CN116801725A - Meat substitute comprising animal myoglobin - Google Patents

Abstract

Described herein are meat substitutes or food ingredients comprising animal myoglobin, genetic constructs comprising nucleic acids encoding the proteins, host cells comprising the genetic constructs, and methods for producing the myoglobin or the Methods for Meat Substitutes.

Description

Meat substitutes containing animal myoglobin

Technical Field

Aspects and embodiments described herein relate to the fields of meat substitutes comprising myoglobin, gene constructs comprising nucleic acids encoding said proteins, host cells comprising said gene constructs, and methods for producing said myoglobin or said meat substitutes.

Background Art

Several meat substitutes have been developed and commercialized. The flavor, color, texture, and nutritional quality of such products are not always optimal because they do not properly mimic "meat." Additionally, they may not have the same nutritional qualities as meat.

Therefore, there is still a need for new inventions that do not have all the disadvantages of existing food products intended to replace meat.

Summary of the invention

[0013] Accordingly, aspects and embodiments of the present invention as described herein address at least some of the problems and needs as discussed herein.

Meat substitutes

In a first aspect, a meat substitute or food ingredient is provided comprising animal myoglobin, preferably mammalian myoglobin (more preferably from mammoth, pig, sheep or cattle, more preferably wherein the mammoth is a woolly mammoth (Mammuthus primigenius) or a steppe mammoth (Mammuthus trogontherii), most preferably wherein the mammoth is a steppe mammoth), or avian myoglobin (preferably from chicken), or fish myoglobin (preferably from tuna), or a myoglobin derived therefrom.

In an embodiment, the meat substitute or food ingredient comprises animal myoglobin from or derived from mammoth, pig, sheep, cow, chicken or tuna, preferably wherein the mammoth is a woolly mammoth (Mammuthus thomsoniae) or a steppe mammoth (Mammuthus thomsoniae), more preferably wherein the mammoth is a steppe mammoth.

When the amino acid sequence of the animal myoglobin is derived from one of mammoth (preferably woolly mammoth or steppe mammoth), pig, sheep, cattle, chicken or tuna by the addition, deletion and/or substitution of at least one amino acid, it can be said that the animal myoglobin is derived from mammoth (preferably woolly mammoth or steppe mammoth, more preferably derived from steppe mammoth), pig, sheep, cattle, chicken or tuna. The present invention also contemplates the addition, deletion and/or substitution of two, three, four, five, six, seven, eight, nine or ten amino acids. Examples of myoglobin amino acid sequences from woolly mammoth, steppe mammoth, pig, sheep, cattle, chicken or tuna have been disclosed later by the given SEQ ID NO. Such animal myoglobin derived from mammoth, woolly mammoth, steppe mammoth, pig, sheep, cattle, chicken or tuna myoglobin can also exert at least detectable levels of animal myoglobin activity, as explained later herein. In the context of the present application, unless explicitly stated otherwise, a mammoth may be any species in the genus Mammoth, such as Woolly Mammoth (common mammoth) or Steppe Mammoth (steppe mammoth).

In embodiments, the myoglobin in the meat substitute or food ingredient is from or derived from mammoth.

In preferred embodiments, the myoglobin in the meat substitute or food ingredient is from or derived from woolly mammoth.

In preferred embodiments, the myoglobin in the meat substitute or food ingredient is from or derived from steppe mammoth.

In an embodiment, the myoglobin in the meat substitute or food ingredient is represented by one of the following amino acid sequences:

-a) has at least 70% sequence identity to SEQ ID NO: 3 and at least one of the following amino acid combinations:

o F at position 30, and/or

o Q at position 65, and/or

o H at position 92, and/or

o H at position 94, and/or

o F at position 30 and Q at position 65, and/or

o F at position 30 and H at position 92, and/or

o F at position 30 and H at position 94, and/or

o Q at position 65 and H at position 92, and/or

o Q at position 65 and H at position 94, and/or

o H at position 92 and H at position 94, and/or

o F at position 30, Q at position 65, and H at position 92, and/or

o F at position 30, Q at position 65, and H at position 94, and/or

o F at position 30 and H at position 92 and H at position 94, and/or

o Q at position 65 and H at position 92 and H at position 94, and/or

o F at position 30 and Q at position 65 and H at position 92 and H at position 94;

-b) having at least 70% sequence identity to SEQ ID NO: 2 or 3, comprising Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 2 or 3: 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, I at position 102, E at position 123 and/or I at position 143;

-c) having at least 70% sequence identity to SEQ ID NO: 1, comprising Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within 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, I at position 102 and/or E at position 123;

-d) having at least 70% identity to SEQ ID NO: 4, 5 or 6, comprising Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 4, 5 or 6: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150;

-e) has at least 70% identity to SEQ ID NO:7, comprises Q or H at position 65 and H at position 94, and optionally has at least one of the following amino acids at the following positions within 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 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 111, I at position 116, A at position 117, E at position 118, A at position 121, S at position 128, K at position 133, S at position 145, and/or F at position 150;

-f) having at least 70% identity to SEQ ID NO: 8, comprising Q or H at position 65 and H at position 94.

SEQ ID NO: 1 represents a part of the amino acid sequence of woolly mammoth myoglobin, SEQ ID NO: 2 represents the amino acid sequence of woolly mammoth; 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, SEQ ID NO: 5 represents the amino acid sequence of bovine (cow) myoglobin, SEQ ID NO: 6 represents the amino acid sequence of porcine myoglobin, SEQ ID NO: 7 represents the amino acid sequence of chicken myoglobin, and SEQ ID NO: 8 represents the amino acid sequence of tuna myoglobin. Obviously, SEQ ID NO: 1 representing a part of the amino acid sequence (i.e., the partial sequence) of woolly mammoth myoglobin is contained in SEQ ID NO: 2 representing the (complete) amino acid sequence of woolly mammoth myoglobin.

SEQ ID NO: 9 is a nucleic acid sequence encoding SEQ ID NO: 1. SEQ ID NO: 10 is a nucleic acid sequence encoding SEQ ID NO: 2, and SEQ ID NO: 11 is a nucleic acid sequence encoding SEQ ID NO: 3. SEQ ID NO: 12 is a nucleic acid sequence encoding SEQ ID NO: 4. SEQ ID NO: 13 is a nucleic acid sequence encoding SEQ ID NO: 5. SEQ ID NO: 14 is a nucleic acid sequence encoding SEQ ID NO: 6. SEQ ID NO: 15 is a nucleic acid sequence encoding SEQ ID NO: 7. SEQ ID NO: 16 is a nucleic acid sequence encoding SEQ ID NO: 8.

Meat substitutes are by definition not meat, not natural meat, and not real meat. Meat substitutes can be considered as non-natural foods or edible products. This means that in the context of the present invention, meat substitutes are not meat from or derived from pigs, sheep, cows, chickens or tuna (or from or derived from mammoths if such animals still existed, which is not the case). Meat substitutes are synonyms for meat replicas or meat analog products or meat-like products.

Meat substitutes may have the same appearance (e.g., patterns, text), form, structure, composition (e.g., similar fat, protein and/or heme iron content), texture, color, flavor, aroma and/or appearance as meat.

Alternatively, it may have an appearance, form, structure, texture, color, flavor, aroma and/or appearance that is different from meat.

Meat substitutes can be formulated as liquid, solid, or semi-solid foods.

Suitable examples of liquid foods include soups.

Suitable examples of solid foods include cell-based meat or cultured meat or plant-based meat. Such plant-based meat may contain vegetable protein, such as soy protein. In addition, they contain animal myoglobin as defined herein. In an embodiment, such animal myoglobin is the only source of heme-containing protein.

Suitable examples of snacks include protein bars or protein shakes.

A food ingredient may be any edible ingredient that can be added to an edible product to obtain a unique edible product. Alternatively, the food ingredient may be consumed as is. The term "food ingredient" may be replaced by the term "food supplement".

Suitable examples of food ingredients containing the myoglobin identified above may be solid (e.g. powder, spice mix) or liquid (e.g. extract) or semi-liquid or semi-solid (e.g. paste or gel or juice or soup or cream) food ingredients that can be sold to customers. Customers can add these food ingredients to their own food to supplement their food with the myoglobin contained herein, thereby creating their own meat substitute.

Suitable examples of solid food supplements include tablets or pills or powders containing the above-mentioned animal myoglobin. The tablets or pills or powders may be formulated with other compounds such as vitamins or minerals.

The inventors have found that the "meat experience" can be mimicked in the meat substitute of the present invention using animal myoglobin as defined herein.

In embodiments, it is contemplated that the meat substitutes of the present invention mimic the appearance (e.g., patterns, text), form, structure, composition (e.g., similar fat, protein, and/or heme iron content), flavor, texture, color, aroma, appearance, and/or nutritional value of meat. This is typically the case when the meat substitute is cell-based meat or cultured meat or plant-based meat. This is primarily due to the presence of animal myoglobin as described herein. Meat substitutes may not have all of the disadvantages of meat. For example, meat substitutes are healthier than meat. In addition, the environmental impact (long-term direct/indirect impact on climate change) of meat substitutes is expected to be smaller than the known impact of meat.

In embodiments, it is contemplated that the meat substitutes of the present invention mimic the appearance (e.g., patterns, text), form, structure, composition (e.g., similar fat, protein, and/or heme iron content), flavor, texture, color, aroma, and/or appearance of meat. This is typically the case when the meat substitute is cell-based meat or cultured meat or plant-based meat. This is primarily due to the presence of animal myoglobin as described herein. Meat substitutes may not have all of the disadvantages of meat. For example, meat substitutes are healthier than meat. In addition, the environmental impact (long-term direct/indirect impact on climate change) of meat substitutes is expected to be smaller than the known impact of meat.

In embodiments, it is contemplated that the meat substitutes of the present invention mimic the composition (e.g., similar fat, protein, and/or heme iron content), flavor, color, and/or aroma of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

In embodiments, the meat substitutes of the present invention are expected to mimic the flavor and/or aroma of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

In embodiments, the meat substitutes of the present invention are expected to mimic the flavor of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

In embodiments, the meat substitutes of the present invention are expected to mimic the aroma of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

The inventors have further discovered that the "meat experience" can be mimicked using the food ingredients described herein.

In embodiments, it is contemplated that the food ingredients of the present invention mimic the composition (e.g., similar fat, protein, and/or heme iron content), flavor, color, aroma, and/or nutritional value of meat without all of its drawbacks, the flavor of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

In embodiments, it is contemplated that the food ingredients of the present invention mimic the composition (e.g., similar fat, protein, and/or heme iron content), flavor, color, and/or aroma of meat without all of its drawbacks, the flavor of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

In embodiments, the food ingredients of the present invention are expected to mimic the flavor, color and/or aroma of meat without all of its drawbacks, the flavor of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

In embodiments, it is contemplated that the food ingredients of the present invention mimic the composition (e.g., similar fat, protein, and/or heme iron content), flavor, and/or aroma of meat without all of its drawbacks, the flavor of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

In embodiments, it is contemplated that the food ingredients of the present invention mimic the composition (e.g., similar fat, protein, and/or heme iron content) and flavor of meat without all of its drawbacks, and the flavor of meat without all of its drawbacks. This is primarily due to the presence of animal myoglobin as described herein.

In embodiments, it is contemplated that the food ingredients of the present invention mimic the composition (e.g., similar fat, protein, and/or heme iron content) of meat, the aroma without all of its drawbacks, the flavor of meat without all of its drawbacks. This is primarily due to the presence of the animal myoglobin described herein.

In the context of the present invention, the meat experience can be simulated when the meat substitute and/or food ingredient produces aroma and/or flavor compounds that are recognizable by humans and the characteristics of certain odorants and/or flavor compounds released when meat is cooked. For example, the heme iron present in myoglobin may catalyze the formation of some odorants. Without being bound by this theory, the formation may be due to lipid oxidation and/or Maillard reactions. This situation can be simulated with the myoglobin of the meat substitute and/or food ingredient of the present invention.

In an embodiment, the meat substitute or food ingredient of the present invention, preferably the raw meat substitute of the present invention, is expected to imitate the bloody and/or metallic taste of meat, preferably raw meat. Preferably, the meat substitute or food ingredient of the present invention is able to imitate the bloody and/or metallic taste of meat to a higher degree than other meat substitutes or food ingredients (i.e., meat substitutes or food ingredients not according to the present invention). In the context of the present invention, raw means not heat-treated, preferably uncooked and not roasted.

In an embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, is intended to imitate the grilled aroma of meat, in particular the aroma of grilled meat. Preferably, the meat substitute or food ingredient of the present invention is able to imitate the grilled aroma of meat to a higher degree than other meat substitutes or food ingredients (i.e., meat substitutes or food ingredients not according to the present invention).

In the context of the present invention, especially in the embodiments of this section, "other meat substitutes" or "meat substitutes not of the present invention" are preferably plant-based burgers containing recombinant soy leghemoglobin (LegH), more preferably the commercially available Impossible Burger.

In an embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, has an aroma characterized by a higher concentration of oxidized lipids, lipid oxidation products, pyrazines and/or pyrroles in the volatile compounds obtained from the (grilled) meat substitute or food ingredient relative to other meat substitutes or food ingredients (i.e. not according to the present invention). Higher preferably means at least 10%, 20%, 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%. Preferred pyrazines are methylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-6-methylpyrazine. The preferred pyrrole is pyrrole. The preferred lipid oxidation products are 2-methylbutanal and 3-methylbutanal.

In an embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, has an aroma characterized by a higher concentration of 2,5-dimethylpyrazine in the volatile compounds obtained from the (grilled) meat substitute or food ingredient relative to other meat substitutes or food ingredients (i.e. not according to the present invention). Higher preferably means at least 10%, 20%, 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%.

In an embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, has an aroma characterized by a higher concentration of 2-ethyl-6-methylpyrazine in the volatile compounds obtained from the (grilled) meat substitute or food ingredient relative to other meat substitutes or food ingredients (i.e. not according to the present invention). Higher preferably means at least 10%, 20%, 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%.

In an embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, has an aroma characterized by a higher concentration of pyrroles in the volatile compounds obtained from the (grilled) meat substitute or food ingredient relative to other meat substitutes or food ingredients (i.e. not according to the present invention). Higher preferably means at least 10%, 20%, 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%.

In an embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, has an aroma characterized by a higher concentration of 2-methylbutanal in the volatile compounds obtained from the (grilled) meat substitute or food ingredient relative to other meat substitutes or food ingredients (i.e. not according to the present invention). Higher preferably means at least 10%, 20%, 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%.

In an embodiment, the meat substitute or food ingredient of the present invention, preferably the grilled meat substitute of the present invention, has an aroma characterized by a higher concentration of 3-methylbutanal in the volatile compounds obtained from the (grilled) meat substitute or food ingredient relative to other meat substitutes or food ingredients (i.e. not according to the present invention). Higher preferably means at least 10%, 20%, 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%.

In Example 2.5, an aromatic analysis of a meat substitute according to the invention is provided. The aroma of meat and meat substitutes and the concentration of volatile compounds can be determined by the method described in this example, i.e. by gas chromatography coupled to mass spectrometry of volatile compounds.

In the context of the present invention, a meat substitute can mimic the meat experience when its color is similar to the color of meat. The color of meat is determined by the concentration of heme-containing proteins and/or the oxidation state in meat. Thus, myoglobin as defined herein will define the color of a meat substitute. The same applies to the food ingredients of the present invention.

In embodiments, the meat substitute or food ingredient of the present invention, preferably the raw meat substitute of the present invention, is expected to mimic the color of meat, preferably the color of raw meat. Without being bound by this theory, the color of the meat substitute is mainly due to the presence of animal myoglobin. Preferably, the meat substitute or food ingredient of the present invention is able to mimic the color of meat to a higher degree than other meat substitutes or food ingredients (i.e., meat substitutes or food ingredients not according to the present invention).

In a preferred embodiment, the color of the meat substitute or food ingredient of the present invention (preferably the raw meat substitute of the present invention) is preferably substantially stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days after storage at 4°C under constant light, more preferably under the conditions of Example 2.4 and measured by the technique of that example. Preferably, the color of the meat substitute or food ingredient of the present invention is stable for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days longer than the color of meat or other meat substitute or food ingredient (i.e. not according to the present invention), more preferably, wherein a change in ΔE from 0% to 10% is defined as stable (see further). Substantially stable for at least X days preferably means that the change in the ΔE of the meat substitute or food ingredient between day 0 and day X is between 0% and 0.5%, 1%, 1.5%, 2%, 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%, wherein the ΔE is determined as in Example 2.4. Most preferably, the ΔE of the meat substitute or food ingredient does not change by more than 10% after storage for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.

Example 2.4 provides a color stability analysis of meat substitutes according to the invention. The color and color stability of meat and meat substitutes can be determined by the method described in this example.

In the context of the present invention, a meat substitute can mimic the meat experience when its composition is similar to that of meat. A meat substitute can be considered to have a composition similar to that of meat when the same or similar components are present and optionally when they are present in the same or similar amounts as those present in meat. For example, a meat substitute may contain fat, protein, and/or heme iron content similar to that of meat.

These animal myoglobins, when formulated into meat substitutes or as food ingredients, are also expected to provide approximately the same amount of total available iron or heme iron as meat. In this context, "approximately" means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more. Typically, the amount of total bioavailable iron or heme iron in the meat substitutes of the present invention does not exceed 120% of the amount of total bioavailable iron or heme iron in real meat. This means that these animal myoglobins can be considered a unique alternative enhancer that can improve the iron status of the population.

It is also expected that these animal myoglobins provide an amount of total bioavailable iron or heme iron that is approximately higher than the amount of total bioavailable iron or heme iron provided in plant-based meat analog products. In this context, "higher" means at least 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% or more.

In a preferred definition, nutritional value refers to the amount of total available iron or heme iron. According to this preferred definition, "nutritional value of simulated meat" should be interpreted as "providing approximately the same amount of total available iron or heme iron as meat", as described above.

Example 2.6 provides a nutritional analysis of a meat substitute according to the invention, evaluating the bioavailability of iron.

In the context of the present invention, total iron is the total amount of bioavailable iron in a meat substitute. Heme iron is the portion of iron that is transported by proteins containing a heme domain. Heme iron is available for absorption by the human body. This protein is myoglobin as defined herein. The availability of iron can be assessed as described in Proulx A.K., et al. (2006)., J.Agric.Food.Chem. [Journal of Agricultural Food Chemistry], (54), 1518-1522) and/or the method in Example 2.6. In short, the ferritin concentration of the preparation is first normalized to the cellular protein concentration. Subsequently, the percentage of normalized ferritin is compared to the percentage of FeSO4 and expressed as a relative biological value (RBV).

In embodiments, the meat substitute does not comprise leghemoglobin produced by bacteria symbiotically in root nodules of soybean plants and/or comprises animal myoglobin as previously defined herein as the only heme-containing protein.

In embodiments, the animal myoglobin disclosed herein may be the only source of heme-containing proteins present in the meat substitute of the present invention. This means that the meat substitute of the present invention may contain other proteins different from the animal myoglobin disclosed herein. Examples of proteins that may be present include soy protein.

In the context of the present invention, the defined heme-containing protein may refer to all proteins or protein subunits that can covalently or non-covalently bind a heme moiety. Heme-containing polypeptides can transport or store oxygen. Some examples of heme-containing proteins include globin, hemoglobin, leghemoglobin.

In another embodiment, the meat substitute does not contain leghemoglobin produced by bacteria symbiotically in the root nodules of soy plants. In an embodiment, the meat substitute does not contain symbiotic hemoglobin. In an embodiment, the meat extract does not contain leghemoglobin. In an embodiment, the meat substitute does not contain proteins produced by bacteria symbiotically in the root nodules of soy plants.

In an embodiment, the meat substitute does not comprise leghemoglobin produced by bacteria symbiotically in root nodules of soybean plants and comprises animal myoglobin as previously defined herein as the only heme-containing protein.

In an embodiment, the meat substitute comprises an amount of animal myoglobin similar to the corresponding real meat. In an embodiment, the range of this amount is from 0.1 to 5% by weight of animal myoglobin. In an 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% by weight or more% by weight of animal myoglobin. The present invention also encompasses an amount of at least 10%, 20%, 30%, 40%, 50% of the final weight of the product. The skilled artisan is aware that this amount may vary depending on the meat substitute.

In one embodiment, the weight percentage of myoglobin in the meat substitute according to the present invention is 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%. .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%.

In one embodiment, the weight percentage of myoglobin in the meat substitute according to the present invention is 0.5% 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%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, or 0.6.

In an embodiment, the food ingredient comprises an amount of animal myoglobin, which may range from 0.1 to 100% by weight of animal myoglobin. In an embodiment, the amount is 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%, 100% of the final weight of the product. The skilled person will appreciate that the amount may vary depending on the food ingredient.

In an embodiment, the meat substitute comprises or is prepared by mixing: 10% to 40% of a protein source, 5% to 30% of a lipid source, 0.1% to 5% of NaCl, 0.1% to 5% of a fiber source, and 0.1% to 5% of myoglobin according to the present invention. In a more preferred embodiment, the meat substitute comprises or is prepared by mixing: 20% to 30% of a protein source, 10% to 20% of a lipid source, 0.5% to 2% of NaCl, 0.5% to 1.5% of a fiber source, and 0.1% to 3% of myoglobin according to the present invention.

Preferably, the protein source is texturized soy protein, pea protein isolate, mung bean protein, textured wheat protein, rice protein or potato protein. In this context, texturized soy protein is a defatted soy flour product, also known as texturized vegetable protein (TVP) or soy meat, which is clear to the skilled person.

Preferably, the lipid source is sunflower oil, coconut oil, canola oil or cocoa butter.

Preferably, the fiber source is methylcellulose or potato starch.

In a more preferred embodiment, the meat substitute comprises or is prepared by mixing: 20% to 30% textured soy protein, 10% to 20% sunflower oil, 0.5% to 2% NaCl, 0.5% to 1.5% methylcellulose, and 0.1% to 10% myoglobin according to the present invention. Even more preferably, according to a more preferred embodiment, the weight percentage of myoglobin in the meat substitute is 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%, 0.85%, 0.8%, 0.75%, 0.7%, 0.65%, 0.6%, 0.55% or 0.5%.

In an embodiment, a meat substitute comprises or is prepared by mixing 20% to 30% textured soy protein, 10% to 20% sunflower oil, 1% to 2% NaCl, 0.5% to 1.5% methylcellulose, 50% to 70% water and 0.2% to 0.8% myoglobin.

In a preferred embodiment, the meat substitute comprises or is prepared by mixing: 23% to 27% textured soy protein, 13% to 17% sunflower oil, 1.25% to 1.75% NaCl, 0.75% to 1.25% methylcellulose, 55% to 60% water and 0.45% to 0.55% myoglobin.

In an embodiment, a meat substitute comprises or is prepared by mixing: 25% textured soy protein, 15% sunflower oil, 1.5% NaCl, 1.0% methylcellulose, 57% water, and 0.5% myoglobin, wherein these weight percentages are rounded to two significant figures.

In an embodiment, a meat substitute comprises or is prepared by mixing: 15% to 35% textured soy protein, 10% to 20% sunflower oil, 0.5% to 2.5% NaCl, 0.5% to 1.5% methylcellulose, 50% to 70% water and 0.5% to 1.5% myoglobin.

In a preferred embodiment, the meat substitute comprises or is prepared by mixing: 22% to 27% textured soy protein, 13% to 17% sunflower oil, 1.25% to 1.75% NaCl, 0.75% to 1.25% methylcellulose, 50% to 60% water and 0.75% to 1.25% myoglobin.

In an embodiment, a meat substitute comprises or is prepared by mixing: 25% textured soy protein, 15% sunflower oil, 1.5% NaCl, 1.0% methylcellulose, 57% water, and 1.0% myoglobin, wherein these weight percentages are rounded to two significant figures.

In the above meat substitutes, % refers to percentage by weight. If the percentages by weight describing the composition of the meat substitute do not add up to 100%, it is assumed that water is added in an amount that reaches the corresponding percentage.

Example 1.4 provides an example of preparing a meat substitute according to the present invention.

In embodiments, the meat substitute contains an amount of bioavailable iron or heme iron similar to the corresponding real meat. In embodiments, the amount ranges from 0.3 to 20 mg bioavailable iron or heme iron per 100 g of meat substitute. In embodiments, the amount ranges from 0.1 mg Up to 16.5mg, 16.17mg, 15.84mg, 15.51mg, 15.18mg, 14.85mg, 14.52mg, 14.19mg, 13.86mg, 13.53mg, 13.2mg, 12.87mg, 12.54mg, 12.21mg, 11.88mg, 11.55mg, 11.22mg, 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, 7.92mg, 7.755mg, 7.59mg, 7.425mg, .26mg, 7.095mg, 6.93mg, 6.765mg, 6.6mg, 6.435mg, 6.27mg, 6.105mg, 5.94mg, 5.775mg, 5.61mg, 5.445mg, 5.28mg, 5.115mg, 4.95mg, 4.785mg, 4.62mg, 4.455mg, 4.29mg, 4.125mg, 3.96mg, 3.795mg, 3.63mg, 3.465mg, 3.3mg, 3.135mg, 2.97mg, 2.805mg, 2.64mg, 2.475mg, 2.31mg, 2.145mg, 1.98mg, 1.815mg or 1.65mg bioavailable iron or heme iron per 100g of meat substitute. In another embodiment, such amount is 0.3, 0.4, 0.5, 0.6, 0.7, 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 . 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, 13.5, 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, 18.5, 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 bioavailable iron or heme iron per 100 g meat substitute.

In embodiments, the amino acid sequence of an animal myoglobin identified previously herein comprises a sequence having 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 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%, 99% or 100% identity or similarity to a sequence identified previously herein, combined with at least one of the following amino acid combinations:

- 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 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 and Q at position 65 and H at position 92 and H at position 94.

In an embodiment, the amino acid sequence of an animal myoglobin identified previously herein comprises a sequence having 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 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%, 99% or 100% identity or similarity to a sequence identified previously herein in combination with Q or H at position 65 and H at position 94 for each identified sequence, preferably further combined with I at position 143.

In an embodiment, the amino acid sequence of an animal myoglobin identified previously herein comprises a sequence having 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 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%, 99% or 100% identity or similarity to a sequence identified previously herein in combination with W or H at position 65, Q at position 92, and H at position 94 for each identified sequence, preferably further combined with I at position 143.

In an embodiment, the amino acid sequence of an animal myoglobin identified previously herein comprises a sequence having 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 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%, 99% or 100% identity or similarity to a sequence identified previously herein in combination with Q or H at position 65, H at position 92, and H at position 94 for each identified sequence, preferably further combined with I at position 143.

In an embodiment, the amino acid sequence of an animal myoglobin identified previously herein comprises a sequence having 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 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%, 99% or 100% identity or similarity to a sequence identified previously herein in combination with H at position 92 for each identified sequence, preferably further combined with I at position 143.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and being combined with Q or H at position 65 and H at position 94, and optionally having at least one of 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, I at position 102 and/or E at position 123. In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or 3 in combination with Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions in SEQ ID NO: 2 or 3: 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, I at position 102, E at position 123 and/or I at position 143. Preferably, the amino acid sequence of the identified animal myoglobin comprises a sequence having 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 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 100% identity or similarity to SEQ ID NO: 1, 2 or 3 in combination with Q or H at position 65 and H at position 94. SEQ ID NO: 1 is a portion of myoglobin from mammoth. SEQ ID NO: 2 and 3 are myoglobin from mammoth. In an embodiment, the meat substitute comprises myoglobin comprising the amino acid sequence SEQ ID NO: 1. In an embodiment, the meat substitute comprises mammoth myoglobin represented by the amino acid sequence SEQ ID NO: 2 or 3. This myoglobin is expected to have attractive properties because this myoglobin is stable to oxidation, especially at low pH values. The color of the meat substitute of the present invention is expected to be stable because the mammoth myoglobin is resistant to oxidation. This anti-oxidative stability also provides additional health properties to the meat substitute of the present invention because it is less likely to be converted into carcinogenic products compared to real meat.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 in combination with Q or H at position 65, Q at position 92 and H at position 94, and optionally having at least one of the following amino acids at the following positions in SEQ ID NO: 2: 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, I at position 102 and/or E at position 123. Preferably, the amino acid sequence of the identified animal myoglobin comprises a sequence having 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 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 100% identity or similarity to SEQ ID NO: 2 in combination with Q or H at position 65, Q at position 92 and H at position 94. SEQ ID NO: 2 is myoglobin from woolly mammoth. In an embodiment, the meat substitute comprises woolly mammoth myoglobin represented by the amino acid sequence SEQ ID NO: 2. This myoglobin is expected to have attractive properties because it is stable to oxidation, especially at low pH values. The color of the meat substitute of the present invention is expected to be stable because the woolly mammoth myoglobin is resistant to oxidation. This anti-oxidative stability also provides additional health properties to the meat substitute of the present invention because it is less likely to be converted into carcinogenic products than real meat.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3 in combination with Q or H at position 65, H at position 92 and H at position 94, and optionally having at least one of the following amino acids at the following positions in SEQ ID NO: 3: 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, I at position 102 and/or E at position 123. Preferably, the amino acid sequence of the identified animal myoglobin comprises a sequence having 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 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 100% identity or similarity to SEQ ID NO: 3 in combination with Q or H at position 65, H at position 92 and H at position 94. SEQ ID NO: 3 is myoglobin from steppe mammoth. In an embodiment, the meat substitute comprises steppe mammoth myoglobin represented by the amino acid sequence SEQ ID NO: 3. This myoglobin is expected to have attractive properties because it is stable to oxidation, especially at low pH values. The color of the meat substitute of the present invention is expected to be stable because the steppe mammoth myoglobin is resistant to oxidation. This anti-oxidative stability also provides additional health properties to the meat substitute of the present invention because it is less likely to be converted into carcinogenic products than real meat.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 combined with F at position 30.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 combined with Q at position 65.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO:3 combined with an H at position 92.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO:3 combined with an H at position 94.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 combined with F at position 30 and Q at position 65.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 combined with an F at position 30 and an H at position 92.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 combined with an F at position 30 and an H at position 94.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 in combination with a Q at position 65 and an H at position 92.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 in combination with a Q at position 65 and an H at position 94.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 in combination with an H at position 92 and an H at position 94.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 in combination with F at position 30, W at position 65 and H at position 92.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 in combination with F at position 30, Q at position 65 and H at position 94.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 in combination with F at position 30, H at position 92, and H at position 94.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 in combination with Q at position 65, H at position 92, and H at position 94.

In an embodiment, a meat substitute or food ingredient comprising myoglobin is represented by an amino acid sequence having at least 70%, 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 to SEQ ID NO: 3 combined with F at position 30, Q at position 65, H at position 92, and H at position 94.

In an embodiment, a meat substitute comprises steppe mammoth myoglobin represented by the amino acid sequence SEQ ID NO: 3. This myoglobin is expected to have attractive properties because this myoglobin is stable to oxidation, especially at low pH values. The color of the meat substitute of the present invention is expected to be stable because the mammoth myoglobin is resistant to oxidation. This antioxidative stability also provides additional health properties for the meat substitute of the present invention because it is less likely to be converted into a carcinogenic product compared to real meat and/or a meat substitute that does not comprise steppe mammoth myoglobin represented by the amino acid sequence SEQ ID NO: 3. Example 2.4 provides a color analysis of steppe mammoth myoglobin according to the present invention and a meat substitute comprising steppe mammoth myoglobin according to the present invention. In addition, Example 2.5 provides an aromatic analysis of a meat substitute, and Example 2.6 provides a nutritional analysis of myoglobin, evaluating the bioavailability of iron.

In an embodiment, the meat substitute does not contain one or more milk proteins selected from the group consisting of β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin, and transferrin, wherein the milk proteins are from woolly mammoth. In another embodiment, the meat substitute does not contain one or more milk proteins selected from the group consisting of β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin, and transferrin, wherein the milk proteins are from mammoth.

In an embodiment, the meat substitute does not comprise one or more milk proteins selected from the group consisting of β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin and transferrin, wherein the milk protein is not produced by a mammal or mammalian cell.

In a more preferred embodiment, the meat substitute does not contain milk proteins such as β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin or transferrin, wherein the milk proteins are from woolly mammoth. In another more preferred embodiment, the meat substitute does not contain milk proteins such as β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin or transferrin, wherein the milk proteins are from mammoth.

In another more preferred embodiment, no milk proteins such as β-casein, κ-casein, α-S1-casein, α-S2-casein, α-lactalbumin, β-lactoglobulin, lactoferrin or transferrin are contained, wherein the milk proteins are not produced by mammals or mammalian cells.

In an embodiment, the meat substitute does not contain kappa-casein from woolly mammoth. In another embodiment, the meat substitute does not contain a protein represented by a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 17. In a further embodiment, the meat substitute does not contain a protein represented by SEQ ID NO: 17.

In an embodiment, the meat substitute does not contain beta-casein from woolly mammoth. In another embodiment, the meat substitute does not contain a protein represented by a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 18. In a further embodiment, the meat substitute does not contain a protein represented by SEQ ID NO: 18.

meat

In a second aspect, there is provided a meat product comprising animal myoglobin, preferably mammalian myoglobin, more preferably from mammoth, pig, sheep or cattle, more preferably wherein the mammoth is a woolly mammoth (common mammoth) or a steppe mammoth (steppe mammoth), preferably a steppe mammoth, or avian myoglobin, preferably from chicken, or fish myoglobin, preferably from tuna, or myoglobin derived therefrom; wherein the animal myoglobin is not produced by the animal or any cell contained therein, the meat product being derived from the animal. In other words, the meat product is derived from an animal but comprises myoglobin that is not produced therein.

In an embodiment, a meat product comprising exogenous myoglobin is provided, the animal myoglobin preferably being exogenous animal myoglobin, more preferably from mammoth, pig, sheep or cattle, more preferably wherein the mammoth is woolly mammoth (common mammoth) or steppe mammoth (steppe mammoth), preferably steppe mammoth, or avian myoglobin, preferably from chicken, or fish myoglobin, preferably from tuna. In this article, exogenous myoglobin is defined as myoglobin that is not endogenously present in the meat.

In an embodiment, the myoglobin in the meat product in the above embodiment is represented by one of the following amino acid sequences:

-a) has at least 70% sequence identity to SEQ ID NO: 3 and at least one of the following amino acid combinations:

o F at position 30, and/or

o Q at position 65, and/or

o H at position 92, and/or

o H at position 94, and/or

o F at position 30 and Q at position 65, and/or

o F at position 30 and H at position 92, and/or

o F at position 30 and H at position 94, and/or

o Q at position 65 and H at position 92, and/or

o Q at position 65 and H at position 94, and/or

o H at position 92 and H at position 94, and/or

o F at position 30, Q at position 65, and H at position 92, and/or

o F at position 30, Q at position 65, and H at position 94, and/or

o F at position 30 and H at position 92 and H at position 94, and/or

o Q at position 65 and H at position 92 and H at position 94, and/or

o F at position 30 and Q at position 65 and H at position 92 and H at position 94;

-b) having at least 70% sequence identity to SEQ ID NO: 2 or 3, comprising Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 2 or 3: 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, I at position 102, E at position 123, and/or I at position 143;

-c) having at least 70% sequence identity to SEQ ID NO: 1, comprising Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within 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, I at position 102 and/or E at position 123;

-d) having at least 70% identity to SEQ ID NO: 4, 5 or 6, comprising Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 4, 5 or 6: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150;

-e) has at least 70% identity to SEQ ID NO:7, comprises Q or H at position 65 and H at position 94, and optionally has at least one of the following amino acids at the following positions within 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 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 111, I at position 116, A at position 117, E at position 118, A at position 121, S at position 128, K at position 133, S at position 145, and/or F at position 150;

-f) having at least 70% identity to SEQ ID NO: 8, comprising Q or H at position 65 and H at position 94.

In an embodiment, a meat product comprising myoglobin is provided, wherein the concentration of myoglobin in the meat product is higher than the average concentration of myoglobin in the meat from which the meat product is derived. Here, the concentration of myoglobin should be interpreted as the total concentration of all myoglobin variants. The average concentration of myoglobin in the meat from which the meat product is derived refers to the average concentration of myoglobin in the meat measured for a number of corresponding animals without the addition of exogenous myoglobin after harvesting.

Myoglobin

In a third aspect, the present invention provides a protein which can be represented by a sequence comprising SEQ ID NO:3.

In a preferred embodiment, a protein is provided which can be represented by a sequence comprising SEQ ID NO: 3, wherein the length of 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 6, 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 ,217,218,219,220,221,222,223,224,2 25, 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, 25 5, 256, 257, 258, 259, 260, 261, 262, 263, 294, 295, 296, 297, 298, 299, or 300 amino acid residues.

In a preferred embodiment, a protein is provided, which can be represented by a sequence consisting of SEQ ID NO: 3 and a terminal sequence at the N-terminus of SEQ ID NO: 3. Preferably, the length of the terminal sequence 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, 25, 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, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135

In an alternative embodiment, a protein is provided which can be represented by a sequence consisting of SEQ ID NO: 3 and a terminal sequence at the C-terminus of SEQ ID NO: 3. Preferably, the length of the terminal sequence 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, 25, 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, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135

In a preferred embodiment, a protein is provided, which may be represented by a sequence comprising or consisting of: SEQ ID NO: 3 and a tag, preferably wherein the tag is a polyhistidine tag.

In a preferred embodiment, the present invention provides a myoglobin, wherein the myoglobin is from steppe mammoth, and preferably wherein the myoglobin can be represented by SEQ ID NO: 3. The myoglobin can be used in the meat substitute, food ingredient or meat product as described above.

In Example 2.2, the structural features of the myoglobin represented by SEQ ID NO: 3 were investigated. In particular, it was shown that SEQ ID NO: 3 unexpectedly corresponds to a higher positive surface charge compared to other myoglobins. Without being bound by this theory, the myoglobin represented by SEQ ID NO: 3 may therefore be characterized by increased stability and reduced aggregation tendency. This would facilitate the use of these myoglobins in meat substitutes, food ingredients or meat products as described above.

The present invention further provides a composition comprising a protein according to the third aspect as described above.

Nucleic Acids

In a fourth aspect, the present invention provides a nucleic acid, wherein the nucleic acid can be represented by a sequence comprising a subsequence encoding a protein represented by SEQ ID NO: 3, preferably wherein the subsequence can be represented by SEQ ID NO: 11.

In a preferred embodiment, a nucleic acid is provided which can be represented by a sequence comprising a subsequence encoding a protein which can be represented by SEQ ID NO: 3, preferably wherein the subsequence can be represented by SEQ ID NO: 11, wherein the length of the nucleic acid is 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515 3. 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523 ,524,525,526,527,528,52 9, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559 ,560,561,562,563,564,56 5, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599 or 600 bases.

In a preferred embodiment, a nucleic acid is provided, which can be represented by a sequence comprising: a subsequence encoding a protein represented by SEQ ID NO: 3, preferably wherein the subsequence can be represented by SEQ ID NO: 11, and a terminal sequence at the 5' end of the subsequence. Preferably, the length of 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, 25, 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135

In a preferred embodiment, a nucleic acid is provided, which can be represented by a sequence comprising: a subsequence encoding a protein represented by SEQ ID NO: 3, preferably wherein the subsequence can be represented by SEQ ID NO: 11, and a terminal sequence at the 3' end of the subsequence. Preferably, the length of 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, 25, 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135

In a preferred embodiment, a nucleic acid is provided, wherein the nucleic acid encodes myoglobin from steppe mammoth, preferably wherein the myoglobin can be represented by SEQ ID NO: 3, and most preferably wherein the nucleic acid can be represented by SEQ ID NO: 11.

The present invention further provides a composition comprising a nucleic acid according to the fourth aspect as described above.

Gene constructs

In a fifth aspect, a genetic construct is provided, comprising a nucleic acid encoding a myoglobin as defined herein before.

In view of this disclosure, "gene construct" as described herein has its customary and ordinary meaning as understood by those skilled in the art. "Gene construct" may also be referred to as "expression cassette" or "expression construct", and refers to a gene or a group of genes, including a gene encoding a protein of interest, which is operably connected to a promoter controlling its expression. The section entitled "General Information" of this application includes more details about "gene construct". "Operably connected" as used herein is further described in the section entitled "General Information" of this application.

The nucleotide sequence encoding animal myoglobin can be derived from any animal myoglobin gene or animal myoglobin coding sequence, preferably an animal myoglobin gene or an animal myoglobin coding sequence from mammoth, pig, sheep, cattle, chicken or tuna; or a mutated animal myoglobin gene or an animal myoglobin coding sequence, preferably from mammoth, pig, sheep, cattle, chicken or tuna.

Thus, in some embodiments, the preferred nucleotide sequence encoding animal myoglobin encodes a polypeptide represented by an amino acid sequence comprising a sequence having 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 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 100% identity or similarity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, and having the corresponding amino acids at the positions identified above herein (i.e., in combination with Q or H at position 65 and H at position 94). Optional amino acid positions are also contemplated.

Thus, in some embodiments, the preferred nucleotide sequence encoding animal myoglobin encodes a polypeptide represented by an amino acid sequence comprising a sequence having 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 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 100% identity or similarity to SEQ ID NO: 3 and having at least one of the following amino acid combinations:

- 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 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 and Q at position 65 and H at position 92 and H at position 94;.

In some embodiments, the nucleotide sequence encoding animal myoglobin present in the gene construct according to the present invention has 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%, 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 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 100% identity with any sequence selected from the group consisting of: SEQ ID NO: 9, 10, 11, 12, 13, 14, 15 or 16.

Descriptions of "identity" or "sequence identity" and "similarity" or "sequence similarity" have been provided under the section entitled "General Information".

In some embodiments, a gene construct as described herein is provided, wherein the nucleotide sequence encoding animal myoglobin is selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide represented by an amino acid sequence comprising a sequence having 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 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 100% sequence identity or similarity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 in combination with Q or H at position 65 and H at position 94. Optional amino acid positions are also contemplated.

(b) a nucleotide sequence having 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%, 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 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 100% sequence identity to SEQ ID NO:9, 10, 11, 12, 13, 14, 15 or 16.

(c) A nucleotide sequence that differs from the nucleotide sequence of (b) due to the degeneracy of the genetic code.

In some embodiments, a gene construct as described herein is provided, wherein the nucleotide sequence encoding animal myoglobin is selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide represented by an amino acid sequence comprising a sequence having 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 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 100% sequence identity or similarity to SEQ ID NO: 3, and having at least one of the following amino acid combinations:

- 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 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 and Q at position 65 and H at position 92 and H at position 94;.

(b) a nucleotide sequence having 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%, 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 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 100% sequence identity to SEQ ID NO:11.

(c) A nucleotide sequence that differs from the nucleotide sequence of (b) due to the degeneracy of the genetic code.

The animal myoglobin encoded by the nucleotide sequences described herein or derived from the animal myoglobin described herein exerts at least a detectable level of animal myoglobin activity as known to those skilled in the art.

The activity of animal myoglobin may be the transport of iron through its heme domain. This activity may be assessed by methods known to those skilled in the art, such as those defined herein above.

In some embodiments, a gene construct as described herein is provided, wherein the nucleotide sequence encoding animal myoglobin is selected from SEQ ID NO: 19, 20, 21, 22, 23 and 24.

Among them, SEQ ID NO: 19 represents a nucleotide sequence encoding steppe mammoth myoglobin as shown in SEQ ID NO: 3, SEQ ID NO: 20 represents a nucleotide sequence encoding sheep myoglobin as shown in SEQ ID NO: 4, SEQ ID NO: 21 represents a nucleotide sequence encoding bovine myoglobin as shown in SEQ ID NO: 5, SEQ ID NO: 22 represents a nucleotide sequence encoding porcine myoglobin as shown in SEQ ID NO: 6, SEQ ID NO: 23 represents a nucleotide sequence encoding chicken myoglobin as shown in SEQ ID NO: 7, and SEQ ID NO: 24 represents a nucleotide sequence encoding tuna myoglobin as shown in SEQ ID NO: 8, wherein these nucleotide sequences are codon-optimized for expression in Pichia pastoris (Komagataella phaffii). For details of codon optimization, see Example 2.1.

In some embodiments, the gene construct further comprises a regulatory region. In an embodiment, the gene construct further comprises a promoter. In an embodiment, the gene construct comprises a promoter, a terminator and an optional signal peptide. The gene construct may also comprise a marker. The signal peptide as used herein preferably promotes the secretion of the expressed myoglobin. The technician knows that, depending on the host cell used, the characteristics of the regulatory region and the marker must be optimized.

In some embodiments, a gene construct as described herein is provided, wherein the gene construct is represented by SEQ ID NO: 25, 26, 27, 28, 29 or 30.

Here, SEQ ID NOs: 25-30 represent gene constructs that are codon-optimized for expression in Pichia pastoris (Phaffia calvaria), comprising nucleotide sequences encoding steppe mammoth myoglobin represented by SEQ ID NOs: 19-24, respectively, and a signal peptide of Saccharomyces cerevisiae mating factor alpha. In other words, SEQ ID NOs: 25-30 correspond to gene constructs expressing steppe mammoth, sheep, cattle, pig, chicken, and tuna myoglobin, respectively. Codon optimization is described in Example 2.1.

For example, if bacteria (preferably E. coli) are used as host cells, the following regulatory regions can be used. Promoters suitable for use in bacteria are lac, trp, tac, T7 (used in examples), phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from rrnB ribosomal RNA operon, ^PL promoter from bacteriophage λ. Terminators suitable for use in bacteria are lac, trp, tac, T7 (used in examples), phoA, ara, xapA, cad, recA, spc, bla, P1 and P2 from rrnB ribosomal RNA operon, ^PL terminator from bacteriophage λ. The preferred promoter used is the T7 promoter and/or the preferred terminator is the T7 terminator. The preferred signal peptide for secretion is E. coli Sec recognition peptide (SecA), E. coli Tet recognition peptide, E. coli dsbA, E. coli phoA, E. coli pelB, E. coli MBP (maltose binding protein). A suitable marker for E. coli is ampicillin. Alternatively, the proBA operon from E. coli strain K12, including its native transcriptional regulatory elements, can be used to facilitate antibiotic-free selection.

In another example, if yeast is used as a host cell, the following regulatory regions may be used. Promoters suitable for use in yeast may be constitutive promoters. Examples of suitable constitutive promoters include: glycolytic promoters selected from FBA1, TPI1, PGK1, PYK1, TDH3, ENO2, HXK2, PGI1, PFK1, PFK2, GPM1 genes or non-glycolytic promoters of the TEF2 gene.

Suitable promoters for yeast may be inducible. If the yeast is Pichia pastoris, the methanol inducible promoter AOX1 is preferred. Otherwise, when the yeast is Saccharomyces cerevisiae, the GAL1 promoter (galactose inducible) may be used. The genes mentioned from which promoters for use in yeast as host cells may also be used to derive terminators for the same yeast. Preferred signal peptides for secretion of Pichia pastoris (and Saccharomyces) include: Saccharomyces cerevisiae alpha mating factor pre-secretory signal peptide, Saccharomyces cerevisiae Ost1 signal peptide, Saccharomyces cerevisiae Aga2 signal peptide, and fusions thereof.

Example 2.1 describes the synthesis of a gene construct encoding myoglobin as defined herein above. In this example, AOX1 was used for expression in Pichia pastoris strain GS115.

In another example, if a filamentous fungus is used as a host cell, the following regulatory regions may be used. The following promoters may be used: Aspergillus niger glucoamylase promoter (glaA), Aspergillus nidulans alcohol dehydrogenase promoter (alcA) or Aspergillus oryzae taka amylase A promoter (amyB), Aspergillus niger alcohol dehydrogenase promoter (adhA), Trichoderma reesei pyruvate kinase promoter (pki) or Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA). The genes mentioned for promoters in filamentous fungi as host cells may also be used to derive terminators for the same filamentous fungi. Preferred signal peptides for secretion by filamentous fungi, preferably Aspergillus, include: Aspergillus niger glucoamylase signal peptide (glaA), Aspergillus niger alpha-galactosidase signal peptide (AglB) and Trichoderma reesei cellobiohydrolase I (CbhI).

A preferred promoter and terminator for use with Aspergillus, more preferably Aspergillus niger, is the Aspergillus niger glucoamylase promoter and glucoamylase terminator.

Expression vector

The gene construct described herein can be placed in an expression vector. Therefore, in another aspect, an expression vector is provided, which comprises the gene construct as described in any one of the above embodiments.

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

Composition

In a further aspect, a composition is provided, comprising the gene construct as described above and/or the expression vector as described above, optionally further comprising one or more components.

Host cells

In another aspect, there is provided a host cell comprising a gene construct as defined herein. The host cell may be a prokaryotic or eukaryotic organism.

The prokaryotic organism may be a bacterium. The bacterium may be a gram-positive/gram-negative bacterium selected from the following list: Absidia, Achromobacter, Acinetobacter, Aerobic Bacillus, Thiamine-lytic Bacillus, Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter, Arzoarcus, Azomonas, Azospirillum, Azotobacter, Bacillus, Beyerlinckia, Bradyrhizobium, Brevibacillus, Burkholderia, Mycoticola, Citrobacter, Clostridium, Comamonas, Cupricobacter, Corynebacterium, Deinococcus, Escherichia, Enterobacter, Flavobacterium, Fusobacterium Pseudomonas, Gossypium, Klebsiella, Lactobacillus, Listeria, Megasphaera, Micrococcus, Mycobacterium, Nocardia, Porphyromonas, Propionibacterium, Pseudomonas, Ralstonia, Rhizobium, Rhodopseudomonas, Rhodospirilla, Rhodococcus, Roseburia, Shewanella, Streptomyces, Xanthomonas, XyIeIIa, Yersinia, Treponema, Vibrio, Streptococcus, Lactococcus, Zymomonas, Staphylococcus, Salmonella, Sphingomonas, Sphingolipids, Neosphingolipids, Brucella and Microoscillator.

Preferred bacteria include Bacillus pallidum, Bacillus thermophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus alkaliphilus, Bacillus brevis, Bacillus thermorubrum, Bacillus ginseng soil brevis, Basel greedy copper bacteria, G.lraustophilus, Gluconobacter oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pelotomaculum thermopropionicum, Pseudomonas zeaxanthinifaciens, Pseudomonas putida, Paracoccus denitrificans, Escherichia coli, Corynebacterium glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium meliloti, Sphingolipids species, Neosphingolipids species, Sphingomonas henshuiensis, and Radiorhizobium. Preferred bacteria are Escherichia coli.

Preferred E. coli strains include: 58, 679, WG1, DH5α, TG1, TOP10, K12 (used in the examples), BL21, BL21 DE3, XL1-Blue, XL10-Gold, TB1, REG-12, W945, HB101, DH1, DP50, AB284, JC9387, AG1, C600, Cavalli Hfr, and Y10.

The eukaryotic organism may be a yeast or a filamentous fungus.

Preferred yeasts include Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kluyveromyces, Schwannella, Yarrowia, Cryptococcus, Debaryomyces, Saccharomyces copsis, Saccharomyces, Wicklowia, Debaryomyces, Hansenula sporangiophora, Ogataea, Kuraishia, Komagataella, Metschnikowia, Pseudomonas, Nakazawaea, Torulella sporangiophora, Buller's Explosive Spore, Rhodotorula, Sporobolomyces. Among yeasts, the species Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha (also known as Ogataea henricii), Yarrowia lipolytica, Candida tropicalis and Pichia pastoris (also known as Phaffia calvaria) are preferred.

Preferred Pichia strains are selected from the following list: Bg09, Bg10, Bg11, Bg12 (exemplary), Bg20, Bg21, Bg22, Bg23, Bg24, Bg25, Bg26, Bg40, Bg43, Bg44, Bg45, Y-11430, X-33, GS115, KM71, SMD1168, SMD1165, MC100-3, most preferably Bgl0 and derivatives.

In Example 2.3, Pichia strain GS115 (his4) was used as the host cell.

Preferred Saccharomyces strains are selected from the following list: S288C, CEN.PK family, CBS 2354, ATCC 2360, ATCC4098, ATCC 4124, ATCC 4126, ATCC 4127, ATCC 4921, ATCC 7754, ATCC 9763, ATCC 20598, ATCC 24855, ATCC 24858, ATCC 24860, ATCC 26422, ATCC 46523, ATCC 56069, ATCC 60222, ATCC 60223, ATCC 60493, ATCC 66348, ATCC 66349, ATCC 96581.

Preferred yeasts are strains of the genus Pichia, more preferably Pichia.

The filamentous fungi may be selected from the following list, which list includes: Acremonium, Agaricus, Aspergillus, Chrysobasidiomycete, Chrysosporium, Coprinus, Cryptococcus, Ustilago, Fusarium, Humicola, Giant Shell, Mucor, Myceliophthora, Neocallinastix, Neurospora, Paecilomyces, Penicillium, Pyrocystis, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermocystis, Thielavia, Toxicodendron, Ustilago and Trichoderma.

Preferred filamentous fungi are selected from the following list: Aspergillus niger, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus vadensis, Penicillium chrysogenum, Penicillium citrinum, Penicillium rubrum, Penicillium oxalicum, Penicillium subrubescens, Roseburia emersonii, Talaromyces emersonii, Cephalosporium chrysogenum, Trichoderma reesei, Aspergillus sojae, and Chrysosporium lucknowense.

Preferred filamentous fungal strains are selected from the following list: Aspergillus niger CBS 513.88, N593, CBS 120.49 (as used in the examples), N402, ATCC 1015 Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, Aspergillus vadensis CBS 113365, CBS 102787, IMI 142717, IBT 24658, CBS 113226, Penicillium chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Wisconsin 54-1255, Penicillium subrubescens CBS 132785, FBCC 1632, Talaromyces emersonii CBS 393.64, Cephalosporium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC56765 or ATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense ATCC44006.

In a preferred embodiment, Aspergillus is used as the filamentous fungus. More preferably, a strain of Aspergillus niger is used.

method

The host cells of the present invention can be used to produce myoglobin. In some embodiments, the myoglobin is from mammoths, preferably from woolly mammoths or steppe mammoths. In some embodiments, the myoglobin is from pigs. In some embodiments, the myoglobin is from sheep. In some embodiments, the myoglobin is from cattle. In some embodiments, the myoglobin is from chickens. In some embodiments, the myoglobin is from tuna. Therefore, in a further aspect, the present invention provides a method for producing myoglobin as defined herein above, the method comprising culturing the host cells of the present invention in a suitable culture medium and optionally recovering the host cells and/or myoglobin. Optionally, the myoglobin produced does not contain a signal peptide as defined elsewhere herein.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from mammoth, preferably myoglobin having at least 70% sequence identity to SEQ ID NO: 1, more preferably myoglobin having at least 70% sequence identity to SEQ ID NO: 1, together with Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within 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, I at position 102 and/or E at position 123, the method comprising culturing a host cell of the present invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from woolly mammoth, preferably myoglobin having at least 70% sequence identity to SEQ ID NO: 2, more preferably myoglobin having at least 70% sequence identity to SEQ ID NO: 2, together with Q or H at position 65, Q at position 92 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 2: 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, I at position 102, E at position 123 and/or I at position 143, the method comprising culturing a host cell of the present invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from steppe mammoth, preferably myoglobin having at least 70% sequence identity to SEQ ID NO: 3, more preferably myoglobin having at least 70% sequence identity to SEQ ID NO: 3, together with Q or H at position 65, H at position 92 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 3: 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, I at position 102, E at position 123 and/or I at position 143, the method comprising culturing a host cell of the present invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from steppe mammoth, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 3, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 3 together with at least one of the following amino acid combinations:

- 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 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;

The method comprises culturing a host cell of the invention in a suitable culture medium and optionally recovering the host cell and/or myoglobin.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from sheep, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 4, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 4, together with Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 4: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150, the method comprising culturing a host cell of the present invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from a cow, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 5, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 5, together with Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 5: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150, the method comprising culturing a host cell of the present invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from a pig, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 6, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 6, together with Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 6: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150, the method comprising culturing a host cell of the present invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from chicken, preferably myoglobin having at least 70% sequence identity to SEQ ID NO: 7, more preferably having at least 70% sequence identity to SEQ ID NO: 7, together with Q or H at position 65 and H at position 94, and optionally in SEQ ID NO: A myoglobin having at least one of the following amino acids at the following positions within NO:7: 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 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, I at position 116, A at position 117, E at position 118, A at position 121, S at position 128, K at position 133, S at position 145 and/or F at position 150, the method comprising culturing the host cell of the invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides a method for producing myoglobin as defined herein above from tuna, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 8, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 8, together with comprising Q or H at position 65 and H at position 94.

The features of this aspect are preferably as described elsewhere herein. The method may hereinafter be referred to alternatively as a method according to the present invention. Suitable cell culture methods for the method according to the present invention are known to the technician and are described below: for example van't Riet, K. and Tramper, J., 1st edition, Basic Bioreactor Design [Basic Bioreactor Design], CRC Press, New York, 1991. Such methods include, but are not limited to, submerged fermentation in liquid culture medium, surface fermentation in liquid culture medium, and solid-state fermentation. Cell culture can be carried out, for example, by culturing in microtiter plates, shake flasks, small benchtop bioreactors, medium-sized bioreactors, and/or large-scale bioreactors in laboratories and/or industrial environments. Suitable cell culture modes include, but are not limited to, continuous, batch, and/or fed-batch fermentation and combinations thereof. In an embodiment, continuous fermentation is used to culture cells. In a preferred embodiment, batch fermentation is used to culture cells. In a more preferred embodiment, fed-batch fermentation is used to culture cells.

In the context of the present invention, "culture medium", hereinafter alternatively referred to as "growth medium", can be interpreted to include situations in which cultured cells are not present as well as situations in which cultured cells are present in the culture medium. "Culture fluid" refers to the culture medium in which cultured cells are present. "Culture supernatant" refers to the culture medium in which cultured cells are not present. "Cell-free extract" refers to a cell lysate that does not contain cell debris. The cell culture as part of the method of the present invention can be carried out under conditions known to the skilled person that are conducive to the production of the introduced myoglobin. These conditions depend not only on the chemical composition of the culture medium, but also on other engineering parameters, including culture duration, temperature, O2 levels in the culture fluid and/or headspace, _CO2 levels in the culture fluid and/or headspace, pH, ionic strength, stirring speed, hydrostatic pressure, etc. Cell culture can be carried out using procedures known in the art, using a culture medium containing appropriate nutrients (e.g., carbon and nitrogen sources) and additional compounds (e.g., inorganic salts and vitamins) (see, e.g., Bennett, W. and Lasure, L., 1st edition, More Gene Manipulations in Fungi, Academic Press, California, 1991). Suitable growth media are available from commercial suppliers or can be prepared using published compositions appropriate for the respective host (e.g., in the catalogs of the Netherlands Collection of Microorganisms (CBS) or the American Type Culture Collection (ATCC)).

The exact composition of the growth medium and the value of the culture engineering parameters are not key features of the present invention. Any growth medium composition can be considered, as long as it allows the production of host cell growth and introduced myoglobin. The growth medium generally contains a carbon source for the growth of the cultured cells. The technician understands that a suitable carbon source can be added to the growth medium from the outside or can already be present in the culture medium. The carbon source can exist or be added alone or in the form of a mixture of multiple carbon sources. Examples of suitable carbon sources known in the art include monosaccharides such as glucose, maltose, sucrose, xylose, arabinose, complex sugars such as maltodextrin, hydrolyzed starch, starch, molasses and second-generation raw materials. Second-generation raw materials are particularly attractive because of their lower carbon footprint. Second-generation raw materials generally include lignocellulosic materials. This material includes any lignocellulose and/or hemicellulose-based materials. This material can be derived from agricultural waste streams, industrial waste streams or municipal waste streams, preferably agricultural waste streams. Examples of suitable materials include (agricultural) biomass, commercial organic matter, municipal solid waste, original biomass such as waste paper and garden waste, or non-original biomass. Common forms of biomass include trees, shrubs and grasses, wheat, wheat straw, bagasse, switchgrass, Japanese pampas grass, corn, corn stover, corn cobs, rape straw, soybean stover, sweet corn, corn kernels, products and byproducts from grain milling (including wet and dry milling), such as corn, wheat and barley milling, often referred to as "bran or fiber", and municipal solid waste. Biomass can also be forage, agricultural residues, forestry residues, municipal solid waste, waste paper, and pulp and paper mill residues. Agricultural biomass includes branches, shrubs, tows, corn and corn stalks, energy crops, forests, fruits, flowers, cereals, forage, herbaceous crops, leaves, bark, needles, logs, roots, young trees, short-term rotation woody crops, shrubs, switch herbs, trees, vegetables, fruits, grapevines, beet pulp, wheat slag, oat husks, and hardwood and softwood (excluding toxic wood), and organic wastes produced in agricultural processing, including agricultural and forestry activities, particularly forestry wood wastes. Agricultural biomass can be any single aforementioned or any combination or mixture thereof. Carbon sources such as organic acids, aldehydes, ketones, esters and alcohols can also be considered. In the method of the present invention, it is also possible to consider the use of a growth medium comprising a combination of multiple different carbon sources. As a non-limiting example, such a medium can combine a carbon source (e.g., organic acid) with a higher degree of oxidation with a carbon source (e.g., alcohol) with a higher degree of reduction. The example of suitable nitrogen source known in the art comprises soybean meal, corn steep liquor, yeast extract, whey protein, egg protein, casein hydrolyzate, urea, ammonia, ammonium salt and nitrate.The example of other suitable compounds known in the art comprises phosphate, sulfate, metal such as magnesium, trace element and vitamin.The definite growth medium requirement will be different according to the host cell, for example, between yeast, bacterium and filamentous fungi, and the requirement will be known to the technician.Therefore, the growth medium can be complete (enriched) medium or minimal medium, i.e., the medium only comprising the necessary components of growth, which depends on the host cell cultivated.

Similar to the composition of the growth medium, the method parameters can specify any values, as long as they allow the host cell growth and the production of the introduced myoglobin. Generally, the values will be different according to the host cell being cultivated and will be known to the technician. Preferably, the method according to the present invention is an oxygen-limited or aerobic method, meaning that the cell culture is carried out under oxygen-limited or aerobic conditions, and more preferably the method is oxygen-limited. Oxygen-limited conditions, also referred to as microaerobic conditions, are culture conditions in which oxygen consumption is limited by oxygen availability. The degree of oxygen restriction depends on the amount and composition of the incoming gas flow and the actual mixing/mass transfer characteristics of the fermentation equipment used. Preferably, under oxygen-limited conditions in liquid culture, the oxygen consumption rate is at least about 5.5mmol/L/h, more preferably at least about 6mmol/L/h, and even more preferably at least about 7mmol/L/h. Aerobic conditions are culture conditions in which oxygen consumption is not limited by oxygen availability.

The cell culture can be carried out at a temperature value that is optimal for the cells, generally in the temperature range of 16-42° C. In some embodiments, the temperature range is between 20-40° C., more preferably between 25-38° C., and most preferably between 28-36° C. In some most preferred embodiments, a temperature value of about 30 or 36° C. is used.

Cell culture can be carried out at a pH value that is optimal for the cells. In some embodiments, the culture pH is about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, about pH 8.5, about pH 9. In preferred embodiments, the pH range is about pH 3.0 to about pH 9, more preferably about pH 3.5 to pH 7. In some most preferred embodiments, a pH value of about 6 is used.

Cell culture can be carried out at a medium ionic strength value that is optimal for the cells, typically in the range of 50 mM-2 M. In some embodiments, the medium ionic strength is in the range of 75 mM-1 M, more preferably between 100 mM-750 mM. In some most preferred embodiments, an ionic strength value of about 100 mM is used.

The cell culture may be carried out for a duration of 14, 13.5, 13, 12.5, 12, 11.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 day, wherein the duration may have a deviation of 20%. Preferably, the cell culture is carried out for a duration of 14, 13.5, 13, 12.5, 12, 11.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 day, wherein the duration may have a deviation of 10%. More preferably, the cell culture is carried out for 5 days, wherein the duration may have a deviation of 20%, most preferably a deviation of 10%.

The methods according to the invention will typically produce at least 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 1 0g/L, 11g/L, 12g/L, 13g/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/L, 75g/L, 100g/L, 200g/L or 300g/L of myoglobin.

In the methods according to the invention, typically at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, preferably at least 20%, more preferably at least 40%, most preferably at least 60% of the carbon source in the growth medium will be converted into myoglobin.

Cell culture can also be carried out by implementing a multi-step, preferably two-step culture method. For example, the production step of myoglobin can be preceded by a cell biomass growth step, in which only limited production or no production is performed. Different steps can be performed using different culture modes and/or different growth media and/or different culture method parameter values, depending on the goal of each step and/or the cells being cultured. The biomass during the production step may or may not be actively growing.

In the context of the methods of the invention, the host cells and/or myoglobin may optionally be recovered from the culture medium. When present intracellularly, the myoglobin may optionally be recovered from the recovered cell biomass. Optionally, the recovered myoglobin is purified. Preferably, the purification of the myoglobin will produce a purity of at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, most preferably producing substantially pure myoglobin.

In an embodiment, the host cell produces myoglobin extracellularly in the method according to the invention. In the method according to this embodiment, myoglobin is transported out of the host cell after it is synthesized in the host cell. The method according to this embodiment can be referred to as an extracellular method according to the invention. In this case, both secretion and extracellular fermentation are considered to be extracellular production.

Without being bound by this theory, the extracellular method according to the present invention has the advantage that downstream processing to recover the produced myoglobin is more convenient, efficient and/or effective. In addition, compared to intracellular methods in which the myoglobin is not transported out of the host cell after its synthesis, the method according to the product can produce a composition comprising high purity myoglobin, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and with minimal downstream processing.

Without being bound by this theory, the extracellular method according to the present invention has the advantage that the optional step of recovering myoglobin does not include lysing the host cells. Therefore, the extracellular method according to the present invention can produce a composition having a low concentration of nucleic acids derived from host cells, as described in the following examples.

Thus, in a further aspect, the present invention provides a method for producing myoglobin as defined herein before, the method comprising culturing a host cell of the invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In embodiments, the extracellular methods according to the invention produce a composition having a myoglobin purity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, most preferably substantially pure myoglobin.

Preferably, purity is measured as the weight percentage of the total protein fraction in the cell-free supernatant obtained at the end of the method or the extracellular method according to the invention.

In embodiments, the extracellular process according to the present invention results in a composition comprising less than or equal 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%, 2.9%, 2.8%, 2. 7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 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 0.1% nucleic acid from host cells.

In an embodiment, the invention provides an extracellular method according to the invention for producing myoglobin as defined herein above from woolly mammoth, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 2, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 2, together with Q or H at position 65, Q at position 92 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 2: 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, I at position 102, E at position 123 and/or I at position 143, the method comprising culturing a host cell of the invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides an extracellular method according to the present invention for producing myoglobin as defined herein before from steppe mammoth, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 3, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 3 together with at least one of the following amino acid combinations:

- 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 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;

The method comprises culturing a host cell of the invention in a suitable culture medium and optionally recovering the host cell and/or myoglobin.

Example 2.3 provides the extracellular production of steppe mammoth myoglobin.

In an embodiment, the invention provides an extracellular method according to the invention for producing myoglobin as defined herein before from a sheep, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 4, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 4, together with Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 4: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150, the method comprising culturing a host cell of the invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the extracellular method according to the present invention is used to produce myoglobin as defined herein above from a dairy cow, preferably a myoglobin having at least 70% sequence identity with SEQ ID NO: 5, more preferably a myoglobin having at least 70% sequence identity with SEQ ID NO: 5, together with Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 5: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150, the method comprising culturing the host cell of the present invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides an extracellular method according to the present invention for producing myoglobin as defined herein above from a pig, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 6, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 6, together with Q or H at position 65 and H at position 94, and optionally having at least one of the following amino acids at the following positions within SEQ ID NO: 6: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, S at position 133, A at position 145 and/or L at position 150, the method comprising culturing a host cell of the present invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the invention provides an extracellular method according to the invention for producing myoglobin as defined herein above from chicken, preferably myoglobin having at least 70% sequence identity to SEQ ID NO: 7, more preferably having at least 70% sequence identity to SEQ ID NO: 7, together with Q or H at position 65 and H at position 94, and optionally in SEQ ID NO: A myoglobin having at least one of the following amino acids at the following positions within NO:7: 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 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, I at position 116, A at position 117, E at position 118, A at position 121, S at position 128, K at position 133, S at position 145 and/or F at position 150, the method comprising culturing the host cell of the invention in a suitable culture medium and optionally recovering the host cell and/or the myoglobin.

In an embodiment, the present invention provides an extracellular method according to the present invention for producing myoglobin as defined herein before from tuna, preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 8, more preferably a myoglobin having at least 70% sequence identity to SEQ ID NO: 8, together with comprising Q or H at position 65 and H at position 94.

The relevant downstream processing technology that may be suitable for recovery and/or purification is not a key feature of the present invention and will depend on whether the myoglobin is accumulated or discharged in the cultured cells. The processing technology and related selection will be known to the skilled person and are discussed, for example, in Wesselingh, J.A and Krijgsman, J., 1st edition, Downstream Processing in Biotechnology, Delft Academic Press, NL, 2013. In a non-limiting example of a recovery method, biomass is recovered from the culture medium using, for example, centrifugation or filtration. If the produced myoglobin accumulates in the cells, it can be recovered and/or purified from the biomass. If it is secreted, it can be recovered from a cell-free culture medium, or if the biomass separation step is skipped, it is directly recovered from the culture fluid. Recovery and/or purification can be carried out according to any conventional recovery or purification method known in the art. Methods for recovering and/or purifying proteins are known to the skilled person and are discussed in standard handbooks, Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, 2001 or Ausubel F. et al., eds., Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York, 2003. Examples of widely used recovery and/or purification methods include chromatography, such as gel filtration chromatography, ion exchange chromatography, immunoaffinity chromatography, metal affinity chromatography, gel filtration chromatography, fractionation using precipitants such as ammonium sulfate and polyethylene glycol, gel electrophoresis and salting out, and dialysis. Preferably, metal affinity chromatography or size exclusion chromatography is used. Recovery and/or purification can optionally be enhanced by linking the enzyme polypeptide to a sequence that facilitates purification, such as a GST domain, using well-known molecular toolbox techniques. Optionally, the sequence that promotes purification and/or the signal peptide that promotes secretion of myoglobin are removed from the final product using techniques known in the art, such as proteolysis by an endopeptidase that targets the linker between the sequence that promotes purification and/or the signal peptide and myoglobin. In some embodiments, the enzyme polypeptide is linked (fused) to a six-histidine peptide, such as a tag provided in a pET23a(+) vector (Genescript Biotech, Leiden, the Netherlands), many of which are commercially available. For example, the six-histidine peptide facilitates purification of the fusion protein, as described in, for example, Gentz et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 86: 821-824 (1989).

In a preferred embodiment, the produced myoglobin is recovered and/or purified from the culture medium. This can be achieved continuously or subsequently during the production process.

In a preferred embodiment, the myoglobin produced is recovered and/or purified from the cultured cells. This can be achieved continuously during the production process by harvesting fractions of the growing cells, or subsequently.

In a preferred embodiment, the cultured host cells used in the method of the present invention are fixed. Immobilization of the cells can be achieved by any means known to those skilled in the art, as discussed in standard manuals, such as Guisan, J.M., Bolivar, J.M., López-Gallego, F., Rocha-Martín, J. (eds.), Immobilization of Enzymes and Cells: Methods and Protocols, Springer US, USA, 2020. In general, host cells can be fixed to a semisolid or solid support by three different methods. The first method involves polymerizing or solidifying a solution containing spores or cells. Examples of polymerizable or solidifiable solutions include alginate, λ-carrageenan, chitosan, polyacrylamide, polyacrylamide-hydrazide, agarose, polypropylene, polyethylene glycol, dimethyl acrylate, polystyrene divinylbenzene, polyvinylbenzene, polyvinyl alcohol, epoxy carriers, cellulose, cellulose acetate, photocrosslinkable resins, prepolymers, polyurethanes, and gelatin. The second method involves cell adsorption onto a support. Examples of such supports include bone char, cork, clay, resin, sand porous alumina beads, porous bricks, porous silica, diatomaceous earth or sawdust. Host cells can colonize and form biofilms on supports. The third method includes using chemical reagents such as glutaraldehyde, o-dianisidine (U.S. Patent No. 3,983,000), polymeric isocyanates (U.S. Patent No. 4,071,409), silanes (U.S. Patent No. 3,519,538 and 3,652,761), hydroxyethyl acrylate, transition metal activated supports, cyanuric chloride, sodium periodate, toluene, etc. to covalently couple host cells to supports. Cultivated host cells can be immobilized at any stage of their growth, for example, after reaching a desired cell density in culture. Suitable culture modes and/or different culture method parameter values will be known to the skilled person and are discussed in standard manuals, e.g. Colin R. Phillips C. R., Poon Y. C., Immobilization of Cells: In Biotechnology Monographs book series (Biotechnology, Vol. 5), Springer-Verlag, Berlin, Germany, 1988; Tampion J., Tampion M. D., Immobilized Cells: Principles and Applications, Cambridge University Press, United Kingdom, 1987. Preferably, the immobilized cells are cultured in a packed bed bioreactor, also known as a plug flow bioreactor or an expanded (fluidized) bed bioreactor. Suitable growth media and recovery and/or purification methods are further discussed elsewhere herein.

In a further aspect, there is provided a method for producing a meat substitute as defined above, the method comprising formulating the myoglobin obtainable by the method defined above into a meat substitute. Depending on the envisaged meat substitute and/or food ingredient, the skilled person will know which formulation is most suitable.

General information

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and are to be read in light of this disclosure.

As used herein, the term "promoter" or "regulatory sequence" refers to a nucleic acid fragment that plays a role in controlling the transcription of one or more coding sequences and is located upstream relative to the transcription direction of the transcription start site of the coding sequence, and is structurally characterized by the presence of a binding site for a DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art that acts directly or indirectly to regulate the amount of transcription of the promoter. A "constitutive promoter" is a promoter that is active under most physiological and developmental conditions. An "inducible" and/or "repressible" promoter is a promoter that is physiologically or developmentally regulated to be induced and/or inhibited, for example by the application of a chemical inducer or an inhibitory signal.

As used herein, the term "operably linked" refers to the connection of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" to another nucleic acid sequence when it is placed in a functional relationship with the other nucleic acid sequence. For example, if a transcription regulatory sequence such as a promoter affects the transcription of a coding sequence, it is operably linked to a coding sequence. Operably linked means that the DNA sequences being linked are generally continuous, and when it is necessary to connect two protein coding regions, they are continuous and in reading frame.

As used herein, a "regulator" or "transcriptional regulator" is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA by binding to a specific DNA sequence.

The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule composed of a chain of amino acids without regard to a particular mode of action, size, 3-dimensional structure or origin.

The term "gene" refers to a DNA fragment comprising a region (transcribed region) operably linked to a suitable regulatory region (e.g., a promoter) that is transcribed into an RNA molecule (e.g., mRNA) in a cell. A gene typically comprises several operably linked segments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end), such as comprising a polyadenylation and/or transcription termination site.

"Gene expression" refers to the process in which a DNA region operably linked to an appropriate regulatory region, particularly a promoter, is transcribed into biologically active RNA, ie, RNA that can be translated into a biologically active protein or peptide.

In the amino acid sequences described herein, amino acids or "residues" are represented by three letter symbols. These 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, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine. The residue can be any proteinogenic amino acid, and can also be any nonproteinogenic amino acid, such as D-amino acids and modified amino acids formed by post-translational modification, and any unnatural amino acid, as described herein.

In the context of the present application, all percentages in the context of concentration or composition refer to percentages by weight, unless otherwise defined.

In the context of the present application, expressions such as "a parameter having a value of at least X, Y or Z" should be interpreted as said parameter having a value of at least X, at least Y or at least Z.

Sequence identity

In the context of the present invention, a nucleic acid molecule, such as a nucleic acid molecule encoding animal myoglobin, is represented by a nucleic acid or nucleotide sequence encoding a protein fragment or a polypeptide or a peptide or a derived peptide.

It should be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct identified herein by a given sequence identification number (SEQ ID NO) is not limited to the specific sequence disclosed. Each coding sequence as identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct or is itself a protein fragment or polypeptide or construct or peptide or derived peptide.

Throughout this application, each time a specific nucleotide sequence SEQ ID NO (taking SEQ ID NO: X as an example) encoding a given protein fragment or polypeptide or peptide or derived peptide is mentioned, it can be replaced by:

i. a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: X;

ii. a nucleotide sequence whose sequence differs from the sequence of the nucleic acid molecule of (i,) due to the degeneracy of the genetic code; or

iii. A nucleotide sequence which encodes an amino acid sequence having at least 60% amino acid identity or similarity with the amino acid sequence encoded by the nucleotide sequence SEQ ID NO:X.

Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

In the present application, each time a specific amino acid sequence SEQ ID NO (taking SEQ ID NO: Y as an example) is mentioned, it can be replaced by: a polypeptide represented by an amino acid sequence comprising a sequence having at least 60% sequence identity or similarity with the amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

Each nucleotide sequence or amino acid sequence described herein, by virtue of its percentage of identity or similarity with a given nucleotide sequence or amino acid sequence, in a further preferred embodiment, has 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%, 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 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, , 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 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 100% identity or similarity.

The terms "homology", "sequence identity" and the like are used interchangeably herein. "Sequence identity" is described herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, the sequence identity is calculated based on the full length of two given SEQ ID NOs or a portion thereof. Its portion preferably represents at least 50%, 60%, 70%, 80%, 90% or 100% of the two SEQ ID NOs. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, determined by the match between strings of such sequences. The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutes with the sequence of a second polypeptide. "Identity" and "similarity" can be easily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modem Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, all of which are incorporated herein by reference.

"Sequence identity" and "sequence similarity" can be determined by comparing two peptides or two nucleotide sequences using an overall or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length preferably use an overall alignment algorithm (e.g., Needleman-Wunsch) to compare, which optimally compares sequences over the entire length, while sequences with very large length differences preferably use a local alignment algorithm to compare (e.g., Smith-Waterman). When sequences (when using default parameters to compare optimally by, for example, program EMBOSS needle or EMBOSS water) share at least a certain minimum percentage of sequence identity (as described below), they can be referred to as "substantially identical" or "substantially similar."

When two sequences have similar lengths, global alignment is applicable to determining sequence identity. When sequences have significantly different total lengths, local alignment is preferred, such as those using the Smith-Waterman algorithm. EMBOSS needle uses the Needleman-Wengsch global alignment algorithm to align two sequences over the entire length (total length), thereby maximizing the number of matches and reducing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, EMBOSS needle and EMBOSS water default parameters are used, with gap open 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 Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).

Alternatively, the percentage similarity or identity can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the invention can be further used as a "query sequence" to search public databases, for example to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10 (incorporated by reference). NBLAST nucleotide searches can be performed with the BLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402 (incorporated by reference) can be utilized. When using BLAST and Gap BLAST programs, the default parameters of each program (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information, which can be accessed on the World Wide Web at www.ncbi.nlm.nih.gov/ .

Optionally, when determining amino acid similarity, the skilled person may also consider so-called "conservative" amino acid substitutions. As used herein, "conservative" amino acid substitutions refer to the interchangeability of residues with similar side chains. The following table provides examples of amino acid residue classes for conservative substitutions.

Alternative conservative amino acid residue substitution categories:

1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

Physical and functional classification of amino acid residues for their substitutability:

For example, a group of amino acids with aliphatic side chains is: glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Val to Ile or Leu.

Gene or coding sequence

The term "gene" refers to a DNA fragment comprising a region (transcribed region) operably linked to a suitable regulatory region (e.g., a promoter) that is transcribed into an RNA molecule (e.g., mRNA) in a cell. A gene typically comprises several operably linked segments, such as a promoter, a 5' leader sequence, a coding region, and a 3' non-translated sequence (3' end), e.g., comprising a polyadenylation and/or transcription termination site. "Gene expression" refers to a process in which a DNA region operably linked to a suitable regulatory region, particularly a promoter, is transcribed into a biologically active RNA, i.e., an RNA that can be translated into a biologically active protein or peptide.

Promoter

As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences and is located upstream in the direction of transcription relative to the transcription start site of the coding sequence and is structurally identified by the presence of a binding site for a DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those of ordinary skill in the art that act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive promoter" is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example by the application of a chemical inducer.

Operatively connected

As used herein, the term "operably linked" refers to the connection of polynucleotide elements in a functional relationship. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked" to another nucleic acid sequence. For example, if a transcriptional regulatory sequence affects the transcription of a coding sequence, it is operably linked to the coding sequence. Operably linked means that the DNA sequences being linked are usually continuous, and when it is necessary to connect two protein coding regions, they are continuous and in reading frame. The connection can be completed by connecting at a convenient restriction site or at an adapter or joint inserted in its place, or by gene synthesis.

Protein and amino acids

The terms "protein" or "polypeptide" or "amino acid sequence" are used interchangeably and refer to molecules consisting of amino acid chains without reference to a specific mode of action, size, 3-dimensional structure or origin. In the amino acid sequences described herein, amino acids or "residues" are represented by three letter symbols. These 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, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine. The residue can be any proteinogenic amino acid, and can also be any non-proteinogenic amino acid, such as D-amino acids and modified amino acids formed by post-translational modifications, as well as any non-natural amino acids.

Gene constructs

Any cloning and/or recombinant DNA techniques known to those skilled in the art can be used to prepare the gene constructs described herein, wherein the nucleotide sequence encoding the myoglobin is expressed in suitable cells (e.g., cultured cells or cells of a multicellular organism), as described in Ausubel et al., "Current Protocols in Molecular Biology," Green Publishing and Wiley Interdisciplinary Publishers, New York (1987) and Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entirety. See also Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site-directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, J.A., et al. (1985) Gene 34:315 (describing cassette mutagenesis).

Expression vector

The phrase "expression vector" or "vector" generally refers to a tool used in molecular biology to obtain gene expression in a cell, for example by introducing a nucleotide sequence capable of affecting the expression of a gene or coding sequence into a host compatible with such a sequence. The expression vector carries a genome that can be stable and maintained free in the cell. In the context of the present invention, a cell may mean a cell including a cell for preparing a construct or a cell in which a construct will be administered. Alternatively, the vector can be integrated into the genome of the cell, for example by homologous recombination or other means.

These expression vectors typically include at least a suitable promoter sequence and an optional transcription termination signal. Other factors that are necessary or helpful to affect expression may also be used as described herein. Nucleic acid or DNA or nucleotide sequences encoding myoglobin are incorporated into a DNA construct that can be introduced into an in vitro cell culture and expressed therein. Specifically, the DNA construct is suitable for replication in a prokaryotic host (e.g., bacteria, e.g., Escherichia coli), or can be introduced into a cultured mammal, plant, insect (e.g., Sf9), yeast, fungi or other eukaryotic cell line.

The DNA construct prepared for introduction into a specific host can include a replication system recognized by the host, an expected DNA fragment encoding a desired polypeptide, and transcription and translation initiation and termination regulatory sequences operably connected to the polypeptide coding fragment. The term "operably connected" has been described herein. For example, if a promoter or enhancer stimulates the transcription of a coding sequence, the promoter or enhancer is operably connected to the sequence. If the DNA of a signal sequence is expressed as a preprotein that participates in polypeptide secretion, the DNA of the signal sequence is operably connected to the DNA of the coding polypeptide. Usually, the DNA sequence operably connected is continuous, and in the case of a signal sequence, it is both continuous and in the reading frame. However, enhancers do not need to be adjacent to the coding sequence that they control its transcription. Connection is by connecting at a convenient restriction site or replacing an adapter or joint inserted therein, or by gene synthesis to complete.

The selection of appropriate promoter sequences generally depends on the host cell selected for expressing the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters well known in the art (see, e.g., Sambrook and Russell, 2001, supra). Transcriptional regulatory sequences generally include heterologous enhancers or promoters recognized by the host. The selection of appropriate promoters depends on the host, but promoters such as trp, lac and bacteriophage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell, 2001, supra). Expression vectors include replication systems and transcription and translation regulatory sequences and insertion sites for polypeptide coding segments. In most cases, the replication system only works in cells (bacterial cells such as Escherichia coli) used to make vectors. Most plasmids and vectors will not replicate in cells infected by the vector. Examples of possible combinations of cell lines and expression vectors are described by Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334 : 31-36. For example, suitable expression vectors can be expressed in yeast (e.g., Saccharomyces cerevisiae), insect cells, such as Sf9 cells, mammalian cells (e.g., CHO cells), and bacterial cells (e.g., E. coli). Thus, the cell can be a prokaryotic or eukaryotic host cell. The cell can be a cell suitable for cultivation in a liquid or solid medium.

Alternatively, the host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal.

The selection of appropriate promoter sequences generally depends on the host cell selected for expressing the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters well known in the art (see, e.g., Sambrook and Russell, 2001, supra). Transcriptional regulatory sequences generally include heterologous enhancers or promoters recognized by the host. The selection of appropriate promoters depends on the host, but promoters such as trp, lac and bacteriophage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell, 2001, supra). Expression vectors include replication systems and transcription and translation regulatory sequences and insertion sites for polypeptide coding segments. In most cases, the replication system only works in cells (bacterial cells such as Escherichia coli) used to make vectors. Most plasmids and vectors will not replicate in cells infected by the vector. Examples of possible combinations of cell lines and expression vectors are described by Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334 : 31-36. For example, suitable expression vectors can be expressed in yeast (e.g., Saccharomyces cerevisiae) and bacterial cells (e.g., Escherichia coli). Thus, the cell can be a prokaryotic or eukaryotic host cell. The cell can be a cell suitable for culture in liquid or solid medium.

Express

Expression can be assessed by any method known to those skilled in the art. For example, expression can be assessed by measuring the level of transgene expression in the transduced tissue at the mRNA or protein level using standard assays known to those skilled in the art, such as qPCR, RNA sequencing, Northern blot analysis, Western blot analysis, mass spectrometry of protein-derived peptides, or ELISA.

Expression can be assessed at any time after administering a gene construct, expression vector or composition as described herein. In some embodiments herein, expression can be assessed after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks or more.

In this document and its claims, the verb "comprise" and its variants are used in their non-limiting sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb "consisting of" can be replaced with "consisting essentially of", meaning that the meat substitutes, genetic constructs, host cells (or methods) described herein can contain one or more additional components (or additional steps) in addition to the specifically identified components, and the one or more additional components do not change the unique characteristics of the invention. In addition, the verb "consisting of" can be replaced with "consisting essentially of", meaning that the method as described herein can contain one or more additional steps in addition to the specifically indicated steps, and the one or more additional steps do not change the unique characteristics of the invention.

Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the element be present. Thus, the indefinite article "a" or "an" generally means "at least one".

As used herein, "at least" a particular value means the particular value or more. For example, "at least 2" is understood to be the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ... and so on.

In addition, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and not necessarily to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein are capable of operating in other sequences than those described or illustrated herein.

When used in conjunction with a numerical value (eg, about 10), the word "about" or "approximately" preferably means that the value may be plus or minus 0.1% of the given value (10).

As used herein, the term "and/or" means that one or more of the stated circumstances may occur alone or in combination with at least one of the stated circumstances, up to and including all of the stated circumstances.

Various embodiments are described herein. Unless otherwise stated, each embodiment as described herein can be combined together.

All patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and to the extent the incorporated material is inconsistent with the explicit disclosure herein, in which case the language of the disclosure controls.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The three-dimensional structure of bovine myoglobin. The structure of wild-type pork Mb was determined by X-ray diffraction. The protein was resolved at 1.30 Å resolution (Krzywda et al., 1998). Shown are the heme, the proximal histidine (His94), and the distal histidine (His65).

Figure 2. Comparison of myoglobin sequences from six animal species. The sequence alignment shows sequences from tuna (Thunnus orientalis), chicken (Gallus gallus), steppe mammoth (Woolly mammoth), pork (Sus scrofa), cattle (Bos taurus), and sheep (Ovis aries). Amino acid conservation levels are indicated by blue shading and scored using BLOSUM62. The proximal histidine (His94, boxed) is present in all six species. Steppe mammoth carries a glutamine (Gln65, boxed) at the position normally occupied by a distal histidine (His65, boxed), a phenylalanine at position 30 (Phe30, boxed), and a histidine at position 92 (His92, boxed).

Figure 3. Models of the three-dimensional structures of myoglobin from the Asian elephant (left) and the steppe mammoth (right). The ribbon view (top) and the surface (bottom) view are shown. The heme molecule is depicted, along with the residue at position 92 (glutamine in elephant, histidine in mammoth). In the surface view, positive charges are shown in dark grey.

Figure 4. Production of extracellular animal Mb in Pichia pastoris. Cell-free supernatants were collected after fermentation with (+) or without (-) methanol induction and concentrated 10-fold by ultrafiltration. The concentrated sample obtained after methanol induction was dark red (A) and showed a protein with the expected molecular weight of Mb after SDS-PAGE analysis (B).

Figure 5. Absence of recombinant DNA in myoglobin preparations obtained by fermentation. The same gel shows two different exposure times (upper and lower panels). The asterisks indicate the primers remaining after the PCR reaction.

Figure 6. Sequence coverage of mammoth myoglobin identified by mass spectrometry. The bar graph (lower panel) shows the intensity of different peptides on a logarithmic scale. The peptide sequences 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 Mb (black circles), bovine Mb (white circles) and commercially available Mb purified from horse muscle (triangles) were recorded at room temperature. (B) Absorption spectra of horse Mb during incubation for 24 hours at pH 5.6 and 25°C. The inset shows the ratio between the absorbance at 580nm (MbO2) and 505nm (MetMb) of recombinant mammoth Mb (black columns), bovine Mb (grey columns) and commercially available horse Mb (white columns). Asterisks indicate statistically significant differences between recombinant Mb and horse Mb: (*) = p < 0.05, (**) = p < 0.01.

Figure 8. Color of laboratory-made meat analogs containing myoglobin. Images (A) and quantification (B) show the effect of adding myoglobin on the color of a plant-based burger.

Figure 9. Color stability of meat analogs containing myoglobin. The images (A) and quantification (B) show the color changes observed over time for plant-based burgers containing different concentrations of purified horse Mb exposed to constant light at 4°C. Commercial burgers containing soy leghemoglobin were used for comparison.

Figure 10. Volatile compounds extracted from laboratory-made meat analogs containing recombinant heme proteins. (A) Quantification of the number of volatile compounds recovered 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 according to Tukey's HSD test. (B) Principal component analysis (PCA) of volatiles from laboratory-made plant-based burgers, either raw (shown in green) or grilled (shown in blue). A commercially available plant burger containing recombinant soybean leghemoglobin (LegH) was used as a reference.

Figure 11. 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 Mb 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) Aroma description of volatile compounds also present in cooked meat (van Ba et al. 2021, The Good Scent Company information system).

Figure 12. Iron bioavailability from myoglobin. Iron uptake by human Caco-2 enterocytes exposed to 0.5 mg/ml, 1.0 mg/ml or 2.0 mg/ml of purified bovine Mb compared to control medium (CM) alone (ferritin content in ng, normalized to total protein content in mg). Asterisks indicate statistically significant differences between CM and treatment: (****) = p < 0.0001.

Figure 13. Effect of recombinant myoglobin on differentiated Caco-2 cell monolayers. Cytotoxicity was measured after 24 h of incubation in the presence of recombinant mammoth or bovine Mb or complete medium (CM) as control. Asterisks indicate statistically significant differences between CM and treatment: (*) = p <0.05; ( ^** ) = p < 0.01.

Examples

Example 1: Production Method A

Example 1.1: General construction of gene constructs and vectors

In some cases, complete vectors and/or genomic integration boxes suitable for expression in Pichia pastoris, Escherichia coli, and Aspergillus niger are synthesized and manufactured by commercial suppliers. In some cases, the construction of vector fragments and/or genomic integration boxes is carried out as follows: PCR is used according to the manufacturer's protocol (using primers designed for introducing compatible overhangs between fragments and commercially available polymerases), followed by assembly of complete vectors and/or genomic integration boxes in vitro using commercially available kits according to the manufacturer's protocol. In some cases, vector fragments are transformed into yeast cells as previously described in the art (Kuijpers et al., 2013), and complete vectors are then isolated using commercially available kits, and complete vector assembly is performed in vivo.

The complete vector comprises the known necessary origin of replication sequences for maintaining the vector in the corresponding organism, the nucleotide sequence to be expressed (selected from: SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16), suitable regulatory elements (at least a promoter and a terminator) for expression in the corresponding organism, and a selection marker that allows selection of correct transformants and maintenance of the vector in the transformed strain. The genomic integration cassette comprises the nucleotide sequence to be expressed (selected from: SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16), suitable regulatory elements (at least a promoter and a terminator) for expression in the corresponding organism, and optionally 5' and 3' regions of suitable length (e.g. 30-3000 bp) that are homologous to the corresponding genomic integration site to promote homologous recombination. In some cases, the genomic integration cassette is fused and/or co-transformed with a suitable selection marker to facilitate selection. In some cases, after conversion, use methods well known in the art (such as counter selection in suitable culture medium, using Cre-Lox recombinase system or using CRISPR/Cas method) to remove the suitable selection marker fused with genomic integration box from the final production strain, to produce unmarked bacterial strain.In some cases, the nucleotide sequence expressed from carrier and/or genomic integration box is operably connected to the sequence encoding suitable signal peptide, to promote the secretion of the fusion protein expressed, and/or be operably connected to the sequence tag, to promote the purification of the fusion protein expressed after its secretion.In some cases, the sequence operably connected, for example, causes the peptide expressed to be fused to the N-terminal of fusion protein.In some cases, the fusion sequence comprises the recognition site of natural or synthetic peptidase, and peptidase helps to cut signal peptide and/or label from fusion protein when or after the fusion protein expressed by cell secretion.

Example 1.2: General strain construction

Pichia pastoris, Escherichia coli and Aspergillus niger strains are transformed using molecular toolbox techniques known in the art. Depending on whether to use selective markers and their types (dominant/auxotrophic), after allowing enough time to observe colony growth, under known suitable growth conditions of each organism, the selection of correct transformants is carried out in suitable known selective growth medium (e.g., comprising antibiotics). The correct transformant in each case is confirmed by diagnostic PCR or other suitable known molecular toolbox methods (e.g., Western blot). In some cases where a genomic integration box is used for transformation, the strain to be transformed has defects in the non-homologous end joining repair mechanism to promote homologous recombination. In some cases where a selective marker is used for transformation, the selective marker is removed as described in Example 1, for example, to produce a marker-free strain. The correctly transformed strain is stored in a glycerol stock solution at -80°C for further use.

Example 1.3: Transformed strains produce myoglobin

The extracellular myoglobin production of the transformed bacterial strain obtained in Example 2 is tested. Cell culture is carried out by culturing in a shake flask under culture conditions that are suitable for each host and contribute to the production of protein, and these conditions are generally known, such as temperature, pH and ionic strength of the growth medium and stirring speed. In some cases, the temperature value ranges from 16°C to 40°C, the pH value ranges from 3 to 7, the ionic strength value ranges from 100 to 1000 mM, and the stirring speed ranges from 100 to 300 rpm. The growth medium suitable for each host culture is generally known and comprises a carbon source and a nitrogen source, as well as additional nutrients (such as inorganic salts and vitamins). Cultures are inoculated from the frozen stock solution of the transformed bacterial strain. After culturing for 12 to 24 hours, the culture fluid is collected and biomass is removed by centrifugation. Then known techniques (such as SDS-PAGE electrophoresis) are used to test the presence of myoglobin produced in the culture supernatant by identifying the correct size band or by using the Western blotting of commercially available antibodies. The result confirms that extracellular myoglobin is produced. The produced myoglobin was then purified from the culture supernatant and purified by chromatographic techniques according to standard protocols. The absence of secretory peptides (cleaved during secretion) was confirmed by mass spectrometry according to standard protein sequencing protocols. The purified myoglobin was stored at -20°C for later use.

Example 1.4: Production of meat substitutes

According to known procedures, the purified myoglobin produced in Example 3 is used to produce muscle tissue and lipid (fat) tissue. In order to produce muscle tissue substitutes, purified myoglobin is cross-linked with pea globulin by transglutaminase. Muscle tissue substitutes are also produced by forming a heated gel of pea globulin, adding purified myoglobin to it and mixing it thoroughly during the cooling of the heated gel to room temperature. Muscle tissue substitutes can also be made by co-extrusion of purified myoglobin and pea globulin. Adipose tissue substitutes are produced by forming a heated gel of pea albumin, oil and lecithin after high pressure homogenization, adding purified myoglobin to it and mixing it thoroughly during the cooling of the heated gel to room temperature. Connective tissue substitutes are produced by extrusion using a zein source according to known procedures. Muscle, fat and connective tissue substitutes are mixed in a meat grinder in the desired proportion to produce meat substitutes. In some cases, the meat substitutes are then cooked.

Example 1.5: Production of purified myoglobin by E. coli

Full-length myoglobin genes from woolly mammoth, steppe mammoth, sheep, cattle, pig, chicken and tuna (SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16) were synthesized and cloned into a modified pET-23a(+) vector containing a T7 promoter and terminator and a C-terminal hexa-His tag (Genscript Biotech, Leiden, The Netherlands). Here, it should be understood that SEQ ID NO: 9 only encodes a portion of myoglobin, but can still be synthesized and cloned into the vector. The ampR marker gene originally present in the vector was replaced by the proBA operon from E. coli strain K12, including its original transcriptional regulatory elements, to facilitate antibiotic-free selection. The correctly assembled plasmid was confirmed by PCR and used to transform a proline auxotrophic E. coli protein production strain (E. coli K12ΔproBA). The transformed strain was incubated overnight at 37°C and 150 rpm (pH 6) in a shake flask containing minimal medium (10.5 g/L K ₂ HPO ₄ , 4.5 g/L KH ₂ PO ₄ , 1.0 g/L (NH ₄ ) ₂ SO ₄ , 0.12 g/L MgSO 4 , 0.5 g/L nitrate, 2 g/L glucose and 5.0 mg/L thiamine'HCl). 500 μl of the overnight culture was transferred to a 1 L shake flask containing 500 mL minimal medium and incubated at 37°C and 150 rpm until an OD600 of 0.4-1 was reached. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the culture at a concentration of 100 μM, and the culture was then incubated at 16°C and 150 rpm for 24 hours. The culture was harvested and centrifuged at 3500 x g (4°C) for 15 minutes. The supernatant was discarded and the pellet was dissolved in 50 mL BugBuster protein extraction reagent (Novagen) containing 1 KU lysozyme/ml (Sigma-Aldrich), 25 U Nuclease and cOmplete ^™ , EDTA-free protease inhibitor cocktail (Roche). The dissolved precipitate was incubated in a shaker at 4°C for 30 min. The centrifugation step was repeated and the cell-free extract (supernatant) was collected and analyzed by SDS-PAGE to confirm the production of myoglobin. The presence of the protein band of the correct size confirmed the production of woolly mammoth, steppe mammoth, sheep, cattle, pig, chicken and tuna myoglobin by the corresponding transformed strain.

For purification, the cell-free extract containing protein soluble fraction is loaded into the HisTrap FF 1mL column (Cytiva, Massachusetts, USA) coupled with the AKTA startup system. The column is balanced with 20mM HEPES, 0.4M NaCl and 20mM imidazole, pH 7.5, 1mL/min flow rate. Protein is eluted with 20mM HEPES, 0.4M NaCl and 400mM imidazole (pH 7.5). The fraction containing myoglobin is collected, concentrated and confirmed by SDS-PAGE and Western blotting using anti-histidine tag antibody (Bio-Rad), which confirms the successful purification of all myoglobin. The purified myoglobin is stored at -20°C for later use.

Example 1.6: Production of purified myoglobin by Aspergillus niger

A synthetic full-length myoglobin expression cassette comprising genes from woolly mammoth, steppe mammoth, sheep, cattle, pig, chicken and tuna (SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16) under the control of the Aspergillus niger glaA promoter and terminator, operably linked to the secretion signal sequence of pectin methylesterase from Aspergillus niger to promote myoglobin secretion. In this context, it is understood that SEQ ID NO: 9 encodes only a portion of myoglobin but may still be included in the expression cassette. The genomic integration cassette was assembled by fusion PCR of the myoglobin expression cassette with the orotidine 5'-phosphate decarboxylase gene sequence (pyrG) from Aspergillus oryzae and 1000 bp at the 5' and 3' ends homologous to the native upstream and downstream sequences of the Aspergillus niger pyrG gene.

The genomic integration cassette was transformed into protoplasts of Aspergillus niger CBS 120.49ΔkusAΔpyrG. Prior to transformation, protoplasts were prepared by overnight shaking flask culture at 30°C and 250 rpm in complete medium (2% (mass/volume) glucose, 6 g/L NaNO ₃ , 1.5 g/L KH ₂ PO ₄ , 0.5 g/L KCl, 0.5 g/L MgSO ₄ *7H ₂ O, 0.2% (mass/volume) tryptone, 0.1% (mass/volume) yeast extract, 0.1% (mass/volume) casamino acids, 0.05% (mass/volume) yeast RNA, and trace elements according to Vishniac (1957)). Mycelia were harvested by filtration and dissolved in PS buffer (0.2 M sodium phosphate buffer, 0.8 M L-sorbitol, pH 6), and 0.5 g of 1% ethanol was added. Pro lyase/g mycelium, then incubate at 30°C and 100rpm. Undigested mycelium is removed by filtration, and protoplasts are collected by mild centrifugation (1500x g, 3°C), washed with SC solution (182.2g L ^-1 sorbitol, 7.35g L ^-1 CaCl ₂ *2H ₂ O) and resuspended in the same solution to a concentration of 10 ⁸ protoplasts/mL. Fresh protoplasts are transformed by: 200 μL protoplast suspension is mixed with 5 μg genomic integration cassette in a mixture containing 20 μL 0.4M ATA (golden tricarboxylic acid ammonium salt) and 100 μL 20% PEG-4000, and then incubated for 10 minutes. Then, 5mL of 1.2M sorbitol solution is added and the mixture is incubated for another 10 minutes. The transformed protoplasts are collected by mild centrifugation and resuspended in 1mL of the same sorbitol solution. Transformed protoplasts were plated on minimal medium agar plates (1.5% (mass/vol) agar, 2% (mass/vol) glucose, 6 g/L NaNO ₃ , 1.5 g/L KH ₂ PO ₄ , 0.5 g/L KCl, 0.5 g/L MgSO ₄ *7H ₂ O, and trace elements according to Vishniac (1957)) and incubated at 30° C. for 4 days (pH 5). DNA was extracted from selected transformants using standard phenol/chloroform extraction. Correct integration of the genomic integration cassette containing the myoglobin gene from woolly mammoth, steppe mammoth, sheep, cattle, pig, chicken and tuna was confirmed by Western blotting.

Spores of the correct transformant were obtained by incubation at 30°C for four days on complete medium agar plates and harvested with 10mL N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer. 2x10 ⁸ spores were used to inoculate a shake flask culture containing 400mL of minimal medium and incubated overnight at 30°C, 250rpm. The mycelium was transferred to a fresh shake flask culture containing 400mL of minimal medium and cultivated for 24 hours at 30°C, 250rpm. Cells were removed by harvesting the culture and then centrifuged at 3200x g for 10 minutes at 4°C. The supernatant was analyzed by SDS-PAGE to confirm myoglobin production. The presence of the protein band of the correct size confirmed that the corresponding transformed strain expressed woolly mammoth, steppe mammoth, sheep, cattle, pig, chicken and tuna myoglobin.

For purification, the culture supernatant containing the soluble protein fraction is loaded onto a HiLoad 16/600 Superdex 75pg column (10mm x 300mm) (Situofan, Massachusetts, USA). The column is balanced with 0.15M ammonium acetate, pH 6.0, and a flow rate of 0.75mL/min. According to standard protocols, the fractions containing myoglobin are collected, concentrated, and confirmed by SDS-PAGE and LC-MS. The results confirm the presence of purified myoglobin without a secretory signal peptide.

Example 1.7. Production of purified myoglobin by Pichia pastoris

A full-length myoglobin expression cassette was synthesized, containing genes from woolly mammoth, steppe mammoth, sheep, cattle, pig, chicken, and tuna (SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16) under the control of the native pgk1 promoter and terminator of Pichia pastoris. The genomic integration cassette was assembled by fusion PCR of the myoglobin expression cassette with the gene encoding the native Pichia pastoris histidine biosynthesis trifunctional protein gene (his4) under the control of its own promoter and terminator and 1000 bp at the 5' and 3' ends homologous to the native upstream and downstream sequences of the Pichia pastoris his4 locus. The genomic integration cassette was transformed into the histidine auxotrophic Pichia pastoris strain Bg12 (BioGrammatics Inc. Carlsbad, CA) without an antibiotic resistance marker using the lithium acetate transformation protocol previously described in Gietz and Woods (2002) (Gietz RD et al., (2002), Methods Enzymol., 350:87-96). Correct transformants were obtained by incubation on 2% w/v agar plates containing synthetic medium (without histidine to facilitate selection) as previously described in Verdyun et al. (1992) (Verduyn C et al. (1992), Yeast, 8: 501-517): ₅ g/L ( _NH4 ) _2SO4 , 3 g/ _{L KH2PO4} , 0.5 _g /L MgSO4.7H2O, 4.5 mg/L ZnSO4.7H2O, 0.3 mg/L _{CoCl2.6H2O , 1 mg} /L _{MnCl2.4H2O , 0.3 mg} /L _CuSO4.5H2O , _4.5 mg/L _CaCl2.H2O , 3 _mg /L _FeSO4.7H2O , _0.4 mg/L _NaMoO4.2H2O O, 1 mg/L H ₃ BO ₃ , 0.1 mg/L KI, 0.05 g/L biotin, 1 mg/L calcium pantothenate, 1 mg/L nicotinic acid, 25 mg/L inositol, 1 mg/thiamine.HCl, 1 mg/L pyridoxine.HCl, 0.2 mg/L p-aminobenzoic acid, and 2% w/v glucose (pH 5) as a carbon source. The plates were incubated at 30° C. for 4 days. Correct integration of the genomic integration cassette containing the myoglobin gene from woolly mammoth, steppe mammoth, sheep, cattle, pig, chicken, and tuna was confirmed by colony PCR after DNA was prepared using a yeast protein kit (Zymo Research, Irvine, CA) according to the manufacturer's protocol. Glycerol stocks of correct transformants were prepared and stored at −80° C.

For myoglobin production, 1L pre-culture shake flask containing 500mL synthetic medium was inoculated with frozen glycerol stock solution and incubated overnight at 30°C and 250rpm. Pre-culture was used to inoculate subsequent shake flask cultures to an initial OD660 of 0.2. Culture was incubated at 30°C and 250rpm for 24 hours. Harvest culture and centrifuged at 3500x g (4°C) for 15 minutes. Discard supernatant, resuspend cell pellet in ice-cold demineralized water and repeat centrifugation step. Discard supernatant and use yeast protein kit (Zuomo Research, Irvine, California) together with mechanical disruption and lysis cells according to the manufacturer's protocol. A mixture containing cell debris and soluble protein was centrifuged at 3500x g (4°C) for 15 minutes. Collect supernatant (cell-free extract) containing soluble protein and analyze by SDS-PAGE to confirm myoglobin production. The presence of protein bands of the correct size confirmed the production of woolly mammoth, steppe mammoth, sheep, cattle, pig, chicken, and tuna myoglobin by the corresponding transformed strains.

For purification, the cell-free extract containing the soluble protein fraction is loaded onto a HiLoad 16/600 Superdex 75pg column (10mm x 300mm) (Situofan, Massachusetts, USA). The column is balanced with 0.15M ammonium acetate, pH 6.0, and a flow rate of 0.75mL/min. According to standard protocols, the fractions containing myoglobin are pooled, concentrated, and confirmed by SDS-PAGE and LC-MS. The results confirm the presence of purified myoglobin.

Example 2: Production Method B

Unless otherwise expressly stated, "mammoth" and "mammoth myoglobin" in Example 2 refer to "steppe mammoth" and "steppe mammoth myoglobin".

Example 2.1. Materials and methods

Sequence analysis

Sequences encoding myoglobin from cattle, jungle fowl, Pacific bluefin tuna, sheep and wild boar were obtained from Uniprot (accession numbers P02192, P02197, P68190, P02190, P02189, respectively). The myoglobin coding sequence from the steppe mammoth (Woody mammoth), which was unknown at the time of filing this application, was obtained by the inventors after extracting DNA from molar samples of the so-called Adycha specimen, Illumina DNA sequencing, merging reads and mapping them against the African steppe elephant (Loxodonta africana) genome (van der Valk et al. 2021).

Multiple sequence alignment was performed using Clustal Omega (Sievers et al. 2011) and visualized using Jalview 2.11.1.4 (Waterhouse et al. 2009).

Protein Modeling

The structure of wild-type deoxymyoglobin from wild boar was retrieved from the RCSB Protein Data Bank (PDB accession number: 1MWD, chain A). The structure of steppe mammoth myoglobin was modeled using the automated protein structure homology modeling server SWISS-MODEL (Waterhouse et al. 2018) using the crystal structure of myoglobin from Elephas maximus (Asian elephant) as a template (PDB accession number: 1EMY). The net surface charge (Z _Mb ) was calculated as the sum of the charges of all ionizable groups at pH 6.5 using published site-specific ionization constants (Mirceta et al. 2013). All protein structures were visualized and figures were generated using DeepView v4.1 (Guex and Peitsch 1997).

Construction of expression plasmid

The myoglobin coding sequence was codon optimized for expression in Pichia pastoris (Phaffia ) (Love et al. 2016) (SEQ ID NOs: 19-24). It was preceded by an optimized sequence of the coding sequence of Saccharomyces cerevisiae mating factor α (SEQ ID NOs: 25-30) chemically synthesized by GenScript. The gene fragment was cloned into the pBDIPp5 vector, downstream of the AOX1 promoter and upstream of the AOX1 terminator, HIS4 selection marker and AOX1 3′ fragment. The vector was amplified in Escherichia coli (DH10B), purified with a plasmid kit (Quiagen) and verified by Sanger sequencing. The expression cassette was generated from the resulting vector by restriction enzyme digestion of the plasmid with BglII (New England Biolabs, cutting upstream of the AOX1 promoter and downstream of the AOX1 3′ fragment) and purified with a QIAquick gel extraction kit (QUiagen).

Strain Engineering

Pichia pastoris (Phaffi's yeast) strain GS115 (his4) is available from Life Technologies. Basically as previously described (Cregg 2007), electroporation is used to carry out cell transformation. In brief, the cells of GS115 strain grow in YPD (1% yeast extract, 2% peptone and 2% D-glucose) culture medium. The cells in the exponential growth phase are hatched for 30 minutes in the YPD culture medium containing 200mM HEPES buffer (pH 8.0) and 25mM dithiothreitol. Then the competent cells are washed with ice-cold 1M sorbitol, and transferred to a sterile electroporation cuvette (Bor-Rad). Then Gene-Pulser (Bor-Rad) electroporator is used to electroporate cells with 1-5 μg linear expression cassette (see 3.3), and is resuspended in the 1mL YPD culture medium containing 1M sorbitol, and then transferred to a sterile 1.5-mL eppendorf tube. The cells were incubated at 28°C without agitation for 3 hours and then plated onto agar plates containing solid MGY medium (minimal glycerol medium: 1.34% yeast nitrogen base (with ammonium sulfate but without amino acids), 2% D-glucose, 4· ^10-5 % biotin, and 2% agar). The plates were incubated at 28°C for up to 4 days.

Transformants that can grow in the absence of histidine were screened for their ability to express Mb. Cells were grown in BMGY medium (1% yeast extract, 2% peptone, 100 mm potassium phosphate buffer (pH 6), 1.34% yeast nitrogen base (with ammonium sulfate but without amino acids), ^4.10-5 % biotin, 1% glycerol) in 50 mL Falcon tubes. 1% methanol was added to exponentially growing cells 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).

The best producing strains were selected for further experiments and stored frozen at -80°C as master cell banks before use. The results shown here were obtained using the PAL-02-02 (mammoth Mb) and PAL-02-03 (bovine Mb) strains.

Recombinant protein production and purification

Cells from the master cell bank were inoculated on YPD agar (1% yeast extract, 2% peptone and 2% D-glucose, 2% agar) plates and incubated at 28°C for 2 days. Seed cultures were then prepared in baffled flasks containing 100mL BMGY culture medium and incubated at 28°C for 24 hours in an orbital shaking incubator. Seed cultures were then used to inoculate 3L spherical glass bottles or glass container fermenters (Sartorius) containing 900ml BMGY culture medium and 0.03% ^FoamAwayTM irradiated AOF (ThermoFisher). The breeding phase was carried out for about 24 hours until all glycerol was consumed. The temperature was then reduced to 26°C, and methanol 1% was added every 12 hours to induce Mb expression. Dissolved oxygen was maintained at above 20% during the entire fermentation period, while pH was maintained at 6 during the breeding phase, and pH was maintained at 5 during the induction phase.

After 96 hours of methanol induction, cells were removed by centrifugation at 4,000 rpm at 4°C. Remaining cells were removed by microfiltration using a nitrocellulose filter with a pore size of 0.45 μm (Millipore). Cell-free supernatants were stored frozen at -20°C. When assayed, cell-free supernatants were further concentrated by using a disposable ultrafiltration centrifuge device with a molecular weight cutoff of 10 kD for a polyethersulfone membrane (Thermo Scientific Pierce Protein Concentrator).

The presence of recombinant DNA in the cell-free supernatant was tested by PCR using oligonucleotides designed to be complementary to the sequence encoding the signal peptide (Sigma). A DNA fragment amplified from genomic DNA of the PAL-02-02 strain (mammoth Mb) was used as a positive control, alongside 1 μl of concentrated cell-free supernatant containing mammoth or bovine Mb. PCR reactions were performed with 2X master mix and standard buffer (New England Biolabs). PCR products were visualized after migration at 60V for 60 min in 2% agarose TAE gel containing ethidium bromide. Purple 1 kb Plus DNA ladder (New England Biolabs) was used to control the size of the amplified fragments.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PA GE)

For protein electrophoresis, 20 μl of concentrated cell-free supernatant was mixed with an equal volume of 2X protein loading buffer (100 mM Tris (pH 6, 8); 4 mM EDTA, 4% SDS, 20% glycerol; 0.02% bromophenol blue, 4% β-mercaptoethanol). The samples were incubated at 95°C for 5 minutes and the appropriate volume was loaded onto a 15% polyacrylamide gel on a vertical micro PROTEAN gel apparatus (Bio-Rad) at 200 V for 40 minutes. Molecular weight markers (PageRuler ^TM prestained protein ladder) were purchased from Thermofisher. After electrophoresis, the proteins in the gel were stained with Coomassie Brilliant Blue G-250 (Bio-Rad).

Mass spectrometry

For MS analysis, the SDS-PAGE gel band corresponding to Mb was cut from the coomassie-stained gel using a scalpel. The samples were rinsed with sterile water and stored at -20 ° C until MS analysis was performed at the VIB Proteome Center (Gent, Belgium). Peptides were generated by digestion overnight with 1 μg trypsin (Promega) at 37 ° C and stored at -20 ° C until LC-MS/MS analysis. The peptides were redissolved in the loading solvent and injected for LC-MS/MS analysis using a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Data analysis was performed using the MaxQuant algorithm (version 2.0.1.0), with default search settings including a false discovery rate of 1% at the peptide spectrum match (PSM), peptide and protein levels. All raw spectral data files were searched against the theoretical protein sequence of mammoth Mb and the following reference proteomes from the Uniprot database: Pseudomonas aeruginosa (database release version 2021_11, containing 5,073 protein sequences), Bos bovis (taxid 9913, database release version 2021_11, containing 37,513 protein sequences) and Sus scrofa (taxid 9823, database release version 2021_11, containing 49,792 protein sequences).

Plant-based meat analogs

Plant-based burgers were made by mixing 25% textured soy protein, 15% sunflower oil, 1.5% NaCl, 1% methylcellulose and 57.5% water. Recombinant myoglobin or commercially available myoglobin (purified from horse muscle, Sigma) was added at a final concentration of 0.5% or 1%, similar to the myoglobin content of white meat and red meat, respectively. Recombinant myoglobin preparations were obtained by lyophilization of cell-free supernatant. For burgers containing 1% Mb, an additional 13.8 g water/100 g burger was added. Commercially available plant-based burgers containing soy leghemoglobin (Impossible Food) were included for comparison in certain assays.

Spectral and color analysis

The absorption spectra were recorded with Synergy microplate reader (BioTek). The absorbance measurements for determining the auto-oxidation rate of myoglobin were performed in quartz cuvettes using an Ultrospec III spectrophotometer (Pharmacia). The color measurements were performed using a portable Miniscan EZ 4500L 45°/0° (Hunterlab, Murnau, Germany), with an observation area size of 8 mm, a light source of D65, and a standard observer of 10° to record L*, a*, and b* values (based on CIELAB color space). The differences in color over time (ΔE) were calculated according to the CIE76 formula:

ΔE-value (-)

Aromatic Analysis

Aroma of plant-based meat substitutes containing different concentrations of recombinant Mb was analyzed using gas chromatography-mass spectrometry (GC-MS) and headspace solid phase microextraction (HS-SPME). The samples were analyzed either raw or after roasting. The headspace volatiles were extracted by divinylbenzene-carboxyl-polydimethylsiloxane (DVB/CAR/PDMS) coated fibers and separated by HP-1ms column and then identified by mass spectrometry. For data analysis, the areas under the peaks of different aromatic compounds were evaluated by one-way ANOVA (analysis of variance), followed by Tukey's HSD (honestly significant difference) post hoc test when statistically significant differences were found. Multivariate statistical analysis was performed using PCA (principal component analysis).

Iron Bioavailability and Bioaccessibility

The assay was performed by ProDigest (Ghent, Belgium). Caco-2 cells (HTB-37; American Type Culture Collection) were seeded in 12 wells coated with 0.1% gelatin at 5x105 cells. Cells were grown for 14 days in complete medium (Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 10mM HEPES and 1x antibiotic-antimycotic), with the medium replaced 3 times a week. 24 hours before stimulation, the cells were washed once with minimal essential medium (MEM) supplemented with 10mM HEPES, 2mM L-glutamine, 1x antibiotic-antimycotic, 11μM hydrocortisone, 0.87μM insulin, 0.02μM sodium selenite, 0.05μM 3,3′,5-triiodo-L-thyronine sodium salt, 20μg/L epidermal growth factor, and further incubated in the medium for 24h. Then, the cells were incubated with three concentrations (0.5, 1 and 2 mg/mL) of bovine Mb (Tebu-bio NV) or supplemented MEM as a negative control (expressed as CM). After incubation at 37°C for 24 hours, the cells were washed twice with ice-cold PBS and lysed with CelLytic ^™ (Sigma Aldrich). Human ferritin levels were determined using a human ferritin ELISA kit (ThermoScientific) according to the manufacturer's instructions. Finally, protein concentrations were determined using a Pierce ^™ BCA protein assay kit (ThermoFisher Scientific) according to the microplate procedure instructions. All assays were performed in triplicate.

In vitro toxicity assay

The assays were performed by ProDigest (Ghent, Belgium). Caco-2 cells were seeded in 24-well plates coated with 0.1% gelatin and cultured as per the ferritin assay. On the day of the toxicity test, cells were incubated with 0.5, 1.0 or 2.0 mg/ml of recombinant mammoth or bovine Mb. Cytotoxicity assays were performed after 24 h of incubation with Mb at 37°C. To evaluate the possible toxicity of the products on Caco-2 cells, the supernatants were subjected to the lactate dehydrogenase (LDH) cytotoxicity assay (Merck Life Science B.V.) according to the manufacturer's instructions. All assays were performed in triplicate. To evaluate the differences between the complete medium control (CM) and the products, a normal one-way ANOVA with Dunnett's multiple comparison test was performed for each time point. (*) indicates a statistically significant difference between CM and the product. (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001 and (****) = p < 0.0001. All statistics were performed using GraphPad Prism version 9.1.2 for Windows (GraphPad Software, San Diego, CA, USA).

Bacterial reverse mutation assay (AMES test)

Genotoxicity assay was performed according to OECD chemical test guide n ° 471. AMES FT mutagenicity test kit (Moltox, Trinova) was used according to the manufacturer's instructions. All assays were performed in triplicate. According to the data provided by the manufacturer, the average revertant counts of each bacterial strain treated with the vehicle were within the expected range. The mutation rate was calculated as the ratio between the number of revertants observed using the test compound and the vehicle alone.

Example 2.2. Structural features of myoglobin

Myoglobin (Mb) is a relatively small globular protein of approximately 17 kD found in cardiac and skeletal muscle. It carries a single reversible heme group that is able to bind oxygen (Figure 1), enabling myoglobin to transport oxygen from the cell surface to the mitochondria. Mb is also the main pigment responsible for the color of meat. In oxidized Mb (oxymyoglobin), the iron atom of the heme group is coordinated to four nitrogen atoms of the porphyrin ring, the so-called “proximal” histidine (His94) in the Mb chain, and to an oxygen molecule. This can be further stabilized by hydrogen bonding between the oxygen molecule and another histidine in the Mb heme binding pocket, the “distal” histidine (His65). In this form, Mb has a typical bright red color. In the absence of oxygen, Mb is dark red (deoxymyoglobin). When the heme iron is oxidized from the ferrous (Fe(II)) to the ferric (Fe(III)) state, it is unable to bind oxygen and Mb is brown (metmyoglobin), as seen in cooked meat. Heme iron oxidation also reduces the affinity of Mb for heme, leading to increased heme loss and subsequent protein unfolding.

The amino acid sequence of Mb is well conserved during evolution and shows relatively little variation between species (Fig. 2). In proboscideans, Mb presents an atypical phenylalanine at position 30 (Phe30). Of note, the Mb sequence of the steppe mammoth (Mammuthu strongotherii) that we generated from the so-called Adycha specimen (dated between 1.2 and 1.0 Ma) is also included in Fig. 2.

In addition to the characteristic Phe30 substitution (Figure 2), we found that Mb from the steppe mammoth (net surface charge at pH 6.5 ( _ZMb ) = 2.67) showed a higher positive surface charge than Mb from, for example, the Asian elephant ( _ZMb at pH 6.5 = 2.11), due to the presence of a histidine residue instead of glutamine at position 92 (His92) (Figures 2, 3). This is reminiscent of the adaptation of Mb to deep-sea diving in cetaceans, where the increased positive surface charge leads to a reduction in attractive interactions between Mb molecules at contact distances, thereby increasing Mb stability and preventing its aggregation. Overall, Mb from the steppe mammoth significantly presents several features that make it particularly attractive. Here, we evaluated the production and characteristics of recombinant Mb from the steppe mammoth along with other extant species.

Example 2.3. Extracellular production of myoglobin in Pichia pastoris

The sequences encoding Mb from steppe mammoth, pig, chicken, cattle, pork and tuna were cloned into the downstream of the methanol-inducible AOX1 promoter of Pichia pastoris and the upstream of the so-called 3' fragment of the AOX1 terminator, histidine prototrophic selection marker and AOX1 gene. Myoglobin is naturally present in the cytoplasm of muscle cells. In order to promote the purification of recombinant protein, we fused the sequence encoding signal peptide to the myoglobin coding sequence frame to target the nascent Mb protein to the secretory pathway for secretion outside the cell. These constructs are used to transform histidine-auxotrophic Pichia pastoris cells, and transformants are selected according to their ability to grow in the absence of histidine. For each construct, up to ten transformants were tested for their ability to produce extracellular animal myoglobin after methanol induction in microtiter plates. The best production clones were further tested in flasks. Methanol induction caused the cell-free supernatant to appear dark red, especially after being concentrated 10 times by ultrafiltration, as expected for samples containing heme proteins (Figure 4A). The presence of a methanol-induced protein with the expected molecular weight (approximately 17 kD) was also confirmed after protein electrophoresis and staining with Coomassie blue (Figure 4B). Recombinant Mb accounted for 75-82% of the total protein in the concentrated cell-free supernatant, while the yield of Mb varied between 0.420 mg and 1.93 g/liter of supernatant.

Since myoglobin is produced extracellularly, there is no need to lyse yeast cells during the purification process. These cells are removed from the fermentation broth by centrifugation and then microfiltration (0.45 μm pore size). Therefore, it is expected that the final product will not contain any recombinant genetic material. To verify this, we designed a PCR test based on the amplification of a short fragment (130 bp) of the recombinant gene from as little as 1 pg of DNA, which corresponds to the sequence encoding the signal peptide (Figure 5). Using this test, we failed to detect any recombinant DNA in the concentrated cell-free supernatant (Figure 5).

To confirm the identity of the recombinant Mb and ensure that the signal peptide was properly processed and removed during protein secretion, we analyzed the mammoth Mb gel bands by liquid chromatography coupled to mass spectrometry (LC-MS/MS) shotgun measurements. We recovered 90.3% of the peptides covering the entire protein sequence and observed complete removal of the signal peptide and the first methionine, as expected (Figure 6).

Example 2.4. Color and color stability of recombinant myoglobin

As mentioned above, Mb plays a central role in meat color. Therefore, in parallel with mass spectrometry analysis, we measured the absorbance of concentrated cell-free supernatants in the UV and visible range and compared it with the absorbance of commercial myoglobin purified from horse muscle. In all cases, characteristic absorption peaks (Soret bands) for the presence of heme were observed (Tang et al., 2004). The Soret bands of bovine and horse Mb were detected at 410 nm, while those of mammoth Mb were slightly red-shifted to 415 nm (Figure 7A). Based on the absorbance ratios at the wavelength maxima representing metmyoglobin, deoxymyoglobin, and oxymyoglobin, respectively (Tang et al., 2004), we observed that the recombinant myoglobin in the concentrated cell-free supernatant was mainly present in the reduced form: 47.3% oxymyoglobin and 10.0% deoxymyoglobin for mammoth Mb, and 48.3% oxymyoglobin and 9.9% deoxymyoglobin for bovine Mb. Notably, the Soret peak of mammoth Mb was consistently observed at slightly higher wavelengths than that of bovine and horse Mb (415 nm and 410 nm, respectively). This is consistent with previous observations of elephant Mb (Tada et al., 1998). To assess the auto-oxidative behavior of Mb, we recorded absorption spectra every two hours during a 24-hour incubation at pH 5.6 and 25°C (Figure 7B). We then examined the ratio between the absorbance at 580 nm (the peak of oxymyoglobin carrying Fe(II)) and the absorbance at 505 nm (the peak of metmyoglobin carrying Fe(III)). At the beginning of the incubation, we observed that this ratio was significantly higher for recombinant mammoth and bovine Mb than for commercially available Mb purified from horse muscle (Figure 7B, inset), indicating that most of the recombinant Mb was present in a reduced form. This difference from horse Mb was maintained over time at acidic pH (Figure 7B, inset). Over the course of 24 h, the absorbance ratio of mammoth Mb decreased by 26.0%, while that of bovine Mb decreased by 38.3%, indicating that the former exhibited a stronger anti-autooxidation capacity.

Meat color is affected by multiple parameters, including Mb concentration, moisture, and fat content. For conventional meats, which are often purchased raw, the red color imparted by oxymyoglobin plays a key role in consumer purchasing decisions. Therefore, we tested the effect of recombinant Mb on meat analogs based on color. Adding bovine or mammoth Mb to plant-based burgers (Figure 8A-A') resulted in a decrease in lightness (L*-value) and an increase in red (a*-value) and yellow (b*-value) (Figure 8B).

Next, we evaluated the color stability of meat analogs containing different amounts of myoglobin, which were stored in a refrigerator and exposed to constant light for 5 days. Commercially available plant-based burgers containing soy legheme protein were used for comparison. Although we observed significant color changes in burgers prepared without Mb, the addition of Mb was associated with greater color stability (Figures 9A-B).

Example 2.5. Aromatic analysis

In addition to influencing meat color, myoglobin is often considered to contribute to the “bloody” or “metallic” smell of raw meat. During meat cooking, aroma formation occurs mainly through lipid oxidation and Maillard reactions. The latter occur between amino groups in proteins and carbonyl groups from reducing sugars and/or lipid oxidation reaction products. Heme iron can influence these reactions and/or their kinetics (van Ba et al., 2012). However, to our knowledge, there are no conclusive data in the literature on the effect of myoglobin on meat aroma formation. Using the above-mentioned laboratory-made burgers, we analyzed the volatile compounds in raw and cooked plant-based burgers containing recombinant Mb by gas chromatography-mass spectrometry.

We found that the addition of Mb to the plant-based burger significantly increased the number of volatile compounds, both in the raw state and after baking (Figure 10A). Notably, the number of volatiles produced in both cases was higher than that of a commercially available plant-based burger containing soy leghemoglobin (Figure 10A), the Impossible Burger. Using principal component analysis (PCA) on the CG-MS data, we observed clear distinctions between the laboratory-based burger and the commercially available plant-based burger containing soy leghemoglobin on the one hand, and between the raw and baked products on the other hand (Figure 10B).

When analyzing the volatile compounds in grilled, laboratory-made plant-based burgers, we found that 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 resulted in an increase in the number of compounds known to impart a “grilled” flavor to grilled meats (van Ba et al., 2012) (Figure 12). The addition of mammoth Mb was consistently associated with higher amounts of these compounds than observed with bovine Mb. This suggests, without being bound by theory, that when used as an ingredient in plant-based meat analogs, lower amounts of mammoth Mb could be added to obtain the same aromatic profile as when using bovine Mb. Additionally, mammoth Mb and bovine Mb may provide different sensory experiences.

Example 2.6. Nutritional analysis

To assess the bioavailability of iron from Mb, we turned to the epithelial intestinal cell line Caco-2, which is commonly used to study human intestinal iron uptake. We measured the formation of intracellular ferritin as a readout of iron uptake by living Caco-2 cell monolayers and therefore an indicator of iron bioavailability (Glahn et al., 1998). We observed that increasing Mb doses resulted in increased iron uptake (Figure 12), indicating that the heme iron carried by Mb is bioavailable to human intestinal cells.

Example 2.7. Security testing

Cytotoxicity

To assess the potential toxicity of recombinant Mb, differentiated Caco-2 cells were treated with mammoth or bovine Mb, purified bovine Mb, or complete culture medium (CM) as a control. To test for potential cytotoxic effects, we then examined the levels of lactate dehydrogenase (LDH) in the culture supernatant. LDH is an oxidoreductase present in all cells that catalyzes the interconversion of pyruvate to lactate and NADH to NAD+. Upon damage to the cell membrane following apoptosis or necrosis, LDH is released into the supernatant and its concentration can be determined by a colorimetric assay based on the reduction of NAD+ to NADH by LDH (Decker and Lohmann-Matthes 1988). We did not observe an increase in cytotoxicity after incubation with mammoth Mb at any of the concentrations tested (Figure 13). In contrast, bovine Mb showed significantly increased toxicity compared to culture medium alone (Figure 13). Therefore, at the high concentrations tested here, mammoth Mb appears to have a better safety profile than bovine Mb.

Genotoxicity

To determine the mutagenic potential of recombinant myoglobin, we used a bacterial reverse mutagenesis assay (AMES assay) with four histidine-requiring S. typhimurium strains (TA98, TA100, TA1535, TA1537) and one tryptophan-requiring E. coli strain (WP2 uvrA) (Ames et al. 1975). Bacteria were exposed to levels of 0.8, 2.5, 8.0, 25.0, 80.0, or 250 μg/ml of mammoth or bovine myoglobin in the presence or absence of exogenous metabolic activation (S9 mix) (Table 1). The positive control material resulted in the expected increase in the number of revertants in the absence and presence of S9, confirming the sensitivity of the test and the activity of the S9 mix. We did not observe any significant mutagenic activity for bovine or mammoth Mb at any of the concentrations tested.

Table 1. Number of revertants and mutation factors after exposure to mammoth myoglobin. The average number of revertants observed in a total of 48 wells per condition is shown in black. The corresponding mutation factor (ratio of revertant counts in the presence of compound compared to vehicle alone) is shown in grey. All assays were performed in triplicate. Conditions in which significant mutagenesis was observed are highlighted in bold. Compounds used as positive controls were ^a 2-nitrofluorene 2 μg/ml, ^b aminoanthracene 4 μg/ml, ^c N4-aminocytidine 100 μg/ml.

Sequence Listing

<110> Paleo B.V.

<120> Meat substitutes

<130> P6099254PCT

<150> EP 20218000.6

<151> 2020-12-31

<150> EP 21174597.1

<151> 2021-05-19

<160> 80

<210> 1

<211> 127

<212> PRT

<213> Common Mammoth (Mammuthus primigenius)

<220>

<223> Partial sequence of mammoth myoglobin

<400> 1

Met Gly Leu Ser Asp Gly Glu Trp Glu Leu Val Leu Lys Thr Trp

1 5 10 15

Gly Lys Val Glu Ala Asp Ile Pro Gly His Gly Leu Glu Val Phe

20 25 30

Val Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp

35 40 45

Lys Phe Lys His Leu Lys Thr Glu Gly Glu Met Lys Ala Ser Glu

50 55 60

Asp Leu Lys Lys Gln Gly Val Thr Val Leu Thr Ala Leu Gly Gly

65 70 75

Ile Leu Lys Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu

80 85 90

Ala Gln Ser His Ala Thr Lys His Lys Ile Pro Ile Lys Tyr Leu

95 100 105

Glu Phe Ile Ser Asp Ala Ile Ile His Val Leu Gln Ser Lys His

110 115 120

Pro Ala Glu Phe Gly Ala Asp

125

<210> 2

<211> 154

<212> PRT

<213> Common Mammoth

<220>

<223> Mammoth Myoglobin

<400> 2

Met Gly Leu Ser Asp Gly Glu Trp Glu Leu Val Leu Lys Thr Trp

1 5 10 15

Gly Lys Val Glu Ala Asp Ile Pro Gly His Gly Leu Glu Val Phe

20 25 30

Val Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp

35 40 45

Lys Phe Lys His Leu Lys Thr Glu Gly Glu Met Lys Ala Ser Glu

50 55 60

Asp Leu Lys Lys Gln Gly Val Thr Val Leu Thr Ala Leu Gly Gly

65 70 75

Ile Leu Lys Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu

80 85 90

Ala Gln Ser His Ala Thr Lys His Lys Ile Pro Ile Lys Tyr Leu

95 100 105

Glu Phe Ile Ser Asp Ala Ile Ile His Val Leu Gln Ser Lys His

110 115 120

Pro Ala Glu Phe Gly Ala Asp Ala Gln Gly Ala Met Lys Lys Ala

125 130 135

Leu Glu Leu Phe Arg Asn Asp Ile Ala Ala Lys Tyr Lys Glu Leu

140 145 150

Gly Phe Gln Gly

<210> 3

<211> 154

<212> PRT

<213> Steppe Mammoth (Mammuthus trogontherii)

<220>

<223> Steppe mammoth myoglobin

<400> 3

Met Gly Leu Ser Asp Gly Glu Trp Glu Leu Val Leu Lys Thr Trp

1 5 10 15

Gly Lys Val Glu Ala Asp Ile Pro Gly His Gly Leu Glu Val Phe

20 25 30

Val Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp

35 40 45

Lys Phe Lys His Leu Lys Thr Glu Gly Glu Met Lys Ala Ser Glu

50 55 60

Asp Leu Lys Lys Gln Gly Val Thr Val Leu Thr Ala Leu Gly Gly

65 70 75

Ile Leu Lys Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu

80 85 90

Ala His Ser His Ala Thr Lys His Lys Ile Pro Ile Lys Tyr Leu

95 100 105

Glu Phe Ile Ser Asp Ala Ile Ile His Val Leu Gln Ser Lys His

110 115 120

Pro Ala Glu Phe Gly Ala Asp Ala Gln Gly Ala Met Lys Lys Ala

125 130 135

Leu Glu Leu Phe Arg Asn Asp Ile Ala Ala Lys Tyr Lys Glu Leu

140 145 150

Gly Phe Gln Gly

<210> 4

<211> 154

<212> PRT

<213> Sheep (Ovis aries)

<220>

<223> Sheep myoglobin

<400> 4

Met Gly Leu Ser Asp Gly Glu Trp Gln Leu Val Leu Asn Ala Trp

1 5 10 15

Gly Lys Val Glu Ala Asp Val Ala Gly His Gly Gln Glu Val Leu

20 25 30

Ile Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp

35 40 45

Lys Phe Lys His Leu Lys Thr Glu Ala Glu Met Lys Ala Ser Glu

50 55 60

Asp Leu Lys Lys His Gly Asn Thr Val Leu Thr Ala Leu Gly Gly

65 70 75

Ile Leu Lys Lys Lys Gly His His Glu Ala Glu Val Lys His Leu

80 85 90

Ala Glu Ser His Ala Asn Lys His Lys Ile Pro Val Lys Tyr Leu

95 100 105

Glu Phe Ile Ser Asp Ala Ile Ile His Val Leu His Ala Lys His

110 115 120

Pro Ser Asp Phe Gly Ala Asp Ala Gln Gly Ala Met Ser Lys Ala

125 130 135

Leu Glu Leu Phe Arg Asn Asp Met Ala Ala Gln Tyr Lys Val Leu

140 145 150

Gly Phe Gln Gly

<210> 5

<211> 154

<212> PRT

<213> Cow (Bos taurus)

<220>

<223> Bovine myoglobin

<400> 5

Met Gly Leu Ser Asp Gly Glu Trp Gln Leu Val Leu Asn Ala Trp

1 5 10 15

Gly Lys Val Glu Ala Asp Val Ala Gly His Gly Gln Glu Val Leu

20 25 30

Ile Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp

35 40 45

Lys Phe Lys His Leu Lys Thr Glu Ala Glu Met Lys Ala Ser Glu

50 55 60

Asp Leu Lys Lys His Gly Asn Thr Val Leu Thr Ala Leu Gly Gly

65 70 75

Ile Leu Lys Lys Lys Gly His His Glu Ala Glu Val Lys His Leu

80 85 90

Ala Glu Ser His Ala Asn Lys His Lys Ile Pro Val Lys Tyr Leu

95 100 105

Glu Phe Ile Ser Asp Ala Ile Ile His Val Leu His Ala Lys His

110 115 120

Pro Ser Asp Phe Gly Ala Asp Ala Gln Ala Ala Met Ser Lys Ala

125 130 135

Leu Glu Leu Phe Arg Asn Asp Met Ala Ala Gln Tyr Lys Val Leu

140 145 150

Gly Phe His Gly

<210> 6

<211> 154

<212> PRT

<213> Wild Boar (Sus scrofa)

<220>

<223> Porcine myoglobin

<400> 6

Met Gly Leu Ser Asp Gly Glu Trp Gln Leu Val Leu Asn Val Trp

1 5 10 15

Gly Lys Val Glu Ala Asp Val Ala Gly His Gly Gln Glu Val Leu

20 25 30

Ile Arg Leu Phe Lys Gly His Pro Glu Thr Leu Glu Lys Phe Asp

35 40 45

Lys Phe Lys His Leu Lys Ser Glu Asp Glu Met Lys Ala Ser Glu

50 55 60

Asp Leu Lys Lys His Gly Asn Thr Val Leu Thr Ala Leu Gly Gly

65 70 75

Ile Leu Lys Lys Lys Gly His His Glu Ala Glu Leu Thr Pro Leu

80 85 90

Ala Gln Ser His Ala Thr Lys His Lys Ile Pro Val Lys Tyr Leu

95 100 105

Glu Phe Ile Ser Glu Ala Ile Ile Gln Val Leu Gln Ser Lys His

110 115 120

Pro Gly Asp Phe Gly Ala Asp Ala Gln Gly Ala Met Ser Lys Ala

125 130 135

Leu Glu Leu Phe Arg Asn Asp Met Ala Ala Lys Tyr Lys Glu Leu

140 145 150

Gly Phe Gln Gly

<210> 7

<211> 154

<212> PRT

<213> Jungle fowl (Gallus gallus)

<220>

<223> Chicken myoglobin

<400> 7

Met Gly Leu Ser Asp Gln Glu Trp Gln Gln Val Leu Thr Ile Trp

1 5 10 15

Gly Lys Val Glu Ala Asp Ile Ala Gly His Gly His Glu Val Leu

20 25 30

Met Arg Leu Phe His Asp His Pro Glu Thr Leu Asp Arg Phe Asp

35 40 45

Lys Phe Lys Gly Leu Lys Thr Pro Asp Gln Met Lys Gly Ser Glu

50 55 60

Asp Leu Lys Lys His Gly Ala Thr Val Leu Thr Gln Leu Gly Lys

65 70 75

Ile Leu Lys Gln Lys Gly Asn His Glu Ser Glu Leu Lys Pro Leu

80 85 90

Ala Gln Thr His Ala Thr Lys His Lys Ile Pro Val Lys Tyr Leu

95 100 105

Glu Phe Ile Ser Glu Val Ile Ile Lys Val Ile Ala Glu Lys His

110 115 120

Ala Ala Asp Phe Gly Ala Asp Ser Gln Ala Ala Met Lys Lys Ala

125 130 135

Leu Glu Leu Phe Arg Asn Asp Met Ala Ser Lys Tyr Lys Glu Phe

140 145 150

Gly Phe Gln Gly

<210> 8

<211> 147

<212> PRT

<213> Pacific bluefin tuna (Thunnus orientalis)

<220>

<223> Tuna myoglobin

<400> 8

Met Ala Asp Phe Asp Ala Val Leu Lys Cys Trp Gly Pro Val Glu

1 5 10 15

Ala Asp Tyr Thr Thr Met Gly Gly Leu Val Leu Thr Arg Leu Phe

20 25 30

Lys Glu His Pro Glu Thr Gln Lys Leu Phe Pro Lys Phe Ala Gly

35 40 45

Ile Ala Gln Ala Asp Ile Ala Gly Asn Ala Ala Ile Ser Ala His

50 55 60

Gly Ala Thr Val Leu Lys Lys Leu Gly Glu Leu Leu Lys Ala Lys

65 70 75

Gly Ser His Ala Ala Ile Leu Lys Pro Leu Ala Asn Ser His Ala

80 85 90

Thr Lys His Lys Ile Pro Ile Asn Asn Phe Lys Leu Ile Ser Glu

95 100 105

Val Leu Val Lys Val Met His Glu Lys Ala Gly Leu Asp Ala Gly

110 115 120

Gln Gln Thr Ala Leu Arg Asn Val Met Gly Ile Ile Ile Ala Asp

125 130 135

Leu Glu Ala Asn Tyr Lys Glu Leu Gly Phe Ser Gly

140 145

<210> 9

<211> 382

<212> DNA

<213> Common Mammoth

<220>

<223> DNA sequence encoding a partial sequence of mammoth myoglobin

<400> 9

atgggactca gcgacgggga atgggagttg gtgttgaaaa cctgggggaa 50

agtggaggct gacatcccgg gccatgggct ggaagtcttc gtcaggctct 100

tcacaggtca tcccgagacc ctggagaagt tcgacaagtt taagcacctg 150

aagacagagg gcgagatgaa ggcctctgag gacctgaaga agcaaggtgt 200

tactgtgctc actgccctgg ggggcatcct caagaagaaa gggcatcacc 250

aggcggagat tcagcccctg gcccagtctc atgccaccaa gcacaagatc 300

cccatcaagt atctggagtt catctcggac gccatcatcc acgttctgca 350

aagcaagcat cctgcggaat ttggcgctga tg 382

<210> 10

<211> 465

<212> DNA

<213> Common Mammoth

<220>

<223> DNA sequence encoding mammoth myoglobin

<400> 10

atgggactca gcgacgggga atgggagttg gtgttgaaaa cctgggggaa 50

agtggaggct gacatcccgg gccatgggct ggaagtcttc gtcaggctct 100

tcacaggtca tcccgagacc ctggagaagt tcgacaagtt taagcacctg 150

aagacagagg gcgagatgaa ggcctccgag gacctgaaga agcaaggtgt 200

tactgtgctc actgccctgg ggggcatcct caagaagaaa gggcatcacc 250

aggcggagat tcagcccctg gcccagtctc atgccaccaa gcacaagatc 300

cccatcaagt atctggagtt catctcggac gccatcatcc acgttctgca 350

aagcaagcat cctgcggaat ttggcgctga tgcccaggga gccatgaaaa 400

aggccttgga gctgttccgg aatgacattg cggccaagta taaggagctg 450

gggttccagg gctag 465

<210> 11

<211> 465

<212> DNA

<213> Steppe Mammoth (Mammuthus trogontherii)

<220>

<223> DNA sequence encoding steppe mammoth myoglobin

<400> 11

atgggactca gcgacgggga atgggagttg gtgttgaaaa cctgggggaa 50

agtggaggct gacatcccgg gccatgggct ggaagtcttc gtcaggctct 100

tcacaggtca tcccgagacc ctggagaagt tcgacaagtt taagcacctg 150

aagacagagg gcgagatgaa ggcctccgag gacctgaaga agcaaggtgt 200

tactgtgctc actgccctgg ggggcatcct caagaagaaa gggcatcacc 250

aggcggagat tcagcccctg gcccattctc atgccaccaa gcacaagatc 300

cccatcaagt atctggagtt catctcggac gccatcatcc acgttctgca 350

aagcaagcat cctgcggaat ttggcgctga tgcccaggga gccatgaaaa 400

aggccttgga gctgttccgg aatgacattg cggccaagta taaggagctg 450

gggttccagg gctag 465

<210> 12

<211> 465

<212> DNA

<213> Sheep (Ovis aries)

<220>

<223> DNA sequence encoding sheep myoglobin

<400> 12

atggggctca gcgacgggga atggcagttg gtgctgaatg cctgggggaa 50

ggtggaggct gatgtcgcag gccatgggca ggaggtcctc atcaggctct 100

tcacaggtca tcccgagacc ctggagaaat ttgacaagtt caagcacctg 150

aagacagagg ctgagatgaa ggcctccgag gacctgaaga agcatggcaa 200

caccgtgctc acggccctag ggggtatcct gaaaaagaag ggtcaccacg 250

aggcggaggt gaagcacctg gccgagtcac acgccaacaa gcacaagatc 300

cctgtcaagt acctggagtt catctcggac gccatcatcc atgttctgca 350

tgccaagcat ccttcagact tcggtgctga tgcacaggga gccatgagca 400

aggccctgga actgttccgg aacgacatgg ctgcccagta caaggtgctg 450

ggcttccagg gctaa 465

<210> 13

<211> 465

<212> DNA

<213> Cow

<220>

<223> DNA sequence encoding bovine myoglobin

<400> 13

atggggctca gcgacgggga atggcagttg gtgctgaatg cctgggggaa 50

ggtggaggct gatgtcgcag gccatgggca ggaggtcctc atcaggctct 100

tcacaggtca tcccgagacc ctggagaaat ttgacaagtt caagcacctg 150

aagacagagg ctgagatgaa ggcctccgag gacctgaaga agcatggcaa 200

cacggtgctc acggccctgg ggggtatcct gaagaaaaag ggtcaccatg 250

aggcagaggt gaagcacctg gccgagtcac atgccaacaa gcacaagatc 300

cctgtcaagt acctggagtt catctcggac gccatcatcc atgttctaca 350

tgccaagcat ccttcagact tcggtgctga tgcccaggct gccatgagca 400

aggccctgga actgttccgg aatgacatgg ctgcccagta caaggtgctg 450

ggcttccatg gctaa 465

<210> 14

<211> 465

<212> DNA

<213> Wild Boar (Sus scrofa)

<220>

<223> DNA sequence encoding porcine myoglobin

<400> 14

atggggctca gcgacgggga atggcagctg gtgctgaacg tctgggggaa 50

ggtggaggct gatgtcgcag gccatgggca ggaggtcctc atcaggctct 100

ttaagggtca ccccgagacc ctggagaaat ttgataagtt taagcacctg 150

aagtcagagg atgagatgaa ggcctctgag gacctgaaga agcacggcaa 200

cacggtgctg actgccctgg ggggcatcct taagaagaag gggcatcatg 250

aggcagagct gacgcccctg gcccaatcgc atgccaccaa gcacaagatc 300

cctgtcaagt acctggagtt catctcagaa gccatcatcc aggttctgca 350

gagcaagcat cctggggact ttggtgctga cgcccaggga gccatgagca 400

aggccctgga actcttccgg aacgacatgg cggccaagta caaggagctg 450

ggcttccagg gctaa 465

<210> 15

<211> 465

<212> DNA

<213> Junglefowl

<220>

<223> DNA sequence encoding chicken myoglobin

<400> 15

atggggctca gcgaccagga gtggcaacaa gtcctcacca tctggggaaa 50

agtggaggcc gacattgctg gccatggaca cgaggttctg atgagacttt 100

tccatgacca ccctgagact ttggatcgct ttgataagtt caaaggcctg 150

aagacccctg atcagatgaa gggctctgaa gatctgaaga aacatggagc 200

tactgtcctc acccagcttg gcaaaatcct gaagcagaag ggtaatcatg 250

agtcagagct gaagcccctg gctcaaaccc atgccacgaa gcacaaaatc 300

ccagtcaaat atctggagtt catttctgaa gtcattatca aggtcattgc 350

tgaaaaacat gccgcagact ttggggccga ttcccaggct gccatgaaga 400

aggctctgga gttgttccga aatgacatgg ccagcaagta caaggagttt 450

ggtttccagg gttag 465

<210> 16

<211> 444

<212> DNA

<213> Pacific bluefin tuna (Thunnus orientalis)

<220>

<223> DNA sequence encoding tuna myoglobin

<400> 16

atggctgact ttgatgcagt tctgaagtgt tggggtccag tggaggcgga 50

ctacaccacc attggaggcc tggttctgac ccgtttattc aaagagcacc 100

ctgagaccca gaagctgttc cccaaattcg ctggcatcgc ccaggctgac 150

atagccggta acgcagctgt ttctgctcat ggtgccactg tgctgaagaa 200

acttggagag ctgctgaagg ccaaaggcag tcacgctgcc atcctaaaac 250

cactggcaaa cagccatgcc actaagcaca agattcccat taataacttc 300

aagctgattt ctgaggtcct tgtgaaggtc atgcatgaga aggcaggact 350

cgatgccggt gggcagacag ccctgaggaa cgtgatgggt atcatcatcg 400

ccgaccttga ggccaactac aaagagctgg gcttctctgg ctga 444

<210> 17

<211> 175

<212> PRT

<213> Common Mammoth

<220>

<223> Mammoth Casein K

<400> 17

Met Lys Gly Phe Leu Leu Val Val Asn Ile Leu Leu Leu Pro Leu

1 5 10 15

Phe Leu Ala Ala Glu Val Gln Asn Gln Glu Glu Ser Arg Cys Leu

20 25 30

Glu Lys Asp Glu Arg Trp Phe Cys Gln Lys Ala Val Lys Tyr Ile

35 40 45

Pro Asn Asp Tyr Val Leu Lys Ser Tyr Tyr Arg Tyr Glu Pro Asn

50 55 60

Tyr Asn Gln Phe Arg Ala Ala Val Pro Ile Asn Asn Pro Tyr Leu

65 70 75

Ile Tyr Leu Tyr Pro Ala Lys Gln Val Ala Val Arg Pro His Thr

80 85 90

Gln Ile Gln Trp Gln Val Pro Ser Asn Ile Tyr Pro Ser Pro Ser

95 100 105

Val Pro His Thr Tyr Leu Lys Pro Pro Phe Ile Ile Pro Pro Lys

110 115 120

Lys Thr Gln Asp Lys Pro Ile Ile Pro Pro Thr Gly Thr Val Ala

125 130 135

Ser Ile Glu Ala Thr Val Glu Pro Lys Val Asn Thr Val Val Asn

140 145 150

Ala Glu Ala Ser Ser Glu Phe Ile Ala Thr Asn Thr Pro Glu Ala

155 160 165

Thr Thr Val Pro Val Ile Ser Pro Gln Ile

170 175

<210> 18

<211> 260

<212> PRT

<213> Common Mammoth

<220>

<223> Mammoth Casein β

<400> 18

Met Lys Val Phe Ile Leu Ala Cys Leu Val Ala Phe Ala Leu Gly

1 5 10 15

Arg Glu Thr Val Glu Asn Leu Ser Ser Ser Glu Ile Arg Gln Phe

20 25 30

Tyr Ser Glu Gln Lys Pro Glu Gly Val Lys His Glu Glu Gln Gln

35 40 45

Arg Glu Asp Glu His Gln Asn Lys Ile Gln Pro Leu Phe Gln Pro

50 55 60

Gln Pro Leu Val Tyr Pro Phe Ala Glu Pro Ile Pro Tyr Thr Val

65 70 75

Phe Pro Pro Asn Ala Ile Pro Leu Ala Gln Pro Ile Val Val Leu

80 85 90

Pro Phe Pro Gln Pro Glu Val Gln Leu Pro Glu Ala Lys Glu Ile

95 100 105

Thr Phe Pro Arg Gln Lys Leu Met Ser Phe Leu Lys Ser Pro Val

110 115 120

Met Pro Phe Phe Asp Pro Gln Pro Asn Leu Gly Thr Asp Leu Glu

125 130 135

Asn Leu His Leu Pro Leu Pro Leu Leu Gln Pro Leu Arg His Gln

140 145 150

Leu His Gln Pro Leu Ala Gln Thr Pro Val Leu Pro Leu Pro Leu

155 160 165

Ser Leu Pro Lys Val Leu Pro Val Pro Gln Gln Val Ile Pro Tyr

170 175 180

Pro Gln Arg Gly Arg Pro Ile Gln Asn Leu Leu Tyr Glu Glu Pro

185 190 195

Leu Leu Asp Pro Thr Arg Lys Ile Tyr Pro Val Ala Gln Pro Leu

200 205 210

Ala Pro Val Tyr Asn Pro Val Ala Tyr Met Ile Gly Ile Pro Cys

215 220 225

Cys Ser Thr Leu Leu Thr Tyr Leu His Gln Ser Ser Arg Ser Gln

230 235 240

Tyr Pro Ile Gln Asn Lys Leu Gly Tyr Leu Ile Ala Met Pro Lys

245 250 255

Lys Val Arg Pro Thr

260

<210> 19

<211> 462

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence encoding steppe mammoth myoglobin

<400> 19

ggtttgtctg atggtgaatg ggagttggtt ttgaagactt ggggtaaagt 50

tgaggctgat attcctggtc atggtttgga ggtttttgtt agattgttca 100

ctggtcatcc agaaactttg gagaagttcg ataagttcaa acatttgaag 150

actgagggtg aaatgaaggc ttctgaggat ttgaaaaagc aaggtgttac 200

tgttttgact gctttgggtg gtattttgaa gaagaaaggt caccatcaag 250

ctgaaattca accattggct cactctcatg ctactaaaca caagattcca 300

attaagtact tggagttcat ttctgatgct attattcacg ttttgcaatc 350

taagcatcca gctgaatttg gtgctgatgc tcaaggtgct atgaaaaagg 400

ctttggagtt gttcagaaac gatattgctg ctaaatacaa ggagttgggt 450

ttccaaggtt aa 462

<210> 20

<211> 462

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence encoding sheep myoglobin

<400> 20

ggtttgtctg atggtgaatg gcaattggtt ttgaacgctt ggggtaaagt 50

tgaggctgat gttgctggtc atggtcaaga agttttgatt agattgttca 100

ctggtcaccc tgaaactttg gagaagtttg ataagttcaa acacttgaag 150

actgaggctg aaatgaaggc ttctgaggat ttgaaaaagc atggtaacac 200

tgttttgact gctttgggtg gtattttgaa gaagaaaggt caccatgaag 250

ctgaggttaa gcacttggct gaatctcacg ctaacaagca caaaattcct 300

gttaagtact tggagttcat ttctgatgct attattcatg ttttgcacgc 350

taagcatcca tctgatttcg gtgctgatgc tcaaggtgct atgtctaagg 400

ctttggaatt gttcagaaac gatatggctg ctcaatacaa ggttttgggt 450

ttccaaggtt aa 462

<210> 21

<211> 462

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence encoding bovine myoglobin

<400> 21

ggtttgtctg atggtgaatg gcaattggtt ttgaacgctt ggggtaaagt 50

tgaggctgat gttgctggtc atggtcaaga ggttttgatt agattgttca 100

ctggtcatcc agaaactttg gagaagtttg ataaattcaa gcacttgaag 150

actgaagctg agatgaaggc ttctgaggat ttgaaaaagc acggtaacac 200

tgttttgact gctttgggtg gtattttgaa gaaaaagggt caccatgagg 250

ctgaagttaa gcacttggct gaatctcatg ctaacaagca caaaattcct 300

gttaagtact tggagttcat ttctgatgct attattcacg ttttgcatgc 350

taagcaccca tctgatttcg gtgctgatgc tcaagctgct atgtctaagg 400

ctttggagtt gtttagaaac gatatggctg ctcaatacaa ggttttgggt 450

ttccacggtt aa 462

<210> 22

<211> 462

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence encoding porcine myoglobin

<400> 22

ggtttgtctg atggtgaatg gcaattggtt ttgaacgttt ggggtaaagt 50

tgaggctgat gttgctggtc atggtcaaga ggttttgatt agattgttca 100

agggtcatcc agaaactttg gagaaatttg ataagttcaa gcacttgaag 150

tctgaagatg agatgaaggc ttctgaggat ttgaagaaac acggtaacac 200

tgttttgact gctttgggtg gtattttgaa aaagaaaggt caccatgaag 250

ctgagttgac tccattggct caatctcatg ctactaagca caagattcca 300

gttaaatact tggagtttat ttctgaggct attattcaag ttttgcaatc 350

taagcatcct ggtgacttcg gtgctgatgc tcaaggtgct atgtctaagg 400

ctttggagtt gttcagaaac gatatggctg ctaagtacaa ggagttgggt 450

ttccaaggtt aa 462

<210> 23

<211> 462

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence encoding chicken myoglobin

<400> 23

ggtttgtctg atcaagaatg gcaacaagtt ttgactattt ggggtaaagt 50

tgaggctgat attgctggtc atggtcacga ggttttgatg agattgttcc 100

atgatcaccc agaaactttg gatagattcg ataagttcaa gggtttgaaa 150

actccagatc aaatgaaggg ttctgaggat ttgaagaaac acggtgctac 200

tgttttgact caattgggta aaattttgaa gcaaaaaggt aaccatgagt 250

ctgaattgaa gccattggct caaactcacg ctactaagca caagattcct 300

gttaagtact tggagttcat ttctgaggtt attattaagg ttatgctga 350

aaagcatgct gctgatttcg gtgctgattc tcaagctgct atgaaaaagg 400

ctttggaatt gttcagaaac gatatggctt ctaaatacaa ggagttcggt 450

tttcaaggtt aa 462

<210> 24

<211> 441

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleotide sequence encoding tuna myoglobin

<400> 24

gctgatttcg atgctgtttt gaagtgttgg ggtccagttg aagctgatta 50

cactactatt ggtggtttgg ttttgactag attgttcaag gagcatccag 100

aaactcaaaa gttgtttcca aagttcgctg gtattgctca agctgatatt 150

gctggtaacg ctgctgtttc tgctcatggt gctactgttt tgaagaaatt 200

gggtgaattg ttgaaggcta aaggttctca tgctgctatt ttgaagccat 250

tggctaactc tcacgctact aaacacaaga ttccaattaa caatttcaag 300

ttgatttctg aggttttggt taaggttatg cacgaaaagg ctggtttgga 350

tgctggtggt caaactgctt tgagaaacgt tatgggtatt attattgctg 400

atttggaagc taactacaag gaattgggtttttctggtta a 441

<210> 25

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<223> Genetic construct for expression of steppe mammoth myoglobin

<400> 25

atgagattcc catctatttt cactgctgtt ttgttcgctg cttcttctgc 50

tttggctgct ccagttaata ctactactga ggatgaaact gctcaaattc 100

cagctgaagc tgttattggt tactctgatt tggagggtga cttcgatgtt 150

gctgttttgc cattctctaa ctctactaat aacggtttgt tgtttattaa 200

cactactatt gcttctattg ctgctaagga agagggtgtt tctttggaga 250

agagagaagc tgaagctggt ttgtctgatg gtgaatggga gttggttttg 300

aagacttggg gtaaagttga ggctgatatt cctggtcatg gtttggaggt 350

ttttgttaga ttgttcactg gtcatccaga aactttggag aagttcgata 400

agttcaaaca tttgaagact gagggtgaaa tgaaggcttc tgaggatttg 450

aaaaagcaag gtgttatactgt tttgactgct ttgggtggta ttttgaagaa 500

gaaaggtcac catcaagctg aaattcaacc attggctcac tctcatgcta 550

ctaaacacaa gattccaatt aagtacttgg agttcatttc tgatgctatt 600

attcacgttt tgcaatctaa gcatccagct gaatttggtg ctgatgctca 650

aggtgctatg aaaaaggctt tggagttgtt cagaaacgat attgctgcta 700

aatacaagga gttgggtttc caaggttaa 729

<210> 26

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<223> Genetic construct for expressing sheep myoglobin

<400> 26

atgagattcc catctatttt cactgctgtt ttgttcgctg cttcttctgc 50

tttggctgct cctgttaata ctactactga ggatgaaact gctcaaattc 100

ctgctgaagc tgttatattggt tattctgatt tggagggtga cttcgatgtt 150

gctgttttgc ctttctctaa ctctactaat aacggtttgt tgttcattaa 200

cactactatt gcttctattg ctgctaagga agagggtgtt tctttggaaa 250

agagagaggc tgaagctggt ttgtctgatg gtgaatggca attggttttg 300

aacgcttggg gtaaagttga ggctgatgtt gctggtcatg gtcaagaagt 350

tttgattaga ttgttcactg gtcaccctga aactttggag aagtttgata 400

agttcaaaca cttgaagact gaggctgaaa tgaaggcttc tgaggatttg 450

aaaaagcatg gtaacactgt tttgactgct ttgggtggta ttttgaagaa 500

gaaaggtcac catgaagctg aggttaagca cttggctgaa tctcacgcta 550

acaagcacaa aattcctgtt aagtacttgg agttcatttc tgatgctatt 600

attcatgttt tgcacgctaa gcatccatct gatttcggtg ctgatgctca 650

aggtgctatg tctaaggctt tggaattgtt cagaaacgat atggctgctc 700

aatacaaggttttgggtttc caaggttaa 729

<210> 27

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<223> Gene construct for expressing bovine myoglobin

<400> 27

atgagattcc cttctatttt cactgctgtt ttgttcgctg cttcttctgc 50

tttggctgct ccagttaaca ctactactga ggatgaaact gctcaaattc 100

cagctgaagc tgttattggt tactctgatt tggagggtga cttcgatgtt 150

gctgttttgc cattctctaa ctctactaac aatggtttgt tgttcattaa 200

cactactatt gcttctattg ctgctaagga ggaaggtgtt tctttggaga 250

agagagaagc tgaggctggt ttgtctgatg gtgaatggca attggttttg 300

aacgcttggg gtaaagttga ggctgatgtt gctggtcatg gtcaagaggt 350

tttgattaga ttgttcactg gtcatccaga aactttggag aagtttgata 400

aattcaagca cttgaagact gaagctgaga tgaaggcttc tgaggatttg 450

aaaaagcacg gtaacactgt tttgactgct ttgggtggta ttttgaagaa 500

aaagggtcac catgaggctg aagttaagca cttggctgaa tctcatgcta 550

acaagcacaa aattcctgtt aagtacttgg agttcatttc tgatgctatt 600

attcacgttt tgcatgctaa gcacccatct gatttcggtg ctgatgctca 650

agctgctatg tctaaggctt tggagttgtt tagaaacgat atggctgctc 700

aatacaaggttttgggtttc cacggttaa 729

<210> 28

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<223> Gene construct for expressing porcine myoglobin

<400> 28

atgagattcc catctatttt cactgctgtt ttgttcgctg cttcttctgc 50

tttggctgct ccagttaaca ctactactga ggatgaaact gctcaaattc 100

cagctgaagc tgttattggt tactctgatt tggagggtga cttcgatgtt 150

gctgttttgc cattctctaa ttctactaac aatggtttgt tgttcattaa 200

cactactatt gcttctattg ctgctaagga agagggtgtt tctttggaga 250

agagagaagc tgaggctggt ttgtctgatg gtgaatggca attggttttg 300

aacgtttggg gtaaagttga ggctgatgtt gctggtcatg gtcaagaggt 350

tttgattaga ttgttcaagg gtcatccaga aactttggag aaatttgata 400

agttcaagca cttgaagtct gaagatgaga tgaaggcttc tgaggatttg 450

aagaaacacg gtaacactgt tttgactgct ttgggtggta ttttgaaaaa 500

gaaaggtcac catgaagctg agttgactcc attggctcaa tctcatgcta 550

ctaagcacaa gattccagtt aaatacttgg agtttatatttc tgaggctatt 600

attcaagttt tgcaatctaa gcatcctggt gacttcggtg ctgatgctca 650

aggtgctatg tctaaggctt tggagttgtt cagaaacgat atggctgcta 700

agtacaagga gttgggtttc caaggttaa 729

<210> 29

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<223> Gene construct for expressing chicken myoglobin

<400> 29

atgagattcc catctatttt cactgctgtt ttgttcgctg cttcttctgc 50

tttggctgct ccagttaaca ctactactga agatgagact gctcaaattc 100

ctgctgaagc tgttatattggt tactctgatt tggagggtga cttcgatgtt 150

gctgttttgc ctttttctaa ctctactaac aatggtttgt tgttcattaa 200

cactactatt gcttctattg ctgctaagga agagggtgtt tctttggaga 250

aaagagaggc tgaagctggt ttgtctgatc aagaatggca acaagttttg 300

actatttggg gtaaagttga ggctgatatt gctggtcatg gtcacgaggt 350

tttgatgaga ttgttccatg atcacccaga aactttggat agattcgata 400

agttcaaggg tttgaaaact ccagatcaaa tgaagggttc tgaggatttg 450

aagaaacacgtgctactgttttgactcaa ttgggtaaaa ttttgaagca 500

aaaaggtaac catgagtctg aattgaagcc attggctcaa actcacgcta 550

ctaagcacaa gattcctgtt aagtacttgg agttcatttc tgaggttattt 600

attaaggtta ttgctgaaaa gcatgctgct gatttcggtg ctgattctca 650

agctgctatg aaaaaggctt tggaattgtt cagaaacgat atggcttcta 700

aatacaagga gttcggtttt caaggttaa 729

<210> 30

<211> 708

<212> DNA

<213> Artificial sequence

<220>

<223> Gene construct for expressing tuna myoglobin

<400> 30

atgagattcc cttctatttt cactgctgtt ttgtttgctg cttcttctgc 50

tttggctgct ccagttaaca ctactactga agatgagact gctcaaattc 100

cagctgaagc tgttattggt tactctgatt tggagggtga cttcgatgtt 150

gctgttttgc cattctctaa ctctactaat aacggtttgt tgtttattaa 200

cactactatt gcttctattg ctgctaagga ggaaggtgtt tctttggaga 250

aaagagaagc tgaggctgct gatttcgatg ctgttttgaa gtgttggggt 300

ccagttgaag ctgattacac tactattggt ggtttggttt tgactagatt 350

gttcaaggag catccagaaa ctcaaaagtt gtttccaaaag ttcgctggta 400

ttgctcaagc tgatattgct ggtaacgctg ctgtttctgc tcatggtgct 450

actgttttga agaaattggg tgaattgttg aaggctaaag gttctcatgc 500

tgctattttg aagccattgg ctaactctca cgctactaaa cacaagattc 550

caattaacaa tttcaagttg atttctgagg ttttggttaa ggttatgcac 600

gaaaaggctg gtttggatgc tggtggtcaa actgctttga gaaacgttat 650

gggtattatt attgctgatt tggaagctaa ctacaaggaa ttgggttttt 700

ctggttaa 708

<210> 31

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 31

Met Gly Leu Ser Asp Gly Glu Trp Glu Leu Val Leu Lys

1 5 10

<210> 32

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 32

Gly Leu Ser Asp Gly Glu Trp Glu Leu Val Leu Lys

1 5 10

<210> 33

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 33

Leu Ser Asp Gly Glu Trp Glu Leu Val Leu Lys

1 5 10

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 34

Lys Thr Trp Gly Lys Val Glu Ala Asp Ile Pro Gly His Gly Leu

1 5 10 15

Glu Val Phe Val Arg

20

<210> 35

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 35

Lys Val Glu Ala Asp Ile Pro Gly His Gly Leu Glu Val Phe

1 5 10

<210> 36

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 36

Lys Val Glu Ala Asp Ile Pro Gly His Gly Leu Glu Val Phe Val

1 5 10 15

Arg

<210> 37

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 37

Ala Asp Ile Pro Gly His Gly Leu Glu Val Phe Val Arg

1 5 10

<210> 38

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 38

Asp Ile Pro Gly His Gly Leu Glu Val Phe Val Arg

1 5 10

<210> 39

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 39

Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu

1 5 10

<210> 40

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 40

Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys

1 5 10

<210> 41

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 41

Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp

1 5 10

<210> 42

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 42

Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp Lys

1 5 10 15

<210> 43

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 43

Arg Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp Lys

1 5 10 15

Phe Lys

<210> 44

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 44

Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys

1 5 10

<210> 45

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 45

Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp Lys

1 5 10

<210> 46

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 46

Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp Lys

1 5 10

<210> 47

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 47

Lys His Leu Lys Thr Glu Gly Glu Met Lys

1 5 10

<210> 48

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 48

Lys Thr Glu Gly Glu Met Lys Ala Ser Glu Asp Leu Lys Lys

1 5 10

<210> 49

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 49

Lys Lys Gln Gly Val Thr Val Leu Thr Ala Leu

1 5 10

<210> 50

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 50

Lys Lys Gln Gly Val Thr Val Leu Thr Ala Leu Gly Gly Ile Leu

1 5 10 15

Lys

<210> 51

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 51

Lys Gln Gly Val Thr Val Leu Thr Ala Leu

1 5 10

<210> 52

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 52

Lys Gln Gly Val Thr Val Leu Thr Ala Leu Gly Gly Ile Leu Lys

1 5 10 15

<210> 53

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 53

Lys Gln Gly Val Thr Val Leu Thr Ala Leu Gly Gly Ile Leu Lys

1 5 10 15

Lys

<210> 54

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 54

Gln Gly Val Thr Val Leu Thr Ala Leu Gly Gly Ile Leu Lys

1 5 10

<210> 55

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 55

Gly Val Thr Val Leu Thr Ala Leu Gly Gly Ile Leu Lys

1 5 10

<210> 56

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 56

Val Leu Thr Ala Leu Gly Gly Ile Leu Lys

1 5 10

<210> 57

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 57

Leu Thr Ala Leu Gly Gly Ile Leu Lys Lys

1 5 10

<210> 58

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 58

Thr Ala Leu Gly Gly Ile Leu Lys Lys

1 5

<210> 59

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 59

Lys Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu

1 5 10

<210> 60

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 60

Lys Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu Ala His

1 5 10 15

<210> 61

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 61

Lys Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu Ala His

1 5 10 15

Ser His Ala Thr Lys

20

<210> 62

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 62

Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu

1 5 10

<210> 63

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 63

Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu Ala His

1 5 10

<210> 64

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 64

Lys Lys Gly His His Gln Ala Glu Ile Gln Pro Leu Ala His Ser

1 5 10 15

His Ala Thr Lys

<210> 65

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 65

Lys Gly His His Gln Ala Glu Ile Gln Pro Leu

1 5 10

<210> 66

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 66

Lys Gly His His Gln Ala Glu Ile Gln Pro Leu Ala His

1 5 10

<210> 67

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 67

Lys Gly His His Gln Ala Glu Ile Gln Pro Leu Ala His Ser His

1 5 10 15

Ala Thr Lys

<210> 68

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 68

His His Gln Ala Glu Ile Gln Pro Leu Ala His Ser His Ala Thr

1 5 10 15

Lys

<210> 69

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 69

His Gln Ala Glu Ile Gln Pro Leu Ala His Ser His Ala Thr Lys

1 5 10 15

<210> 70

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 70

Ala Glu Ile Gln Pro Leu Ala His Ser His Ala Thr Lys

1 5 10

<210> 71

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 71

Lys Tyr Leu Glu Phe Ile Ser Asp Ala Ile Ile His

1 5 10

<210> 72

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 72

Lys Tyr Leu Glu Phe Ile Ser Asp Ala Ile Ile His Val Leu Gln

1 5 10 15

Ser Lys

<210> 73

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 73

Phe Ile Ser Asp Ala Ile Ile His Val Leu Gln Ser Lys

1 5 10

<210> 74

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 74

Asp Ala Ile Ile His Val Leu Gln Ser Lys

1 5 10

<210> 75

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 75

Lys His Pro Ala Glu Phe Gly Ala Asp Ala Gln

1 5 10

<210> 76

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 76

Lys His Pro Ala Glu Phe Gly Ala Asp Ala Gln Gly Ala Met Lys

1 5 10 15

<210> 77

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 77

Lys His Pro Ala Glu Phe Gly Ala Asp Ala Gln Gly Ala Met Lys

1 5 10 15

Lys

<210> 78

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 78

Pro Ala Glu Phe Gly Ala Asp Ala Gln Gly Ala Met Lys

1 5 10

<210> 79

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 79

Pro Ala Glu Phe Gly Ala Asp Ala Gln Gly Ala Met Lys Lys

1 5 10

<210> 80

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> Peptides in MS Analysis

<400> 80

Lys Ala Leu Glu Leu Phe Arg Asn Asp Ile Ala Ala Lys

1 5 10

Claims (21)

1. A meat substitute or food component comprising animal myoglobin from or derived from a ragon, pig, sheep, cow, chicken or tuna.

2. The meat substitute or food ingredient of claim 1, wherein the myoglobin is derived from or from grassland mammalia, mao-mammalia, mao Mengma.

3. The meat substitute or food ingredient of claim 1 or 2, wherein the animal myoglobin is represented by one of the following amino acid sequences:

-a) a sequence identical to SEQ ID NO:3, and having at least 70% sequence identity, and having at least one of the following amino acid combinations:

F at o position 30, and/or

Q at o position 65, and/or

H at omicron position 92, and/or

H at omicron 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 and Q at position 65 and H at position 92 and H at position 94;

-b) a sequence identical to SEQ ID NO:2 or 3, comprising Q or H at position 65 and H at position 94, and optionally having at least 70% sequence identity to SEQ ID NO:2 or 3 having at least one of the following amino acids at the following positions within 2 or 3: 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, I at position 102, E at position 123, and/or I at position 143;

-c) a sequence identical to SEQ ID NO:1, comprising Q or H at position 65 and H at position 94, and optionally at least 70% sequence identity to SEQ ID NO:1 has at least one of the following amino acids at the following positions within 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, I at position 102, and/or E at position 123;

-d) a sequence identical to SEQ ID NO: 4. 5 or 6, comprising Q or H at position 65 and H at position 94, and optionally at least 70% identity to SEQ ID NO: 4. 5 or 6 has at least one of the following amino acids at the following positions within: n at position 13, Q at position 27, I at position 31, N at position 67, a at position 128, S at position 133, a at position 145, and/or L at position 150;

-e) a sequence identical to SEQ ID NO:7, comprising Q or H at position 65 and H at position 94, and optionally at least 70% identity to SEQ ID NO:7 has at least one of the following amino acids at the following positions within 7: 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 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, I at position 116, a at position 117, E at position 118, a at position 121, S at position 128, K at position 133, S at position 145, and/or F at position 150. Or (b)

-f) a sequence corresponding to SEQ ID NO:8 has at least 70% identity, comprising Q or H at position 65 and H at position 94.

4. The meat substitute or food ingredient of any one of claims 1 to 3, which does not comprise leghemoglobin produced by bacteria symbiotic in soybean plant nodules and/or which comprises myoglobin as defined in any one of claims 1 to 3 as the sole heme-containing protein.

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

6. The food ingredient of any one of claims 1-4, wherein the food ingredient mimics the composition, flavor, color, aroma, and/or nutritional value of meat.

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

8. The genetic construct of claim 7, wherein the nucleic acid is selected from the group consisting of:

(a) A nucleotide sequence encoding a polypeptide represented by an amino acid sequence comprising a sequence having at least 75% sequence identity or similarity to an amino acid sequence as defined in claim 3 a), b), c) or d)

(b) A nucleotide sequence which hybridizes with SEQ ID NO: 9. 10, 11, 12, 13, 14, 15, 16 has at least 60% sequence identity; and

(c) A nucleotide sequence whose sequence differs from the sequence of the nucleotide sequence of (b) due to the degeneracy of the genetic code.

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

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

11. The genetic construct of any one of claims 6 to 10, wherein the nucleotide sequence consists of SEQ ID NO: 19. 20, 21, 22, 23 or 24.

12. The genetic construct of claim 11, wherein the genetic construct consists of SEQ ID NO: 25. 26, 27, 28, 29 or 30.

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

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

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

16. A process for the production of myoglobin as defined in any one of claims 1 to 3, which comprises culturing a host cell as defined in claim 14 or 15 in a suitable medium and optionally recovering the host cell and/or the myoglobin.

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

18. The method of claim 16 or 17, wherein the recovered myoglobin is purified, preferably substantially purified.

19. The method of any one of claims 16-18, wherein the host cell produces the myoglobin extracellular.

20. A method for producing a meat substitute or food ingredient as defined in any one of claims 1 to 6, the method comprising formulating myoglobin obtainable from the method of any one of claims 16 to 19 into the meat substitute or food ingredient.

21. A protein, wherein the protein can consist of a polypeptide comprising SEQ ID NO: 3.