02197, P68190, and P02 190, P02189). The sequence encoding myoglobin from M. gontherii was, at the time of filing this application, was unknown, but DNA extraction from the molar concentration sample from the so-called Adycha specimen, Illumina DNA sequencing, matching the reads and identifying them as African savanna elephants (Loxodonta africana) genome (van der Valk et obtained by the inventors after mapping against ,

[0274] Multiple sequence alignments were performed using Clustal Omega (Sievers et al. al. 2011) and Jalview 2.11.1.4 (Waterhouse The visualization was performed using the MRI software (se et al. 2009).

[0275] Protein modeling The structure of wild-type deoxymyoglobin from wild boar (Sus scrofa) was analyzed by RCS. B Retrieved from the Protein Data Bank (PDB accession number: 1MWD, A The structure of step mammoth myoglobin was Using the crystal structure of myoglobin from the elephant (Elephant elegans) as a template, we performed autoprotein assembly. The homology modeling server SWISS-MODEL (Waterhouse et al. The net surface charge was modeled using the PDB accession number 1EMY. (Z Mb ) is ionizable at pH 6.5 using published site-specific ionization constants. The charge was calculated as the sum of the charges of all the functional groups (Mirceta et al. 2013). All protein structures were visualized using DeepView v4.1 (Guex and Pe The drawings were created using itsch 1997).

[0276] Construction of expression plasmids The myoglobin coding sequence was cloned into Pichia pastoris Development in Komagataella phaffii The codons were optimized for expression (Love et al. 2016) (SEQ ID NOs: 19-2 4) Saccharomyces cerevisiae ae) an optimized sequence preceded by the coding sequence of mating factor alpha (SEQ ID NO:2 5–30) were chemically synthesized using GenScript. downstream of the AOX1 release factor, HIS4 selection marker and AOX1 3' fragment. The vector was cloned upstream into the pBDIPp5 vector. ) (DH10B) and purified using a Plasmid kit (Quiagen). The expression cassette was inserted into the AOX1 promoter and was verified by Sanger sequencing. BglII (New England Transfection) which cleaves upstream from the 3′ fragment of AOX1 and downstream from the 3′ fragment of AOX1 Generated from the resulting vector by restriction of the plasmid using ELISA kits (Biolabs) Then, the QIAquick Gel Extraction Kit (QUiagen) was used. It was purified.

[0277] Stock Engineering Pichia pastoris (Komagataella paphii) Komagataella phaffii GS115 strain (his4) was cultured in Life Cell transformation was performed essentially as previously described. This was done using electroporation, as previously described (Cregg 2007). Cells of the 15 strains were cultured in YPD (1% yeast extract, 2% peptone, and 2% D-glucose) medium. Cells in the exponential growth phase were grown in 200 mM HEPES buffer ( Incubate for 30 min in YPD medium containing 25 mM dithiothreitol (pH 8.0). Competent cells were then washed with ice-cold 1 M sorbitol and resuspended in sterile electrophoresis gel. The mixture was then transferred to a gas-perforated cuvette (Bio-Rad). (Rad) Electroporate 1-5 µg of linear expression cassette (see 3.3) The cells were electroporated using PBS and resuspended in 1 mL of 1 M sorbitol-containing YPD medium. The cells were transferred to a sterile 1.5 mL Eppendorf tube and incubated at 28°C without shaking for 3 hours. After incubation, the cells were cultured in solid NGY medium (minimal glycerol medium: no amino acids, sulfated amino acids). 41% yeast nitrogen base containing ammonium, 1.34%, 2% D-glucose, and 2% agar 0 -5 The plates were incubated at 28°C for up to 4 min. The mixture was incubated for 1 day.

[0278] Transformants capable of growth in the absence of histidine were screened for their ability to express Mb. The cells were cultured in BMGY medium (1% yeast extract, 2% peptone, 100 mM phosphate buffer). Potassium buffer (pH 6), amino acid-free, ammonium sulfate-containing, 1.34% yeast nitrogen base Agent, 4 10 -5 1% biotin, 1% glycerol) in a 50mL Falcon tube Exponentially growing cells were grown in 1% methanol to induce Mb expression. Samples were collected 24, 48, 72, and 96 hours after the start of methanol induction. and analyzed by SDS-PAGE to assess Mb levels (see below). .

[0279] The best producing strains were selected for further experiments and stored in a master cell bank at -80 °C before use. The results presented herein demonstrate that the PAL-02-02 (Mammoth Mb) strain and It was obtained using the PAL-02-03 (bovine Mb) strain.

[0280] Recombinant protein production and purification Cells from the master cell bank were plated on YPD agar (1% yeast extract, 2% peptone and 2% D-glucose, 2% agar) and incubate at 28°C for 2 days. The seed culture was then transferred to a baffled flask containing 100 mL of BMGY medium. The mixture was prepared in a sco and incubated at 28°C for 24 hours in an orbital shaking incubator. The seed culture was then used to inoculate 0.03% FoamAway™ irradiated AOF ( 3 L of spherical glass containing 900 ml of BMGY medium (ThermoFisher) The glycerol was inoculated into a flask or a glass vessel fermenter (Sartorius). The growth phase was carried out for approximately 24 hours until all of the spores were consumed. The temperature was then reduced to 26°C. Mb expression was induced by adding 1% methanol every 12 hours. Dissolved oxygen was maintained throughout the fermentation. The pH was maintained above 20% during the growth phase and at pH 6 during the induction phase and pH 5 during the induction phase.

[0281] After 96 hours of methanol induction, cells were removed by centrifugation at 4,000 rpm at 4°C. The remaining cells were filtered through a nitrocellulose filter (Millip) with a pore size of 0.45 μm. The cell-free supernatant was stored frozen at -20°C. When required for assay, the cell-free supernatant was diluted with a polyethylene glycol monolayer with a molecular weight cutoff of 10 kD. Thermosulfone membrane (Thermo Scientific Pierce Protein Disposable ultrafiltration centrifuges equipped with centrifuge concentrators are used. It was further concentrated by

[0282] The presence of recombinant DNA in the cell-free supernatant was confirmed by PCR using a sequence complementary to the sequence encoding the signal peptide. Tested by PCR using targeted oligonucleotides (Sigma) The DNA fragment amplified from the genomic DNA of the PAL-02-02 strain (Mammoth Mb) was Concentrated cell-free supernatant containing mammoth or bovine Mb was used as a positive control. The PCR reaction was carried out in standard buffer (New Eng) according to the manufacturer's instructions. OneTaq® Quick-Load ( PCR products were purified using ethidium bromide-containing 2x master mix. Visualization was performed on a 50% agarose-TAE gel after running at 60V for 60 minutes. -Load® Purple 1kb Plus DNA Ladder (Ne The size of the amplified fragments was controlled using a PCR primer set (England Biolabs).

[0283] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) For protein electrophoresis, 20 μl of cell-free supernatant was added to an equal volume of 2× protein load. Buffer (100 mM Tris (pH 6.8), 4 mM EDTA, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 4% β-mercaptoethanol The sample was incubated at 95°C for 5 minutes, and an appropriate amount was then transferred to a vertical mini-PROT 200 V for 15% polyacrylamide gels in an EAN gel apparatus (Bio-Rad) The gel was loaded with 1000 ml of ... d Protein Ladder) was purchased from Thermofisher. After the run, proteins in the gel were visualized with Coomassie Brilliant Blue G-250 (Bio-Rad) ) and stained.

[0284] mass spectrometry For MS analysis, SDS-PAGE gel bands corresponding to Mb were Coomassie stained. The sample was then rinsed with sterile water and subjected to VIB Proteomics. -2 until processing for MS analysis at cs Core (Gent, Belgium). The peptides were purified by lysis with 1 μg of trypsin (Promega) at 37°C overnight. The peptides were purified by digestion and stored at -20°C until LC-MS / MS analysis. The mixture was redissolved in a solvent and analyzed by a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The samples were injected for LC-MS / MS analysis using MaxQuant. The algorithm (version 2.0.1.0) was used, and the default search settings were peptide spectrum Pseudomonas mutans match (PSM), false discovery rate set at 1% at peptide and protein level All raw spectral data files were compiled from Mammoth Mb and Uniprot data. The following reference proteomes in the database were identified: Komagataella paphii (Ko magataella phaffii) (Database publication version of 2021_11, 5, 073 protein sequences), cattle (Bos taurus) (taxid 99 13, 2021_11 database release version, containing 37,513 protein sequences) , and wild boar (Sus scrofa) (taxid 9823, 2021_11 The theoretical protein sequences of the published version of the database (containing 49,792 protein sequences) We searched for it together.

[0285] Plant-based meat analogs Textured soy protein 25%, sunflower oil 15%, NaCl 1.5%, Prepare a plant-based burger by mixing 1% chill cellulose and 57.5% water. The final concentrations were 0.5% and 1%, which are the same as the myoglobin contents of white and red meat, respectively. recombinant myoglobin or commercially available myoglobin (purified from horse muscle, Sigma) The recombinant myoglobin preparation was obtained by lyophilization of the cell-free supernatant. For burgers containing b, add 13.8g of additional water per 100g of burger. A commercially available plant-based burger (Impossibl) containing soy leghemoglobin was added. e Food) was included for comparison in some assays.

[0286] Spectroscopy and color analysis Absorption spectra were recorded on a Synergy microplate reader (BioTek). Absorbance measurements for determining the rate of myoglobin autoxidation were performed using an Ultrospec II Color measurements were performed in quartz cuvettes using a spectrophotometer (Pharmacia). , with an 8mm viewing area size, D65 illuminant, and L (based on the CIELAB color space) * value, a * value and b * Portable Minisc with 10° standard observation device for registering values an EZ4500L 45° / 0°(Hunterlab,Murnau,Germa The color difference over time (ΔE) was calculated using the CIE 76 formula.

number

[0287] aroma analysis The aroma from plant-based meat substitutes containing different concentrations of recombinant Mb was analyzed using headspace solid-phase microarrays. Gas chromatography-mass spectrometry (GC-MS) with high-speed extraction (HS-SPME) The samples were analyzed either as is or after grinding. Headspace volatiles The material is divinylbenzene-carboxene-polydimethylsiloxane (DVB / CAR / P DMS) coated fiber and separated on a HP-1ms column. The areas under the peaks of the different aromatic compounds were then analyzed by mass spectrometry. One-way ANOVA (analysis of variance) was performed, and when statistically significant differences were found, HSD(Honestly Significant Difference)po of y Multivariate statistical analysis was performed using PCA (principal component analysis). It was.

[0288] Iron bioavailability and bioaccessibility Assays were performed using ProDigest (Gent, Belgium). o-2 cells (HTB-37, American Type Culture Colle 5 × 105 cells were plated in 12 wells coated with 0.1% gelatin. Cells were seeded in complete medium (20% heat-inactivated fetal bovine serum (FBS), 10 mM Dulbecco's modified Eagle's medium (DM) supplemented with HEPES and 1× antibiotic-antimycotic The cells were grown in EM for 14 days with 3 medium changes per week. 24 hours before stimulation, the cells were incubated at 10 1 mM HEPES, 2 mM L-glutamine, 1x antibiotic-antimycotic, 11 μM hydrazine Isocortisone, 0.87 μM insulin, 0.02 μM sodium selenite, 0. 0.5 μM 3,3',5-Triiodo-L-thyronine sodium salt, 20 μg / L epidermis Wash once with growth factor-supplemented minimum essential medium (MEM) and incubate in this medium for an additional 24 hours. The cells were then incubated with three concentrations (0.5, 1, and 2 mg / mL) of bovine serum albumin. Supplemented MEM with Mb (Tebu-bio NV) or as a negative control After 24 hours of incubation at 37°C, The cells were washed twice with ice-cold PBS and incubated with CelLytic™ (Sigma Aldrich). The human ferritin levels were measured using human ferritin E according to the manufacturer's instructions. The results were determined using a LISA kit (ThermoScientific). Pierce™ BCA Protein A according to the instructions for the cloning procedure. The protein was analyzed using a PCR assay kit (ThermoFisher Scientific). Protein concentrations were determined. All assays were performed in triplicate.

[0289] In vitro toxicity assay The assay was performed using ProDigest (Gent, Belgium). O-2 cells were cultured for ferritin assay and coated with 0.1% gelatin. On the day of the toxicity test, cells were seeded into 24-well plates containing 0.5, 1.0, or were incubated with 2.0 mg / ml of recombinant mammoth or bovine Mb. The activity assay was performed 24 hours after incubation with Mb at 37°C. To evaluate the possible toxicity induced by the product on O-2 cells, lactate decomposition was performed. LDH cytotoxicity assay (Merck Life Science BV) .) was performed on the supernatant according to the manufacturer's instructions. All assays were performed in triplicate. Dunnett's multiple comparisons were performed to assess differences between the total medium control (CM) and the products. Ordinary one-way ANOVA with a variance test was performed for each time point separately. * ) is a commercial This represents a statistically significant difference between the product and the * )=p<0.05, ( ** )=p<0.01 , ( *** )=p<0.001, and ( **** )=p<0.0001. All statistics are GraphPad Prism for Windows version 9.1.2 (GraphPad S This was performed using the software (San Diego, CA, USA).

[0290] Bacterial reverse mutation assay (AMES assay) Genotoxicity assays were performed according to OECD guidelines for chemical assays no. 471. AM ES FT Mutagenicity Test Kit(Moltox,Trino (va) was used according to the manufacturer's instructions. All assays were performed in triplicate. The average revertant count for each treated strain was calculated based on the data provided by the manufacturer. Mutation rates were observed with the test compound and vehicle alone. The ratio was calculated as the ratio between the number of revertants detected.

[0291] Example 2.2. Structural Features of Myoglobin Myoglobin (Mb) is a relatively small globular protein of about 17 kD, found in the heart and Found in skeletal muscle. It has a single heme group capable of reversible oxygen binding ( Figure 1), myoglobin can transport oxygen from the cell surface to the mitochondria. Mb is also the main pigment responsible for the color of meat. In robin, the iron ion of the heme group is attached to the so-called "proximal" histidine ( It is coordinated by four nitrogen atoms of the porphyrin ring (His94) and one oxygen molecule. This is due to the fact that the oxygen molecule in the heme-binding pocket of Mb is bound to another histidine, that is, it is further stabilized by hydrogen bonding with the "distal" histidine (His65). In this form, Mb typically has a bright red color. In the presence of ATP, Mb turns dark red (deoxymyoglobin). The heme iron is converted to ferrous oxide (Fe( When oxidized from the ferric oxide (Fe(II)) state to the ferric oxide (Fe(III)) state, it is unable to bind oxygen. Myoglobin does not react with the serotonin, and Mb shows a brown color (metmyoglobin), as seen in cooked meat. Heme iron oxidation also reduces the affinity of Mb for heme, leading to increased heme loss and subsequent This leads to protein denaturation.

[0292] The amino acid sequence of Mb has been highly conserved during evolution, with relatively little variation between species. In proboscideans, Mb has an atypical phenylalanine at position 30 (Fig. 2). It represents nin (Phe30). Surprisingly, the steppe mammoth (Mammuthus The Mb sequence from the genus Pseudomonas trongotherii dates to 1.2–1.0 Ma. The Adycha specimens were generated by us and are also included in FIG. 2.

[0293] In addition to the characteristic Phe30 substitution (Fig. 2), the inventors identified the steppe mammoth-derived Mb However, the presence of a histidine residue (His92) instead of glutamine at position 92 results in For example, Mb from Asian elephants (Z at pH 6.5) Mb =2.11) (Net surface charge at pH 6.5 (Z Mb )=2.67) (Figure 2, 3) This is a remnant of the adaptation of Mb to deep-sea diving in cetaceans, where positive The increase in surface charge reduces the attractive interaction between Mb molecules at the contact distance, increasing Mb stability. This leads to an increase in the number of spleens and the prevention of their aggregation. Mb derived from the rhesus exhibits several particularly attractive features. The authors produced and characterized recombinant Mb from the steppe mammoth, next to other extant species. Evaluate.

[0294] Example 2.3. Myogation in Pichia pastoris Extracellular production of Robin Encoding Mb from steppe mammoth, pig, chicken, cow, pig, and tuna The sequence was analyzed by methanol induction of Pichia pastoris. downstream of the AOX1 promoter and AOX1 release factor, a histidine prototrophic selectable marker and AOX1 gene, the so-called 3' fragment of which is cloned upstream. To facilitate recombinant protein purification, The authors targeted the nascent Mb protein to the secretory pathway for export outside the cell. To achieve this, the sequence encoding the signal peptide was inserted in frame with the myoglobin coding sequence. These constructs were used to generate histidine auxotrophic Pichia pastoris. Pichia pastoris cells were transformed and the transformants were isolated using histidine-containing nucleotides. For each construct, 10 clones were selected for their ability to grow in the absence of ribosomal DNA. Transformants up to 1000 were cultured in microplates containing extracellular myoglobin after methanol induction. The best producing clones were further tested in flasks. Methanol derivatization is particularly limited as considered for samples containing heme proteins. After 10-fold concentration by ultrafiltration, this led to a cell-free supernatant that exhibited a dark red color (Figure 4A). The presence of a methanol-induced protein with a predicted molecular weight of 100 kD was also confirmed by protein electrophoresis. This was also confirmed after centrifugation and staining with Coomassie blue (Fig. 4B). In the cell-free supernatant, the total protein accounted for 75-82% of the total protein. The total yield varied from 0.420 mg to 1.93 g per liter of supernatant.

[0295] Because myoglobin is produced extracellularly, yeast cells must be lysed during the purification process. These cells were isolated from the fermentation broth by centrifugation followed by microfiltration (0.45 μm pores). Therefore, the final product does not contain any recombinant genetic material. To verify this, we performed a small amount of DNA (1 pg) , a short fragment of the recombinant gene (130 bp) corresponding to the sequence encoding the signal peptide A PCR test was designed based on the amplification of the concentrated cell-free Neither recombinant DNA was detected in the supernatant (Fig. 5).

[0296] To confirm the identity of the recombinant Mb, and that the signal peptide was correctly processed, To confirm that the protein was removed during secretion, we performed a quantification assay coupled to mass spectrometry. The mammoth was identified by liquid chromatography-mass spectrometry (LC-MS / MS) shotgun measurements. The Mb gel bands were analyzed. We found that the complete protein sequence was covered by 90.3% of the original sequence. The peptide was recovered, and complete removal of the signal peptide and the first methionine was observed, as expected. This was observed (Figure 6).

[0297] Example 2.4. Color and Color Stability of Recombinant Myoglobin As discussed above, Mb plays a central role in meat color. In parallel, we therefore investigated the performance of concentrated cell-free supernatants in the ultraviolet and visible ranges. The absorbance of the myoglobin was measured and compared with that of commercially available myoglobin purified from horse muscle. In all cases, the characteristic absorbance peak (Soret band) of the presence of heme was observed. The Soret band was observed in cattle and horses (Tang et al. 2004). b was detected at 410 nm, whereas mammoth Mb was detected at 415 nm. Metmyoglobin, deoxymyoglobin, and oxmyoglobin were slightly shifted to red (Fig. 7A). Based on the ratio of absorbance at each representative wavelength maximum for myoglobin (Tan 2004), we have demonstrated that recombinant myogins in concentrated cell-free supernatants Robin was found mainly in the reduced form, while mammoth Mb was found in 47.3% of the oxime. Myoglobin and 10.0% deoxymyoglobin, and 48.3% for bovine Mb. oxymyoglobin and 9.9% deoxymyoglobin. The Soret peak of guinea pig Mb is at a slightly higher wavelength than that of bovine and equine Mb. This was consistently observed in elephant Mb (415 nm vs. 410 nm, respectively). This is consistent with previous observations on the Mb self-report (Tada et al. 1998). To evaluate the autoxidation behavior, we performed an in vitro autoxidation experiment at pH 5.6 and 25°C for 24 hours. During the incubation, the absorption spectrum was recorded every 2 hours (Fig. 7B). They found that the peak values ​​for oxymyoglobin, which carries Fe(II), were 580 nm and Absorbance at 505 nm (peak value for metmyoglobin, carrying Fe(III)) At the start of incubation, we determined that this ratio was The results showed that the concentrations of guanosine and bovine Mb were significantly higher than those of commercially available Mb purified from horse muscle. We observed that a larger fraction of the recombinant Mb was present in the reduced form (Fig. 7B, inset). This difference from horse Mb was maintained over time at acidic pH (Fig. 7B, insert). Over the course of 24 hours, the absorbance ratio increased to 26.0% for mammoth Mb and 26.0% for For bovine Mb, it was reduced by 38.3%, the former showing greater resistance to autooxidation He suggested that.

[0298] Meat color is influenced by several parameters, including Mb concentration, moisture content, and fat content. Conventional meat, typically purchased raw, is enriched with oxymyoglobin. The red color given to the product plays a key role in the purchase decision of the buyer. Therefore, the effect of recombinant Mb on meat, which is similar in color, was tested. Addition of thrombus Mb to plant-based burgers (Figure 8A-A') resulted in a decrease in brightness (L * -value) , reddish (a * -value) and yellowness (b * -value) increased (Figure 8B).

[0299] Next, we tested various amounts of microorganisms stored in a refrigerator and exposed to constant light for 5 days. The color stability of meat analogs containing soybean heme protein was evaluated. A commercially available plant-based burger prepared without Mb was included for comparison. A significant color change was observed for the burgers, whereas the addition of Mb resulted in a greater color stability. This was related to the quality of the results (Figure 9A-B).

[0300] Example 2.5. Aroma Analysis Besides its role in meat color, myoglobin is generally responsible for the "bloody" appearance of raw meat. During meat cooking, the formation of aromas is essentially Typically, it is produced by lipid oxidation and the Maillard reaction, which occurs when amino groups in proteins are converted to fatty acids. and carbonyl groups from the reaction products of reduced sugars and / or lipid oxidation. Iron can affect these reactions and / or their kinetics (van B a et al. 2012). As far as the inventors know, there are no studies on aroma formation in meat. There are no conclusive data in the literature on the effect of myoglobin on Using such a laboratory-made burger, the inventors performed a gas chromatograph coupled to mass spectrometry. Volatiles from raw and cooked plant-based burgers containing recombinant Mb were analyzed by The mixture was analyzed.

[0301] The inventors have demonstrated that the addition of Mb to plant-based burgers increases the protein content both raw and after grilling. We also found that the number of volatile compounds increased significantly (Figure 10A). The number of volatiles produced was significantly higher in a commercial plant-based bar containing soybean leghemoglobin. (Figure 10A), i.e., the Impossible Burger. In both cases, the results were high. Principal component analysis (PCA) was used for the CG-MS data. On the other hand, the inventors have developed a method for producing a lab-based burger containing soybean leghemoglobin. On the one hand, there is a clear difference between commercially available plant-based burgers and, on the other hand, between raw and roasted products. Separation was observed (Figure 10B).

[0302] By analyzing the volatile compounds from grilled laboratory-made plant-based burgers, we found: The addition of Mb was associated with the presence of oxidized lipids, lipid oxidation products, and pyrazines. In particular, the presence of steppe mammoth or bovine Mb may contribute to the "roasted" taste of grilled meat. Feeding oxidized meat led to increased levels of known compounds (van Ba ​​et al. (2012) (Fig. 12). The addition of mammoth Mb was significantly greater than that observed with bovine Mb. This is consistent with the theory that the amount of these compounds is higher than that of the other compounds. When used as an ingredient in plant-based meat analogues without This suggests that smaller amounts of mammoth Mb can be added to obtain the same aroma characteristics. In addition, mammoth Mb and cow Mb may provide different sensations.

[0303] Example 2.6. Nutritional Analysis To evaluate the bioavailability of iron derived from Mb, we investigated the iron uptake in the human intestinal tract. We then moved to the Caco-2 epithelial-like intestinal cell line, which is commonly used to study inflammatory bowel disease. used intracellular ferric iron as a readout of iron uptake by living Caco-2 cell monolayers. The formation of iron was measured and therefore an index of iron bioavailability (Glahn et al. We found that increasing the dose of Mb resulted in increased iron uptake. We observed that heme iron transported by Mb was transferred to human intestinal cells (Fig. 12). It has been shown that it is biologically feasible.

[0304] Example 2.7. Safety Test cytotoxicity To assess the potential toxicity of recombinant Mb, differentiated Caco-2 cells were cultured in mammoth or were treated with bovine Mycobacterium brevis, purified bovine Mycobacterium brevis, or complete medium (CM) as a control. To test for potential cytotoxic effects, we dehydrated the culture supernatant with lactate. The levels of the enzyme LDH were examined. LDH is the enzyme that converts NADH to NAD+. A redox reaction present in all cells that catalyzes the interconversion of pyruvate to lactate together with Upon cell membrane damage following apoptosis or necrosis, LDH is released into the supernatant. The concentration was determined by a colorimetric assay based on the reduction of NAD+ to NADH by LDH. (Decker and Lohmann Matthes 1988). We found no interaction with mammoth Mb at any of the concentrations tested. No increase in cytotoxicity was observed upon incubation (Figure 13). In contrast, bovine Mb showed significantly increased toxicity compared to medium alone (Fig. 13). At these concentrations, mammoth Mb therefore appears to have a better safety profile than bovine Mb. be.

[0305] Genotoxicity To determine the mutagenic potential of recombinant myoglobin, we used bacterial counter-injection Natural Mutagenesis Test (AMES Test) and Salmonella Typhimurium Four histidine-requiring strains of T. typhimurium (TA98, TA100, TA 1535, TA1537) and one of Escherichia coli A tryptophan-requiring strain (WP2 uvrA) was used ( Ames et al. 1975 ) Bacteria were exposed to mammoth or bovine myoglobin at 0.8, 2.5, 8.0, 25.0, and 80. At levels of 0 or 250 μg / ml, with or without exogenous metabolic activation (S9 mix) The positive control substance showed no reversion in both the absence and presence of S9 (Table 1). The expected increase in the number of mutants occurred, confirming the sensitivity of the test and the activity of the S9 mix. The present invention does not show any efficacy for bovine or mammoth Mb at any of the concentrations tested. No significant mutagenic activity was observed.

[0306] [Table 4]

Claims

1. Animal myoglobin from mammoth, pig, sheep, cow, chicken, or tuna; or A meat substitute or food ingredient containing animal myoglobin derived therefrom.

2. The myoglobin is from a steppe mammoth, a woolly mammoth, or any of these.

2. The meat substitute or food ingredient of claim 1, derived from

3. The animal myoglobin has the following amino acid sequence: a) A sequence that contains at least 70% sequence identity with SEQ ID NO: 3 and contains the following amino acid combinations: Se, that is F at position 30, and / or Q at position 65, and / or H at position 92, and / or H at position 94, and / or F in position 30 and Q in position 65, and / or F at position 30 and H at position 92, and / or F at position 30 and H at position 94, and / or Q at position 65 and H at position 92, and / or Q at position 65 and H at position 94, and / or H at position 92 and H at position 94, and / or F at position 30 and Q at position 65 and H at position 92, and / or F at position 30 and Q at position 65 and H at position 94, and / or F at position 30 and H at position 92 and H at position 94, and / or Q at position 65 and H at position 92 and H at position 94, and / or F at position 30, Q at position 65, H at position 92, and H at position 94 an amino acid sequence having at least one of: b) at least 70% sequence identity with SEQ ID NO: 2 or 3, Q or H at position 65 and H at position 94, and optionally at the following positions in SEQ ID NO: 2 or 3: The following amino acids: E at position 9, K at position 13, T at position 14, P at position 27, L at position 31, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88, I at position 102, E at position 123, and / or E at position 143 an amino acid sequence having at least one of I: c) at least 70% sequence identity with SEQ ID NO: 1, Q or H at position 65, and 94 H, and optionally the following amino acids at the following positions in SEQ ID NO: 1: E at position 9, K at position 13, T at position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q, I at position 102, and / or E at position 123. amino acid sequence, d) at least 70% identity to SEQ ID NO: 4, 5 or 6, Q or H at position 65 and H at position 94, and optionally the following in SEQ ID NO: 4, 5, or 6: The following amino acids are present: N at position 13, Q at position 27, I at position 31, N at position 7, A at position 128, S at position 133, A at position 145, and / or position 150 an amino acid sequence having at least one of the following Ls: e) at least 70% identity to SEQ ID NO: 7, Q or H at position 65, and Q or H at position 94 and optionally the following amino acids at the following positions in SEQ ID NO: 7: , Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, M, H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 3, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, T at position 93, V at position 111, and I at position 117, A at position 118, A at position 121, S at position 128, and S at position 133 at least one of K at position 145, S at position 146, and / or F at position 150; an amino acid sequence, or f) at least 70% identity to SEQ ID NO: 8, Q or H at position 65, and Q or H at position 94 Amino acid sequence containing H 3. The meat substitute or food ingredient of claim 1 or 2, represented by one of:

4. Leghemoglobin produced by bacteria living in symbiosis in root nodules of soybean plants The heme-containing protein according to any one of claims 1 to 3, which does not contain heme and / or is the only heme-containing protein. The meat substitute according to any one of claims 1 to 3, comprising myoglobin as defined in the preceding paragraph. or food ingredients.

5. The meat substitute has the characteristics, form, structure, composition, flavor, texture, color, aroma, appearance and / or texture of meat. The meat substitute according to any one of claims 1 to 4, wherein the meat substitute mimics the nutritional value of the meat.

6. 10. The food ingredient of claim 9, wherein the food ingredient mimics the composition, flavor, color, aroma, and / or nutritional value of meat.

5. The food ingredient according to any one of 1 to 4.

7. A genetic vector comprising a nucleic acid encoding a myoglobin as defined in any one of claims 1 to 4. Child constructs.

8. The nucleic acid (a) an amino acid sequence at least equal to that defined in claim 3 a), b), c), or d); and the amino acid sequence is represented by an amino acid sequence containing a sequence having at least 75% sequence identity or sequence similarity. a nucleotide sequence encoding a polypeptide to be (b) the nucleotide sequences of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, and 16; a nucleotide sequence having at least 60% sequence identity; (c) the sequence of the nucleotide sequence is, due to the degeneracy of the genetic code, the nucleotide sequence of (b) the nucleotide sequence is different from the sequence of The genetic construct of claim 7, selected from the group consisting of:

9. The genetic construct of claim 7 or 8, further comprising a promoter.

10. A signal peptide, preferably a signal peptide that promotes secretion of the expressed myoglobin. The genetic construct according to any one of claims 7 to 9, further comprising a

11. The nucleotide sequence is represented by SEQ ID NO: 19, 20, 21, 22, 23 or 24. The gene construct according to any one of claims 6 to 10.

12. The genetic construct is selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, and 30. The genetic construct of claim 11, which is represented by:

13. A vector comprising a genetic construct as defined in any one of claims 7 to 12. Kutar.

14. A host comprising a genetic construct as defined in any one of claims 7 to 12. Principal cell.

15. 15. The host cell of claim 14, which is a prokaryote or a eukaryote.

16. Cultivating the host cell according to claim 14 or 15 in a suitable medium, and optionally The host cell and / or myoglobin as defined in any one of claims 1 to 3 and recovering said myoglobin.

17. The method of claim 16, wherein the produced myoglobin does not contain a signal peptide. Law.

18. The recovered myoglobin is purified, preferably substantially purified.

18. The method according to claim 16 or 17.

19. Any one of claims 16 to 18, wherein the host cell produces the myoglobin extracellularly.

10. The method according to claim 1.

20. The myoglobin obtainable from the method according to any one of claims 16 to 19 is substituted 10. The method of claim 1, further comprising incorporating the compound into meat or food ingredients. A method for producing the meat substitute or food ingredient.

21. A protein, wherein the protein can be represented by a sequence comprising SEQ ID NO:3.

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