阅读说明：本技术 藻类中过量产生原卟啉ix的方法及由此产生的组合物 (Method for overproducing protoporphyrin IX in algae and compositions produced thereby ) 是由 米勒·特朗 约翰·迪顿 布劳克·亚当斯 迈克尔·玛菲尔德 阿曼达·隆格 奥斯卡·冈萨雷斯 于 2019-11-07 设计创作，主要内容包括：本文提供了由过量产生原卟啉IX(PPIX)的藻类生产组合物的组合物和方法。还提供了使过量生产PPIX的藻类生长的方法,从藻类培养物和组合物中分离含有PPIX的部分的方法以及用由藻类生产的PPIX制备食品的方法。本文提供了藻株和选择过量产生PPIX的藻株的方法。还提供了组合物,包括可食用的组合物,可食用的组合物包括由藻类产生的PPIX。(Provided herein are compositions and methods for producing compositions from algae that overproduce protoporphyrin ix (ppix). Also provided are methods of growing PPIX overproducing algae, methods of separating PPIX-containing fractions from algae cultures and compositions, and methods of making food products with PPIX produced by algae. Provided herein are algal strains and methods of selecting algal strains that overproduce PPIX. Also provided are compositions, including edible compositions, including PPIX produced by algae.)

1. A composition comprising a preparation from an algal strain, wherein the algal strain overexpresses or accumulates protoporphyrin ix (ppix).

2. The composition of claim 1, wherein the agent is biomass from the algal strain.

3. The composition of claim 2, wherein the preparation is fractionated biomass from the algal strain.

4. The composition of claim 3, wherein the fractionated biomass comprises a PPIX-rich fraction.

5. The composition of claim 4, wherein said PPIX-enriched fraction further comprises a protein-enriched fraction.

6. The composition of claim 1, wherein the preparation is an extracellular fraction of the algal culture.

7. The composition of any one of claims 1-6, wherein the formulation is red or red-like in color.

8. The composition of any of claims 1-7, wherein the formulation contains more PPIX than the amount of heme.

9. The composition of any one of claims 1-8, wherein the formulation contains less than about 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% heme.

10. The composition of any one of claims 1-9, wherein the formulation contains less than about 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% heme protein.

11. The composition of any one of claims 1-10, wherein the formulation does not contain a detectable amount of heme protein.

12. The composition of any one of claims 1-11, wherein the formulation does not contain a detectable amount of heme.

13. The composition of any one of claims 1-4, wherein the formulation does not contain a detectable amount of protein.

14. The composition of any one of claims 1-13, wherein the formulation has an amount of protoporphyrin IX that is greater than the content of chlorophyll.

15. The composition of any one of claims 1-4 or 6, wherein said formulation provides at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the total protein content to said edible composition.

16. The composition of any one of claims 1-15, wherein the formulation provides vitamin a, beta carotene, and combinations thereof to the composition.

17. The composition of claim 16, wherein the vitamin a, beta carotene, or combination thereof is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the daily recommended demand.

18. The composition of any one of claims 1-17, wherein the formulation provides less than about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.5%, 2%, 5%, or 10% of the total saturated fat present in the composition.

19. The composition of any one of claims 1-18, wherein the formulation provides at least about 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 125mg, 150mg, 175mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, or 500mg of omega-3 fatty acid to the composition.

20. The composition of any one of claims 1-19, wherein the composition has a red or red-like color derived from the formulation.

21. The composition of any one of claims 1-20, wherein the composition has a meat or meat-like flavor derived from the formulation.

22. The composition of any one of claims 1-21, wherein the composition has a meat or meat-like texture derived from the formulation.

23. The composition of any one of claims 1-22, wherein the algae is Chlamydomonas.

24. The composition of claim 23, wherein the algae is chlamydomonas reinhardtii.

25. The composition of claim 23, wherein the Chlamydomonas is strain CC-125 or a derivative thereof deposited at the collection center of Chlamydomonas university of Minnesota.

26. The composition of claim 23, wherein the algae has reduced or absent ferrochelatase activity or expression of ferrochelatase.

27. A food product comprising the composition of any one of claims 1-26.

28. The food product of claim 27, wherein the food product comprises clean meat, cultured meat, synthetic meat, plant-based meat, or non-animal cell-based meat.

29. The food product of any one of claims 27-28, wherein the food product is selected from the group consisting of a beef-like food product, a fish-like product, a chicken-like product, a pork-like product, and a meat replica.

30. The food product of any one of claims 27-29, wherein the food product is a strict vegetarian, a vegetarian, or a gluten-free food product.

31. An edible ingredient comprising the composition of any one of claims 1-26.

32. The edible component of claim 31 wherein the edible component is part of a finished product and wherein the finished product has a red or red-like color derived from the component.

33. The comestible ingredient of claim 31 wherein the comestible ingredient is part of a finished product, and wherein the finished product has a meat or meat-like flavor derived from the ingredient.

34. The edible component of claim 31 wherein the edible component is part of a finished product and wherein the finished product has the appearance of blood derived from the component.

35. The comestible ingredient of any of claims 31-34 wherein the finished product is an ingredient for a hamburger, sausage, kabob, sliced meat, ground meat product, or meatball.

36. The comestible ingredient of any of claims 31-34 wherein the comestible composition is part of a finished product, and wherein the finished product is an animal feed.

37. The edible ingredient of any one of claims 31-36 wherein the edible ingredient is combined with a protein source, a fat source, a carbohydrate, a starch, a thickener, a vitamin, a mineral, or any combination thereof.

38. The comestible ingredient of claim 35 wherein the protein source is a textured wheat protein, a textured soy protein, a fungal protein, or an algal protein.

39. The comestible ingredient of any of claims 37 or 38 wherein the finished product is free of animal protein.

40. The edible ingredient of any one of claims 37-39 wherein the fat source comprises at least one of refined coconut oil or sunflower oil.

41. The edible ingredient of any one of claims 37-40 further comprising at least one of potato starch, methyl cellulose, water, or a flavor, wherein the flavor is selected from at least one of yeast extract, garlic powder, onion powder, and salt.

42. A meat substitute comprising the composition of any one of claims 1 to 26 or the comestible ingredient of any one of claims 31 to 41.

43. The meat substitute of claim 42, further comprising:

(a) 0.01% -5% (by weight of the meat replica matrix) of a non-animal protoporphyrin IX;

(b) a compound selected from the group consisting of glucose, ribose, fructose, lactose, xylose, arabinose, glucose-6-phosphate, maltose, and galactose, and any combination thereof;

(c) at least 1.5mM of a compound selected from the group consisting of cysteine, cystine, thiamine, methionine, and any combination thereof; and

(d) one or more proteins selected from the group consisting of plant proteins, fungal proteins and algal proteins,

wherein the meat substitute is a ground beef-like food product free of animal products, and wherein cooking the ground beef-like food product results in the production of at least two volatile compounds having a beef-related aroma.

44. A method of producing a protoporphyrin IX composition, the method comprising:

(a) growing a population of algae containing algae as a overproducer of protoporphyrin IX in culture; and

(b) isolating the protoporphyrin IX composition from the culture.

45. The method of claim 44, wherein the growing step comprises culturing the algae under aerobic fermentation conditions.

46. The method of claim 44 or claim 45, wherein said algae comprise chloroplasts.

47. The method of claim 46, wherein said biosynthesis of protoporphyrin IX occurs in the chloroplast.

48. The method of any one of claims 44-47, wherein said algae lacks the ability to produce chlorophyll.

49. The method of any one of claims 44-48, wherein said algae lacks the ability to produce functional Mg chelatase.

50. The method of any one of claims 44-49, wherein the algae has a reduction or absence of one or more of CHLD, CHLI1, CHLI2, or CHLH 1.

51. The method of any one of claims 44-50, wherein said algae has a reduced or absent of functional light-dependent protochlorophyllin.

52. The method of any one of claims 44-51, wherein said algae has a reduction or absence of functional light-independent ortho-chlorophyllin.

53. The method of any one of claims 44-52, wherein said algae is reduced or deficient in ChlB, ChlL or ChlN.

54. The method of any one of claims 44-53, wherein said algae overexpresses one or more of glutamyl-tRNA reductase, glutamate-1-semialdehyde aminotransferase, alanine dehydrogenase, porphobilinogen deaminase, UPG III synthase, UPG III decarboxylase, CPG oxidase, and PPG oxidase.

55. The method of any one of claims 44-54, wherein the algae are produced by mating to produce a produced algal strain, and wherein the color of the produced algal strain is red or red-like.

56. The method of any one of claims 44-54, wherein said algae are generated by mutagenesis.

57. The method of any one of claims 44-56, wherein the algae is red or red-like in color.

58. The method of any one of claims 44-57, wherein the isolated protoporphyrin IX composition is algal biomass.

59. The method of claim 58, wherein the algal biomass is fractionated.

60. The method of claim 59, wherein the algal biomass is fractionated to produce a protein-rich fraction comprising protoporphyrin IX.

61. The method of any one of claims 44-57, wherein the isolated protoporphyrin IX composition is isolated from an extracellular medium of the algal culture.

62. The method of any one of claims 44-61, wherein the isolated protoporphyrin IX composition is isolated from an algal protein.

63. The method of any one of claims 44-62, wherein said algae is deficient in carotenoids.

64. The method of any one of claims 44-63, wherein the algae is Chlamydomonas.

65. The method of any one of claims 44-64, wherein the algae is Chlamydomonas reinhardtii.

66. The method of claim 64, wherein the Chlamydomonas is strain CC-125 deposited at the collection center of Chlamydomonas university of Minnesota or a derivative thereof.

67. A method as claimed in any one of claims 44 to 66, wherein, following mating with another alga, progeny of an algal strain over-expressing protoporphyrin IX grow faster than their parent algal strains.

68. The method of any one of claims 44-66, wherein the algal strain that overexpresses protoporphyrin IX is produced by mating a carotenoid-deficient strain with a strain exhibiting a red or reddish color.

69. The method of any one of claims 44-66, wherein said protoporphyrin IX overexpressing algae is produced by mutagenesis of a first starting line and selection of a second line that grows faster in the dark than said first starting line.

70. The method of any one of claims 44-66, wherein the protoporphyrin IX-overexpressing algae is produced by mutagenizing a first starting strain and selecting from the mutagenized first strain a second strain lacking one or more carotenoids.

71. The method of any one of claims 44-70, wherein said algae lacks functional ferrochelatase.

72. The method of any one of claims 44-70, wherein the algae has a reduced amount or activity of ferrochelatase.

73. The method of any one of claims 44-70, wherein the algae has a reduced amount of heme or is deficient in heme as compared to a wild-type strain.

74. A composition comprising protoporphyrin IX, produced by the method of any one of claims 43-70.

75. The composition of claim 74, wherein the composition does not contain detectable levels of heme, hemopexin, or a combination thereof.

76. The method of any one of claims 44-73, further comprising the step of: (a) culturing the algal strain under dark conditions, wherein the strain produces no or reduced chlorophyll production, and (b) collecting a portion of the algal culture that is red or red-like in color to produce a protoporphyrin IX composition.

77. The method of claim 76, wherein said algae is Chlamydomonas.

78. The method of claim 77, wherein said algae is Chlamydomonas reinhardtii.

79. The method of claim 76, wherein said algae appears red or red-like when grown in said dark conditions.

80. The method of claim 76, wherein the harvested portion is extracellular medium from the algal culture.

81. The method of claim 76, wherein the collected portion is biomass or fractionated biomass from the algal culture.

82. The method of claim 76, wherein said algae are grown under aerobic fermentation conditions.

83. The method of claim 76, wherein the algae are grown to a density of greater than about 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 75g/L, 100g/L, 125g/L, or 150 g/L.

84. The method of claim 76, wherein said algae grow on acetate as a reducing carbon source.

85. The method of claim 76, wherein said algae grow on sugar as a reducing carbon source.

86. The method of claim 76, wherein said algal culture is supplemented with iron during said culturing step.

87. The method of claim 76, wherein the algal culture is inoculated at a density greater than about 0.1g/L, 1.0g/L, 5.0g/L, 10g/L, 20g/L, 50g/L, 80g/L, or 100 g/L.

88. The method of claim 76, further comprising fractionating the collected portion, wherein the fractionating removes substantially all or a majority of a component selected from the group consisting of carotenoids, starch, and protein from the collected portion.

89. The method of claim 76, further comprising fractionating the collected portion, wherein the fractionating removes substantially all or a majority of heme, hemopexin, or a combination thereof from the collected portion.

90. The method of claim 76, further comprising fractionating the collected fraction, wherein the fractionating produces a protein-rich fraction.

91. The method of claim 76, wherein said algae is deficient or reduced in one or more of magntochelase, magnoporphyrinogen IX, a chlorophyllin ester, and chlorophyll.

92. The method of claim 76, wherein said algae is deficient or reduced in ferrochelatase.

93. The method of any one of claims 44-73 or 76-92, wherein said algae is not a transgenic line.

94. A cleaned meat product produced by the method of any of claims 76-93, wherein the method further comprises combining a collected portion with a composition that produces cleaned meat, wherein the collected portion provides the cleaned meat product with a red or red-like color.

95. The cleaned meat product of claim 94, wherein the collected portion is a PPIX-enriched fraction or a purified PPIX.

96. An edible ingredient produced by the method of any one of claims 44-73 or 76-93, wherein the protoporphyrin IX composition imparts a meat or meat-like flavor, a meat or meat-like texture, a meat or meat-like odor, or any combination thereof, to the edible ingredient.

97. The edible ingredient of claim 96 wherein the edible ingredient is incorporated into a finished product selected from the group consisting of a beef-like food product, a fish-like product, a chicken-like product, a pork-like product, and a meat replica.

98. The edible ingredient of any one of claims 96-97 wherein the edible ingredient is a strict vegetarian, a vegetarian or a gluten-free.

99. The edible ingredient of any one of claims 96-98 wherein the edible ingredient is free of animal protein.

100. The edible ingredient of any one of claims 96-99 wherein the edible ingredient does not contain any transgenic components.

Background

With the advent of industrial animal husbandry, the consumption of animal meat continues to increase. Heretofore, animal husbandry accounts for over 18% of greenhouse gas production and is one of the major causes of climate change. In addition to land use, the animal husbandry also requires large amounts of fresh water, a limited resource that is becoming increasingly difficult to obtain. It is estimated that 1799 gallons of fresh water is required to produce one pound of beef, while 576 gallons of water is required to produce one pound of pork. In contrast, 216 gallons of fresh water are required to produce 1 pound of soybeans or 108 gallons to produce 1 pound of corn. The strength of the fresh water required to produce the meat of the animal is a result of the inefficiency of the water required for the growth of the plants consumed by the animal and the conversion of the food consumed by the animal into actual meat.

In order to address the sustainability and ethical issues related to meat consumption by animals, the food industry has been actively attempting to develop plant-based alternatives that have a taste, feel, and odor similar to meat products. However, many current plant-based alternatives do not penetrate the larger food and consumer markets. Typically, these substitutes consist of plant-based materials that are extruded to create a firm texture to improve mouthfeel, and then mixed with various flavors and aroma-forming compounds to improve the taste and odor of these products. Unfortunately, these alternatives are attractive to consumers who have been working on a vegetarian/vegetarian lifestyle, rather than those more accustomed to eating meat. To improve the sustainability of the food ecosystem, products must be developed that are attractive to consumers who currently prefer meat. By producing the next generation of plant-based products, the contribution of greenhouse gases and the water demand generated by animal husbandry can be greatly reduced.

Recent advances have shown the potential to use heme-containing proteins purified from host organisms to bring the flavor and aroma of products closer to meat. It is believed that heme from heme-containing proteins is responsible for imparting flavor and aroma to the "meat" of meat products. However, available sources of hemoproteins are expensive and technically intensive, limiting their utility. For example, the hemopexin leghemoglobin has been extracted from soybean roots, but this process has proven to be expensive, making it less economical to incorporate into meat substitutes. Yeast, pichia, has been engineered to express heme-binding proteins, such as the additional 8 enzymatic pathways used to produce heme molecules. This approach still requires that the hemopexin be purified from the expression host before it can be incorporated into the final product, which limits the positive effects that can be produced due to economic constraints. In addition to being economically undesirable, the product is genetically modified and is therefore less attractive to many consumers who choose to consume non-genetically engineered foods. Thus, there is a need for edible products incorporating heme-containing proteins as described herein.

Disclosure of Invention

Provided herein are compositions and methods of producing these compositions that provide a new source of flavor, color, mouthfeel, taste, odor, texture, and nutrition for food and food ingredients, as well as other uses, such as animal feed. Provided herein are compositions and methods for producing such compositions from protoporphyrin IX overproducing algae. Thus, in one exemplary aspect, the invention provides a composition comprising a preparation from an algal strain, wherein the algal strain overexpresses or accumulates protoporphyrin ix (ppix). In some embodiments, the preparation is biomass from an algal strain. In some embodiments, the preparation is fractionated biomass from an algal strain. In these embodiments, it is contemplated that the fractionated biomass comprises a PPIX-rich fraction (fraction). Further, in these embodiments, it is also contemplated that the PPIX-enriched fraction further comprises a protein-enriched fraction. In some embodiments, wherein the agent is an extracellular fraction (extracellular fraction) from an algal culture.

In some embodiments, the color of the formulation is red or red-like. Alternatively and/or additionally, the formulation contains more PPIX than the amount of heme. Alternatively and/or additionally, the formulation comprises less than about 1%, about 0.5%, about 0.1%, about 0.05%, about 0.01%, about 0.005%, or about 0.001% heme. Alternatively and/or additionally, the formulation comprises less than about 1%, about 0.5%, about 0.1%, about 0.05%, about 0.01%, about 0.005%, or about 0.001% heme protein.

In some embodiments, the formulation does not contain a detectable amount of heme protein. Alternatively and/or additionally, the formulation does not contain a detectable amount of heme. Alternatively and/or additionally, the formulation does not contain a detectable amount of protein. In some embodiments, the protoporphyrin IX content of the formulation is greater than the chlorophyll content.

In some embodiments, the formulation provides at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the total protein content to the edible composition. Alternatively and/or additionally, the formulation provides vitamin a, beta carotene, or a combination thereof to the composition. In these embodiments, it is preferred that vitamin a, beta carotene, or a combination thereof be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the daily recommended demand. In some embodiments, the formulation provides less than about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.5%, 2%, 5%, or 10% of the total saturated fat in the composition. Alternatively and/or additionally, the formulation provides at least about 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 125mg, 150mg, 175mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, or 500mg of omega-3 fatty acid to the composition.

In some embodiments, the composition has a red or red-like color derived from the formulation. Alternatively and/or additionally, the composition has a meat or meat-like flavour derived from the preparation. Alternatively and/or additionally, the composition has a meat or meat-like texture derived from the formulation.

In some embodiments, the algae is Chlamydomonas sp. Alternatively, the algae is Chlamydomonas reinhardtii (Chlamydomonas reinhardtii). In some embodiments, the Chlamydomonas is strain CC-125 or a derivative thereof deposited at the collection center of Chlamydomonas university of Minnesota. In some embodiments, the algae has reduced or absent ferrochelatase activity or expression of ferrochelatase.

Another aspect of the present invention includes a food product comprising the composition described herein. In some embodiments, the food product comprises clean meat, cultured meat, synthetic meat, plant-based meat, or non-animal cell-based meat. Alternatively and/or additionally, the food product is selected from the group consisting of a beef-like food product, a fish-like product, a chicken-like product, a pork-like product and a meat replica. Alternatively and/or additionally, the food product is strictly vegetarian, vegetarian or gluten-free.

Another aspect of the present invention includes edible ingredients comprising the compositions described herein. In some embodiments, the ingredient is part of a finished product, wherein the finished product has a red or red-like color derived from the ingredient. In some embodiments, the ingredient is part of a finished product, wherein the finished product has a red or red-like color derived from the ingredient. Alternatively and/or additionally, the component is part of a finished product, wherein the finished product has the Hasan appearance of blood derived from the component. In some embodiments, the finished product is a component of a hamburger, fish substitute, sausage, kebab, fillet, ground meat product, or meat ball. In some embodiments, the edible composition is part of a finished product, and wherein the finished product is an animal feed.

In some embodiments, the edible ingredient is combined with a protein source, a fat source, a carbohydrate, a starch, a thickener, a vitamin, a mineral, or any combination thereof. In some embodiments, the protein source is a textured wheat protein, a textured soy protein, a fungal protein, or an algal protein. In these embodiments, it is contemplated that the finished product is free of animal protein. In some embodiments, the fat source comprises at least one of refined coconut oil or sunflower oil. In some embodiments, the edible composition further comprises at least one of potato starch, methyl cellulose, water, and a flavor, wherein the flavor is selected from at least one of yeast extract, garlic powder, onion powder, and salt.

Another aspect of the invention includes a meat substitute comprising a composition or edible ingredient described herein. In some embodiments, the meat substitute further comprises (a) 0.01% -5% (by weight of the meat replica matrix) of a non-animal protoporphyrin IX, (b) a compound selected from the group consisting of glucose, ribose, fructose, lactose, xylose, arabinose, glucose-6-phosphate, maltose, and galactose, and any combination thereof, (c) at least 1.5mM of a compound selected from the group consisting of cysteine, cystine, thiamine, methionine, and any combination thereof, and (d) one or more proteins selected from the group consisting of plant proteins, fungal proteins, and algal proteins. Preferably, the meat substitute is a ground beef-like food product that is free of animal products; wherein cooking the ground beef-like food product results in the production of at least two volatile compounds having beef-related aromas.

Another aspect of the invention includes a method of producing a protoporphyrin IX composition. The method comprises the steps of growing a population of algae containing algae that are producers of protoporphyrin IX excess; and isolating the protoporphyrin IX composition from the culture. In some embodiments, the culturing step comprises culturing the algal culture under aerobic fermentation conditions. In some embodiments, the algae contains chloroplasts. In these embodiments, the biosynthesis of protoporphyrin IX occurs in the chloroplast.

In some embodiments, the algae lacks the ability to produce chlorophyll. Alternatively and/or additionally, the algae lacks the ability to produce functional magnesium chelatases. Alternatively and/or additionally, the algae is reduced or deficient in ChlD1, ChlD2, or ChlDH. Alternatively and/or additionally, the algae has a reduced or absent of functional light-dependent orthochlorophyllin. Alternatively and/or additionally, the algae has a reduced or absent of functional light independent orthochlorophyllin. Alternatively and/or additionally, the algae is reduced or deficient in ChlB, ChlL, or ChlN. Alternatively and/or additionally, the algae overexpresses one or more of glutamyl-tRNA reductase, glutamate-1-semialdehyde aminotransferase, ALA dehydrogenase, porphobilinogen deaminase, UPG III synthase, UPG III decarboxylase, CPG oxidase, and PPG oxidase.

In some embodiments, the algae is produced by mating, and wherein the color of the resulting algal strain is red or red-like. Alternatively and/or additionally, the algae are generated by mutagenesis. In some embodiments, the color of the algae is red or red-like. In some embodiments, the isolated protoporphyrin IX composition is algal biomass. In these embodiments, it is contemplated that the algal biomass is fractionated. Alternatively and/or additionally, the algal biomass is fractionated to produce a protein-enriched fraction comprising protoporphyrin IX.

In some embodiments, the isolated protoporphyrin IX composition is isolated from the extracellular medium of an algal culture. Alternatively and/or additionally, the isolated protoporphyrin IX composition is isolated from algal proteins. In some embodiments, the algae is deficient in carotenoids. In some embodiments, the algae is chlamydomonas. In some embodiments, the algae is chlamydomonas reinhardtii. In some embodiments, the Chlamydomonas is strain CC-125 or a derivative thereof deposited at the collection center of Chlamydomonas university of Minnesota.

In some embodiments, progeny of an over-expressed protoporphyrin IX algal strain grow faster than its parent algal strain upon mating with another alga. Alternatively and/or additionally, the protoporphyrin IX algal strain is produced by mating a carotenoid deficient strain with a strain exhibiting a red or reddish color. Alternatively and/or additionally, the alga overexpressing protoporphyrin IX is generated by mutagenesis of a first starting strain and selection of a second strain that grows faster in the dark than the first starting strain. Alternatively and/or additionally, the protoporphyrin IX overexpressing algae is produced by mutagenizing a first starting strain and selecting from the mutagenized first strain a second strain lacking one or more carotenoids.

In some embodiments, the algae lacks functional ferrochelatase. Alternatively and/or additionally, the amount or activity of algal ferrochelatase is decreased. Alternatively and/or additionally, the amount of heme in the algae is reduced or deficient compared to a wild-type strain.

In some embodiments, the method further comprises the steps of a) culturing the algal strain under dark conditions, wherein the strain produces no or reduced chlorophyll production, and (b) collecting a portion of the algal culture that is red or red-like in color to produce the protoporphyrin IX composition. Preferably, the algae are of the genus Chlamydomonas. In some embodiments, the algae is chlamydomonas reinhardtii. In some embodiments, the algae appears red or red-like when grown in dark conditions.

In some embodiments, wherein the collected fraction is extracellular medium from an algal culture. Alternatively and/or additionally, the collected fraction is biomass from an algal culture or fractionated biomass. In some embodiments, the algae are grown under aerobic fermentation conditions. Alternatively and/or additionally, the algae are grown to a density of greater than about 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 75g/L, 100g/L, 125g/L, or 150 g/L. Alternatively and/or additionally, the algae grow with acetate as a reducing carbon source. Alternatively and/or additionally, wherein the algae grow on sugar as a reducing carbon source. Alternatively and/or additionally, the algae culture is supplemented with iron during the culturing step. In some embodiments, the algal culture is inoculated at a density greater than about 0.1g/L, 1.0g/L, 5.0g/L, 10g/L, 20g/L, 50g/L, 80g/L, or 100 g/L.

In some embodiments, the method further comprises fractionating the collected portion, wherein fractionating removes substantially all or a majority of a component selected from the group consisting of carotenoids, starch, and protein from the collected portion. Optionally and/or additionally, the method further comprises fractionating the collected fraction, wherein the fractionating removes substantially all or a majority of the heme, hemopexin, or combination thereof from the collected fraction. Optionally and/or additionally, the method further comprises fractionating the collected fraction, wherein the fractionating produces a protein-enriched fraction.

In some embodiments, the algae lacks or has reduced one or more of a magnesium chelatase, magnesium protoporphyrinogen IX, a protoporphyrinate ester, a chlorophyllide ester, and chlorophyll. Alternatively and/or additionally, ferrochelatase in algae is deficient or reduced. In some embodiments, the algae are not transgenic strains.

Another aspect of the present invention includes a clean meat (clean meat) product produced by the methods described herein, and the methods further comprise combining the collected portion with a composition for making clean meat, wherein the collected portion provides a red or red-like color to the clean meat product. In some embodiments, the collected fraction is a PPIX-rich fraction or a purified PPIX.

Another aspect of the invention includes an edible ingredient produced by the methods described herein, and the protoporphyrin IX composition imparts a meat or meat-like flavor, a meat or meat-like texture, a meat or meat-like odor, or any combination thereof, to the edible ingredient. In some embodiments, the edible ingredient is incorporated into a finished product selected from the group consisting of beef-like (beef-like) food, fish-like products, chicken-like products, pork-like products, and meat replicas. Alternatively and/or additionally, the edible ingredient is strictly vegetarian, vegetarian or gluten-free. Alternatively and/or additionally, the comestible ingredients are animal protein free. Alternatively and/or additionally, the edible ingredient does not contain any transgenic components.

Another aspect of the invention includes protoporphyrin IX-containing compositions produced by the methods described herein. In some embodiments, the composition does not contain detectable levels of heme, heme-binding protein, or a combination thereof.

Drawings

Fig. 1 is a schematic diagram showing an exemplary pathway for heme production in algae. This exemplary pathway can be used by wild type algae to produce chlorophyll, but it can also be used to produce protoporphyrin ix (ppix).

Fig. 2 is a schematic diagram showing an exemplary fractionation of PPIX-overexpressing algae, showing the rich red color of the extract.

FIG. 3 is a schematic showing hamburgers made with 0.01 grams, 0.1 grams, 1.0 grams, and 5.0 grams of PPIX-rich algae.

FIG. 4 is a schematic diagram showing the mixture of ingredients of a plant-based hamburger without heme-rich algae, with PPIX-rich algae added, or with heme-rich algae forming a hamburger before and after cooking.

FIG. 5 is a schematic diagram showing an example of a meat-free "tuna" rich in PPIX.

FIG. 6 shows a partial sequence alignment of wild-type green algae and red algae having a CHLH gene mutation (the upper sequence (Seq _1) is a partial nucleic acid sequence (residue 1621-1679 of SEQ ID NO:27) and a partial amino acid sequence (residue 451-460 of SEQ ID NO: 28) of the CHLH gene of the green algae and the lower sequence (Seq _2) is a partial nucleic acid sequence (residue 1621-1680 of SEQ ID NO:129) and a partial amino acid sequence (residue 451-460 of SEQ ID NO:152) (asterisk) of the CHLH gene of the red algae having a mutation as shown in the figure, the wild-type chlorophyll nucleic acid sequence (SEQ ID NO:27) has thiamine inserted at position 1678 causing proline to change to serine at position 560 of the wild-type chlorophyll amino acid sequence of SEQ ID NO: 28.

Detailed Description

Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein, as will become apparent to those skilled in the art upon reading the present disclosure and so forth. Furthermore, to the extent that the terms "including," include, "" having, "" has, "" with, "or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term" comprising.

The term "about" or "approximately" means within an acceptable error range for the particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" may refer to 1 or more than 1 standard deviation in the practice of a given value. Where particular values are described in the application and claims, the term "about" should be assumed to mean an acceptable error range for the particular value, unless otherwise specified.

As used herein, "absence" or "lack of" or "reduction" of one or more genes and/or enzymes includes, for example, mutation or deletion of a gene sequence, reduction or lack of expression of a gene (RNA and/or protein), and/or lack of accumulation or stability of a gene product (RNA and/or protein).

As used herein, "overexpression" and "overexpression" of an enzyme or gene includes, for example, an increase in expression of the gene (RNA and/or protein) and/or an increase in accumulation or stability of the gene product (RNA and/or protein). Such overexpression can include changes in regulatory regions and/or gene sequences, as well as copy number, genomic location, and post-translational modifications.

As used herein, the term "engineered algae" is used to refer to algae that contain one or more genetic modifications. In some cases, when an engineered alga integrates a heterologous nucleic acid into its genome by recombinant techniques, it is also a recombinantly modified organism. In other cases, the engineered algae are not recombinantly modified organisms (e.g., they are modified by ultraviolet, chemical, or radiation mutagenesis). In some cases, algae that are not recombinant modified organisms are referred to as non-GMO and components from such algae may be referred to as non-GMO components.

As used herein, the term "genetic modification" is used to refer to any manipulation of the genetic material of an organism that does not occur under natural conditions. Genetic modifications may include modifications by mutagenesis (e.g., ultraviolet, x-ray, gamma ray, and chemical exposure). Genetic modification may include gene editing. In some cases, genetic modification can be performed by recombinant techniques. As used herein, "recombinantly modified organism" is used to refer to an organism that has integrated a heterologous nucleic acid (e.g., a recombinant nucleic acid) into its genome by recombinant techniques. Methods for performing such manipulations are known to those of ordinary skill in the art and include, but are not limited to, techniques for transforming cells having a nucleic acid sequence of interest with a vector. Included within the definition are various forms of gene editing in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or "molecular scissors". These nucleases generate site-specific Double Strand Breaks (DSBs) at desired locations in the genome. The induced double-strand break is repaired by non-homologous end joining (NHEJ) or Homologous Recombination (HR), resulting in targeted mutation (i.e., editing).

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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Provided herein are methods of selecting, growing, and incorporating algae that overexpress protoporphyrin ix (ppix) molecules in food and animal feed ingredients and products. These products can include non-transgenic and plant-based replacement foods. It is well known that algae produce many chemical compounds that cause these aquatic organisms to appear in different colors. These compounds include, but are not limited to, chlorophyll to turn algae green, beta-carotene to give algae a yellow or orange color, astaxanthin to give algae a red color, or other various pigments (such as phycocyanin to turn algae blue). Although each of the above compounds has been added to food products, to date no product has incorporated PPIX overproducing algae to impart a red and/or meaty taste and odor.

Provided herein are strains, methods, and compositions using PPIX overproducing algae. In some embodiments, the color of the algal strain when grown is red or red-like. As used herein, in some embodiments, a red-like color may be any color or any mixture of colors having wavelengths between 590 nanometers and 750 nanometers. Alternatively and/or additionally, in some embodiments, a red-like color can be defined as any color in RGB (r.g.b) having an r value between 255 and 80 and a g or b value between 0 and 80. In some embodiments, formulations of overproduced PPIX prepared from algal cultures impart a pink or red color when incorporated into foods and other edible products. In some embodiments, the PPIX overproducing formulations prepared from algal cultures impart a "meat" flavor, odor and/or texture when incorporated into foods and other edible products.

Without being limited by theory, the heme pathway is a biochemical pathway that branches from the chlorophyll biochemical pathway, as shown in fig. 1. Briefly, this pathway begins with the conversion of glutamate tRNA, glutamate tRNA reductase and GSA aminotransferase to 5-aminolevulinic acid (ALA). Subsequently, ALA is converted to porphobilinogen by ALA dehydrogenase. Subsequently, porphobilinogen is converted to hydroxymethylcholine by porphobilinogen deaminase. Next, hydroxymethylcholine is converted to uroporphyrinogen III by UPG III synthase. Next, uroporphyrinogen III is converted to coproporphyrinogen by UPGIII decarboxylase. Next, coproporphyrinogen is converted to protoporphyrinogen IX by CPG oxidase. Next, protoporphyrinogen IX is converted to protoporphyrin IX by PPG oxidase. Protoporphyrin IX can shuttle to the chlorophyll production pathway or switch to heme B. Finally, protoporphyrin IX is converted to heme B by ferrochelatase, which attaches iron to protoporphyrin IX.

By reducing metabolic flux to chlorophyll, flux to PPIX and heme B can be increased. By reducing or eliminating ferrochelatase, this pathway produces PPIX, but the conversion of PPIX to heme is reduced or eliminated.

In some embodiments herein, algal strains used in the methods and compositions produced thereby have a reduced metabolic flux to chlorophyll and an increased metabolic flux to heme B. In some embodiments herein, the engineered algal strain comprises genetic modifications in the ferrochelatase enzyme, such as in one or more of the nucleotide sequences (e.g., SEQ ID NO: 7) and/or amino acid sequences (e.g., SEQ ID NO: 8), and genetic modifications comprising one or more regulatory regions (such as those of SEQ ID NO:114, 115), exons (such as those of SEQ ID NO: 116-.

In some embodiments, the algal strain is one in which chlorophyll and carotenoid synthesis is reduced. In some embodiments, the algal strain is deficient or reduced in the amount of chlorophyll. In some embodiments, the algal strain has a lack or reduction in a function, amount, or activity of a ferrochelatase enzyme of at least 10%, at least 20%, at least 30%, at least 40%, at least 50% as compared to a wild-type algae. In some embodiments, the amount of heme B accumulated by the algal strain is deficient or decreased, and the PPIX accumulated by the algal strain is increased. In some embodiments, the algal strain is red or red-like in color.

In some embodiments, the algal strain lacks one or more enzymes of the chlorophyll biosynthetic pathway. These defects include, but are not limited to, gene deletions, mutations, and other alterations that result in a lack of expression or functional deficiency of the enzyme. In some embodiments, the algal strain lacks magnesium chelatase, which is the first step in the conversion of protoporphyrin IX to chlorophyll. In some embodiments, the algal strain lacks a light-dependent ortho-chlorophyllin ester that converts an ortho-chlorophyllin ester to chlorophyll. In some embodiments, the algal strain lacks a light-independent ortho-chlorophyllin reductase, which converts an ortho-chlorophyllin ester to a chlorophyllin ester in the dark. In some embodiments, the algal strain lacks one or more of ChlB, ChlL, or ChlN. In some embodiments, the algal strain is deficient or reduced in one or more of magnesium chelatase, magnesium protoporphyrinogen IX, a protoporphyrinate ester, a chlorophyllide ester, and chlorophyll.

In some embodiments, the algal strain lacks one or more of the magnesium chelatase subunits, CHLD, CHLH, and CHLI. These subunits are also referred to by the gene name CHLD1 (also known as CHlD1) corresponding to the CHLD subunit, CHLH1 (also known as CHlH1) corresponding to the CHLH subunit, and CHLI1 and CHLI2 corresponding to the CHLI subunit, encoded by two genes CHLI1 and CHLI2 (also known as CHlI1 and CHlI 2).

In some embodiments, the algal strain lacks one or more of CHLD1, CHLH1, CHLI1, CHLI2, or portions thereof (including genetic modifications in one or more introns, exons, regulatory regions, or the entire gene sequence, such as genetic modifications of one or more of SEQ ID NOs: 45-69, 70-88, 89-113, or 130-150). For example, a red algae strain has a genetic modification in the CHLH locus. This modification deletes a single base pair in CHLH, resulting in a frameshift in the CHLH open reading frame and/or the generation of a stop codon, allowing the translation of the protein into a truncated form, as compared to green algal strains. FIG. 6 shows a partial sequence alignment of wild-type green algae and red algae with a mutation in the CHHLH gene (the upper sequence (Seq-1) being part of the nucleic acid sequence (residue 1621-1679 of SEQ ID NO:27) and part of the amino acid sequence (residue 451-460 of SEQ ID NO: 28) of the CHLH gene of the green algae and the lower sequence (Seq-2) being part of the nucleic acid sequence (residue 1621-1680 of SEQ ID NO:129) and part of the amino acid sequence (residue 451-460 of SEQ ID NO:152) (asterisk.) nucleic acid sequences of additional genes that can be altered in this algal strain are provided herein.

In some embodiments, engineered algal strains used with the methods and compositions herein include genetic modifications of a ferrochelatase gene that reduce or lack the production of ferrochelatase and also have modifications in one or more enzymes from PPIX to chlorophyll (e.g., CHLD, CHLI1, CHLI2, and/or CHLH).

In some embodiments, the algal strain overexpresses one or more enzymes such that the balance of pathways favor production of PPIX. In some embodiments, the algal strain overexpresses one or more of glutamyl-tRNA reductase, glutamate-1-semialdehyde aminotransferase, ALA dehydrogenase, porphobilinogen deaminase, UPG III synthase, UPG III decarboxylase, CPG oxidase, and PPG oxidase. In some embodiments, the algal strain overexpresses one or more such enzymes, and also reduces the amount or activity of ferrochelatase. In some embodiments, the algal strain has an improved ability to produce ALA, the rate-limiting precursor of heme B synthesis, and optionally, a reduced amount or activity of ferrochelatase. In some embodiments, the algal strain lacks the ability to produce a functional ferrochelatase gene, which is the enzyme responsible for the conversion of protoporphyrin IX to heme B. In some embodiments, the algal strain has an improved ability to produce a UPG III synthase, a UPG III decarboxylase, a CPG oxidase, or a PPG oxidase. In some embodiments, the algal strain has an increased amount of protoporphyrin IX as compared to a wild-type strain.

In some embodiments, the algal strain produces a carotenoid or a precursor of a carotenoid. Without being bound by theory, carotenoids impart color and can affect the visual appearance of plant-based substitutes. Exemplary carotenoids include, but are not limited to, gamma-carotene, beta cryptoxanthin, zeaxanthin, authoxanthin, lutein, pro-lycopene, and lycopene.

In some embodiments, the algal strain lacks a carotenoid or a precursor of a carotenoid. Defects in carotenoid biosynthesis can occur due to mutations, such as mutations that affect carotenoid biosynthesis (e.g., mutations in the phytoene synthase gene).

In some embodiments, algal strains used in the methods herein and for preparing PPIX-containing compositions are selected or identified based on one or more phenotypes and/or genotypes. In some embodiments, algal strains for overproduction of PPIX may be produced by a mating process. In some embodiments, algal strains used for overproduction of PPIX may be generated by random mutagenesis (such as uv mutagenesis). In some embodiments, algal strains used for overproduction of PPIX may be generated by chemical mutation with compounds that cause DNA changes.

In some embodiments, modification, such as precisely engineered nucleases (such as by CRISPR-CAS nucleases), targeting to alter expression of one or more components may be produced by gene editing. These nucleases can be used to generate insertions, deletions, mutations, and substitutions of one or more nucleotides or nucleotide regions to alter the expression of one or more pathway enzymes in the pathway, thereby reducing chlorophyll and/or increasing production or accumulation of PPIX. After the modification has been produced, the algal strains grow and/or mate such that the nuclease and associated guide nucleic acid are removed and the remaining algal strains do not retain the nuclease and associated editing system. In some embodiments, nucleases, such as CRISPR-CAS nucleases, are used to modify components of the chlorophyll pathway such that chlorophyll expression and/or accumulation is reduced or abolished (aberrated). In some embodiments, nucleases (such as CRISPR-CAS nucleases) are used to modify components of the chlorophyll pathway such that PPIX expression and/or accumulation is increased. In some embodiments, the gene encoding ferrochelatase is genetically modified with a nuclease (such as CRISPR-CAS nuclease), for example, by genetically modifying the nucleic acid sequence of SEQ ID NO: 114-128 or a modification that reduces or abolishes expression of one or more genes; or a modification which abolishes, truncates or causes a frame shift of the gene encoding the enzyme (such as SEQ ID NO: 116-122), and/or a modification which changes or truncates the expressed protein (such as the change of amino acid SEQ ID NO: 8). In some embodiments, nucleases (such as CRISPR-CAS nuclease) are used to make modifications in one or more of CHLD, CHLI1, CHLI2, or CHLH1, resulting in PPIX-rich algal strains. These modifications are made by designing the guide RNA to include one or more point mutations, insertions, deletions or combinations thereof that modify one or more of SEQ ID NOS 45-113, 130-150 or 153. In some embodiments, these genetic modifications in more than one target sequence, such as the ferrochelatase sequence and genetic modifications in another chlorophyll pathway sequence (e.g., CHLD, CHLI1, CHLI2, or CHLH1), are incorporated by simultaneous or sequential nuclease engineering and/or by mating engineered algal strains containing the genetic modifications.

There are several families of engineered nucleases used in gene editing, such as, but not limited to, meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-Cas systems, and ARCUS. However, it should be understood that any known gene editing system utilizing engineered nucleases can be used in the methods described herein. Thus, in some embodiments, the PPIX-overproducing algal strain may be genetically modified by using techniques such as CRISPR-Cas systems (e.g., CRISPR-Cas9) or by using zinc finger nucleases.

CRISPR (clustered regularly interspaced short palindromic repeats) is an acronym for a locus comprising a plurality of short direct repeats of a base sequence. Prokaryotic CRISPR/Cas systems have been engineered for gene editing (silencing, enhancing or altering specific genes) in eukaryotes (see, e.g., Cong, Science,15:339(6121): 819-. By transfecting the cell with an element comprising a Cas gene and a specifically designed CRISPR, the nucleic acid sequence can be cleaved and modified at any desired position. Methods of using CRISPR/Cas systems to prepare compositions for use in genome editing are described in detail in US pub.no.2016/0340661, US pub.no.2016/0340662, US pub.no.2016/0354487, US pub.no.2016/0355796, US pub.no.2016/0355797, and WO2014/018423, which are specifically incorporated herein by reference in their entirety.

Zinc Finger Nucleases (ZFNs) are artificial restriction enzymes produced by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be designed to target specific desired DNA sequences, which enables zinc finger nucleases to target unique sequences in complex genomes. By exploiting endogenous DNA repair mechanisms, these agents can be used to precisely alter the genome of higher organisms. The most common cleavage domain is the type IIS enzyme Fok 1. Fok1 catalyzes double-strand breaks in DNA, with a recognition site of 9 nucleotides on one strand and 13 nucleotides on the other. See, e.g., U.S. Pat. No.5,356,802; 5,436,150 and 5,487,994; and Li et al Proc., Natl.Acad.Sci.USA 89(1992): 4275-; li et al Proc Natl Acad Sci USA 90:2764 + 2768 (1993); kim et al Proc Natl Acad Sci USA 91:883-887(1994 a); kim et al J. biol. chem.269:31,978-31,982(1994b), which is incorporated herein by reference in its entirety. One or more of these enzymes (or enzymatically functional fragments thereof) may be used as a source of cleavage domains.

Methods of algae selection include, but are not limited to, genetic or phenotypic screening for defects, mutations, and changes in the chlorophyll biosynthesis pathway and/or chlorophyll accumulation, and genetic or phenotypic screening for increased expression and/or accumulation of PPIX, PPIX biosynthetic intermediates, and heme biosynthetic enzymes. In some embodiments, algal strains used in the methods herein and for preparing PPIX-containing compositions are selected or identified based on their spectral distribution and/or their red or red-like color. In some embodiments, the algal strain used in the methods herein and for preparing the PPIX-containing compositions is selected or identified based on its growth rate under dark conditions. In some embodiments, the selection is based on the growth rate under dark conditions and the appearance or enhancement of red or red-like color when grown under dark conditions. In some embodiments, algal strains are selected that have an insufficient or reduced amount of carotenoid production or accumulation.

In some embodiments, the algal strains are mated to bind or enhance properties that contribute to PPIX production, PPIX accumulation, chlorophyll reduction, and/or carotenoid reduction. In some embodiments, an algal strain having rapid growth under dark conditions (e.g., faster than a wild-type algal strain) is mated with an algal strain exhibiting a red color or a red-like color. Thus, this algal strain is not a transgenic line. In some embodiments, an algal strain lacking carotenoid production or accumulation is mated with an algal strain exhibiting a red color or a red-like color. This resulting algae is expected to be an algal strain with protoporphyrin IX overexpressed, which grows faster than its parent algal strain after mating with another algae.

In some embodiments, algal strains are mutagenized, and then new algal strains are selected or identified that exhibit one or more of the characteristics of increased PPIX production, PPIX accumulation, chlorophyll reduction, and/or carotenoid reduction. In some embodiments, the algal strains are generated by mutagenesis of a first starting line and selection of a second line that grows faster in the dark than the first starting line. In some embodiments, the algal strain is generated by mutagenesis of a first starting strain and selection of a second strain lacking one or more carotenoids.

Algae for use in compositions and methods

In the compositions and methods provided herein for producing PPIX and PPIX-containing compositions, algal strains having a PPIX biosynthetic pathway are used. In some embodiments, the PPIX overproducing algal strain is the phylum chlorella (green algae). In some embodiments, the green algae is selected from the group consisting of chlamydomonas, dunaliella, haematococcus, chlorella, and scenedesmus. In some embodiments, the chlamydomonas is chlamydomonas reinhardtii. In various embodiments, the green algae can be a green algae, chlamydomonas reinhardtii 137c, or a psbA deficient chlamydomonas reinhardtii strain. In some embodiments, the selected host is Chlamydomonas reinhardtii, such as described in Rasala and Mayfield, Bioeng Bugs, (2011)2(1): 50-4; rasala, et al, Plant Biotechnol j. (2011) May 2, PMID 21535358; coragliotti, et al, Mol Biotechnol (2011)48(1) 60-75; specht, et al, Biotechnol Lett, (2010)32(10): 1373-83; rasala, et al, plantatbbiotechnol j. (2010)8(6) 719-33; mulo, et al, Biochim Biophys Acta, (2011) May 2, PMID: 21565160; and Bonente, et al, Photosynth Res. (2011) May 6, PMID: 21547493; USPub.No. 2012/0309939; US pub.no. 2010/0129394; and intl.pub.no.wo2012/170125. All of the foregoing references are incorporated herein by reference in their entirety for all purposes.

In some embodiments, the PPIX overproducing algal strain is a unicellular algae. Exemplary and additional species of microalgae of interest include, but are not limited to, Aspergillus orientalis (Achnanthes orientalis), Alternaria grisea (Agmenellum), Aphanothece gracilis (Amphiprorora hyalina), Alternaria coffeveri (Amphiobacillus coffeformis), Alternaria coffeveri (Amphiobacillus linea), Alternaria coffeveri spot (Amphiobacillus coffeformis punctata), Alternaria coffeveri (Amphiobacillus coffeformis), Alternaria coffeveri thin (Amphiobacillus coffeformis tenuis), Alternaria delavayi, Alternaria cathita, Alternaria sp (Amphiobacillus sp), Alternaria sp, Bodeck sp, Graria sp, chlamydomonas sp. (Chloromycedomonass p.), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), Chlorella anomala (Chlorella anatrata), Chlorella antarctica (Chlorella antarctica), Chlorella aureoviridis (Chlorella aureoviridis), Chlorella candica, Chlorella cystokiniana (Chlorella capsulata), Chlorella dehydrata (Chlorella desaccata), Chlorella ellipsoidea (Chlorella ellipsoidea), Chlorella pumila (Chlorella emersonii), Chlorella fusca (Chlorella fusca), Chlorella fusca var fusca (Chlorella fusca) var. Vacuoide), Chlorella vulgara (Chlorella viridis), Chlorella viridis (Chlorella viridis) strain (Chlorella viridis), Chlorella viridis (Chlorella viridis) strain (Chlorella viridis), Chlorella viridis (Chlorella viridis strain (Chlorella viridis) and Chlorella viridis (Chlorella viridis), Chlorella viridis strain (Chlorella viridis) or Chlorella viridis strain (Chlorella vulgaris, Chlorella viridis) s strain (Chlorella viridis) s strain, Chlorella viridis strain, Chlorella vulgaris, Chlorella viridis strain (Chlorella viridis strain, Chlorella vulgaris strain, Chlorella viridis strain, Chlorella strain (Chlorella strain, Chlorella viridis strain (Chlorella viridis strain, chlorella minutissima (Chlorella minutissima), Chlorella mutant (Chlorella minutissima), Chlorella nocturna (Chlorella noctuina), Chlorella bardawil (Chlorella parva), Chlorella photophilus (Chlorella phophila), Chlorella prevenii (Chlorella pringshimii), Chlorella primula (Chlorella protothecoides), Chlorella protothecoides, Chlorella acidifera (Chlorella protothecoides), Chlorella regularis (Chlorella regularis) and Chlorella regularis (Chlorella regularis) Chlorella vulgaris (Chlorella vulgaris), Chlorella vulgaris var vulgaris (Chlorella vulgaris f. tertia), Chlorella vulgaris autotrophic variants (Chlorella vulgaris var. autotropica), Chlorella vulgaris green variants (Chlorella vulgaris. viridis), Chlorella vulgaris variants (Chlorella vulgaris. var. vulgaris), Chlorella vulgaris crude variants (Chlorella vulgaris. var. vulgaris. vulgare), Chlorella vulgaris crude variants (Chlorella vulgaris. var. vulgare f. tida), Chlorella vulgaris green variants (Chlorella vulgaris. var. vulgare), Chlorella vulgaris green variants (Chlorella vulgaris. var. vulgare), Chlorella vulgaris, Chlorella flava (Chlorella vulgaris. var. viridis), Chlorella vulgaris (Chlorella vulgaris), Chlorella flava (Chlorella vulgaris), Chlorella vulgaris (Chlorella vulgaris), Chlorella vulgaris, Chlorella (Chlorella sp., Chlorella sp., Chlorella sp Cyclotella meyersinia (Cyclotella meneghiniana), Cyclotella sp (Cyclotella sp.), Dunaliella sp (Dunaliella sp.), Dunaliella bainiensis (Dunaliella bardawil), Dunaliella biguata (Dunaliella bioculata), Dunaliella granulosa (Dunaliella grandiflora), Dunaliella ternifolia (Dunaliella maritima), Dunaliella minutissima (Dunaliella maritima), Dunaliella biguai (Dunaliella viridis), Dunaliella prorectina (Dunaliella salina), Dunaliella salina (Dunaliella salina), Dunaliella terrestra (Dunaliella viridis), Dunaliella viridis (Dunaliella viridans), Dunaliella viridiplella viridans (Dunaliella viridans), Dunaliella viridans (Dunaliella viridis), Dunaliella viridans, Dunaliella salina, Dunaliella viridans (Dunaliella viridis), Dunaliella viridis, The genus chrysophyces sp (gloothamnion sp.), phaeocystis sp (hymenomonassypium sp.), chrysophyces gulfw (galbanum), chrysophyces globiformis (isochrysophyces galbana), chrysophyces globulus (isochrysophyces galbana), lepidoptera (leponicalis), microscopia (micrantium), microcystis micrantium (micrantium) (utlb 2614), monochorium (monoraphium minutum), monochorium (monoraphium sp.), microsphericium sp, Nannochloropsis sp (Nannochloropsis sp.), synechocystis (Nannochloropsis sp.), porphyridium (Nannochloropsis sp), porphyridium (nepheloides sp), porphyridium (porphyridium), porphyridium sp), porphyridium (porphyridium), porphyridium sp), porphyridium (porphyridium), porphyridium sp), porphyridium (porphyridium), porphyridium (porphyridium sp), porphyridium (porphyridium), porphyridium sp), porphyridium (porphyridium), porphyridium (porphyridium), porphyridium sp), porphyridium (, The genera consisting of Sphingomonas gracilis (Nitzschia fructinum), Nitzschia hantzeri (Nitzschia hantzschia), Hippocampus (Nitzschia inconsicularia), Nitzschia intermedia (Nitzschia intermedia), Nitzschia microcephala (Nitzschia microcephala), Nitzschia microzyme (Nitzschia pusilla), Nitzschia ellipsoidea (Nitzschia pusilla), Nitzschia pusilla monoides (Nitzschia pusilla), Nitzschia quadrata (Nitzschia quadrata), Nitzschia sp), Physicolopsis sp Coccolithospermum sp. (Pleurochrysis sp.), Prochloranthus weinmii (Protothecahikerhami), Progesteronema gordonii (Prototheca stagnora), Prototheca podorferi (Prototheca portoriensis), Prokania sanguinea (Prototheca mori), Prototheca fuliginosus (Prototheca grandiflora), Prototheca peltatus (Prototheca grandiflora), Pyrococcus sp. (Pyramethonas sp.), Pyracanthus (Pyroborhizus), Chrysophytum capsulatum (Sarcina nodysophyte), Scenedesmus sp. (Scenedesmus armatus), Schizochytrium sp.), Spirophyceae (Spirophyceae), Tetrasella sp., Tetrastigma, Tetrasella sp. (Tetrasella sp.), Tetrasella sp., Tetrastigmaea (Tetrasella sp.), Tetrastigmatis sp., Tetrastigmaea (Tetrastigmaea sp.), and Tetrastigmatis sp.). In some embodiments, the algae is a chlamydomonas species. In some embodiments, the algae is chlamydomonas reinhardtii. In some embodiments, the algae are derivatives of a strain of chlamydomonas viridis, made by mutagenesis or mating with another strain of algae. In some embodiments, the Chlamydomonas is strain CC-125 or a derivative thereof deposited at the collection center of Chlamydomonas university of Minnesota.

Culture method for overproducing PPIX strains

Methods of culturing algae in liquid culture media include a variety of options, including ponds, ditches, small laboratory systems, and closed and partially closed bioreactor systems. Algae can also grow directly in water, for example, in the ocean, in the sea, in lakes, in rivers, in reservoirs, and the like.

In some embodiments, PPIX overproducing algae useful in the methods and compositions provided herein are grown in controlled culture systems (such as small scale laboratory systems, large scale systems, and/or closed and partially closed bioreactor systems). Small laboratory systems refer to cultures having a volume of less than about 6 liters, and can range from about 1 milliliter or less to about 6 liters. Large scale culture refers to a culture with a growth volume of greater than about 6 liters, and can range from about 6 liters to about 200 liters, and even larger scale system footprints of 5 to 2500 square meters, or greater. The large scale culture system may comprise a liquid culture system of about 10000 to about 20000 liters and up to about 1000000 liters.

Culture systems for use with methods of producing the compositions herein include closed structures (such as bioreactors) in which the environment is more tightly controlled than open or semi-closed systems. A photobioreactor is a bioreactor that incorporates some type of light source to provide photonic energy input to the reactor. The term bioreactor may refer to a system that is closed to the environment and has no direct exchange of gases and contaminants with the environment. A bioreactor can be described as a closed culture vessel and, in the case of a photobioreactor, an illuminated culture vessel designed for controlled biomass production of liquid cell suspension cultures.

In some embodiments, the algae used in the methods and compositions provided herein are grown in a fermentation vessel. In some embodiments, the vessel is a stainless steel fermentation vessel. In some embodiments, the algae are grown under heterotrophic conditions whereby one or more carbon sources are provided to the culture. In some embodiments, the algae are grown under aerobic and heterotrophic conditions. In some embodiments, the algae is grown to a density of greater than or about 10g/L, about 20g/L, about 30g/L, about 40g/L, about 50g/L, about 75g/L, about 100g/L, about 125g/L, or about 150 g/L.

In some embodiments, the algae is inoculated from the seed tank to an initial density of greater than about 0.1g/L, about 1.0g/L, about 5.0g/L, about 10.0g/L, about 20.0g/L, about 50g/L, about 80g/L, or about 100 g/L. Once inoculated, algae grow heterotrophically using an aerobic fermentation process. During this process, the algae are fed nutrients to maintain their growth. In some embodiments, these nutrients include a reduced carbon source. Exemplary aerobic fermentation processes and/or reducing carbon sources include, but are not limited to, acetate, glucose, sucrose, fructose, glycerol, and other types of sugars (e.g., dextrose (dextrose), maltose, galactose, sucrose, ribose, etc.). In some embodiments, the algal culture is supplemented with iron.

In some embodiments, the algae are cultured under dark conditions. Preferably, the dark condition has a brightness of less than 1000lux, less than 750lux, less than 500lux, less than 400lux, less than 300lux, less than 200lux, less than 100 lux. In some embodiments, the algal chlorophyll production cultured under dark conditions is deficient or reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% compared to the algae cultured under dark conditions. In some embodiments, the algae grown under dark conditions are supplemented with one or more nutrients. In some embodiments, algae grown under dark conditions are grown in the presence of a reduced carbon source, such as acetate, glucose, sucrose, fructose, glycerol, or other types of sugars (e.g., dextrose, maltose, galactose, sucrose, ribose, etc.). In some embodiments, algae grown under dark conditions are grown in the presence of iron, or are supplemented with iron.

Formulations and products containing PPIX

The PPIX-overproducing algal strains and cultures (such as those described herein) can be used in a variety of forms and formulations. In some embodiments, a composition comprising PPIX is prepared from a culture of algae over-producing PPIX, wherein the composition is red or red-like in color.

In some embodiments, the PPIX-containing composition is prepared from biomass isolated from cultured algae. In some embodiments, the biomass is further fractionated to remove one or more components. In some embodiments, the biomass is fractionated to remove starch. In some embodiments, the biomass is fractionated to remove proteins. In some embodiments, the biomass is fractionated or otherwise processed to remove carotenoids. In some embodiments, the biomass is fractionated or otherwise processed to enrich certain components. In some embodiments, the fractionated or treated biomass is rich in PPIX. In some embodiments, the fractionated or treated biomass is enriched in protein, or protein and PPIX. In some embodiments, the fractionation or treatment enhances the red or red-like color of the formulation. The fractionated or treated biomass can be enriched in protein content such that the composition is about 10% protein, greater than about 10% protein, or greater than about 20%, about 30%, about 40%, or about 50% protein.

In some embodiments, the biomass is fractionated or otherwise treated to remove or reduce any heme content, and optionally to enrich the PPIX. Thus, in some embodiments, a fraction or composition may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50% more PPIX than the amount of heme. Such fractionation may include the isolation of PPIX from heme. For example, hemopexin and protein-associated heme may be isolated from PPIX, which is not a protein-coupled or protein-associated compound. Both free heme and protein-related heme can be isolated from PPIX based on the binding of heme to iron. PPIX contains no iron moieties and therefore this feature can be used to isolate PPIX from heme containing fractions. In some embodiments, the algal biomass herein is fractionated or otherwise treated such that the heme content is reduced, such as to less than 1%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than a detectable level in the PPIX-containing fraction. Alternatively, the algal biomass herein is fractionated or otherwise treated such that the heme protein content is reduced, such as to less than 1%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than a detectable level in the fraction containing PPIX. In some embodiments, the algal biomass or fractionated biomass is produced by a ferrochelatase deficient strain or a strain that does not produce or accumulate heme, such that the biomass or fraction has little or no heme.

In some embodiments, the PPIX-containing composition is a PPIX-containing liquid prepared from a culture medium of cultured algae. In some embodiments, the PPIX-containing composition is prepared from PPIX (extracellular fraction) found extracellularly in an algal culture. In some embodiments, the algae culture is lysed or otherwise treated to release PPIX from the cells. In some embodiments, the liquid containing PPIX is further fractionated to remove one or more components. In some embodiments, the liquid containing PPIX is fractionated to remove starch. In some embodiments, the PPIX-containing liquor is fractionated to remove proteins. In some embodiments, the PPIX-containing liquor is fractionated or otherwise treated to remove carotenoids. In some embodiments, the PPIX-containing liquor is fractionated or otherwise treated to enrich certain components. In some embodiments, the fractionated or treated liquid containing PPIX is rich in PPIX. In some embodiments, the fractionation or treatment enhances the red or red-like color of the formulation.

In some embodiments, the PPIX-containing liquor is fractionated or otherwise treated to remove or reduce any heme content and optionally to enrich the PPIX. Such fractionation can include the isolation of PPIX from heme. For example, a heme-binding protein and a heme-related protein in a liquid may be separated from PPIX, which is not a protein-coupled or protein-related compound. Both free heme and protein-related heme can be isolated from PPIX based on the binding of heme to iron. PPIX contains no iron moieties, and therefore this feature can be used to isolate PPIX from heme containing fractions. In some embodiments, the PPIX-containing liquid is fractionated or otherwise treated such that the heme content is reduced, e.g., to less than 1%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001%, or less than levels typically detectable in PPIX-containing fractions. In some embodiments, the PPIX-containing liquid is produced by a ferrochelatase deficient strain or a strain that does not produce or accumulate heme, such that the PPIX-containing liquid has little or no heme content.

In some embodiments, the biomass or PPIX-containing composition is a PPIX-containing liquid, and/or a fractionated PPIX-containing composition or PPIX-containing liquid containing about 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, or greater than 10% protoporphyrin IX by total weight percent. Further, in some embodiments, the biomass or PPIX-containing composition is a PPIX-containing liquid, and/or a fractionated PPIX-containing composition or PPIX-containing liquid containing protoporphyrin IX at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher than chlorophyll.

Compositions containing PPIX, including biomass, liquids, and fractionated preparations, can be further processed. Such processing may include concentration, drying, lyophilization and freezing. In various embodiments, for example, a PPIX-containing composition may be combined with additional components and ingredients to produce an edible product. In some embodiments, the PPIX-containing composition imparts a red or red-like color to the edible product. In some embodiments, the PPIX-containing compositions impart meat-like characteristics to the edible product, such as meat-like taste, aroma, and/or texture. In some embodiments, the PPIX-containing compositions provide a blood appearance to edible products (such as meat replicas, beef-like products, chicken-like products, and the like).

In some embodiments, the PPIX-containing composition is a PPIX-containing liquid, and/or a fractionated PPIX-containing composition or PPIX-containing liquid that provides at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% protein to an edible composition. In some embodiments, the PPIX-containing composition is a PPIX-containing liquid, and/or a fractionated PPIX-containing composition or PPIX-containing liquid that provides greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% protein in an edible product. In some embodiments, the PPIX-containing composition is a PPIX-containing liquid and/or a recommended daily dose of omega-3 fatty acids or a portion thereof is provided to an edible product, e.g., a fractionated PPIX-containing composition or PPIX-containing liquid that adds at least about 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 125mg, 150mg, 175mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, or 500mg omega-3 fatty acids to an edible composition.

In some embodiments, the PPIX-containing composition is a PPIX-containing liquid, and/or a fractionated PPIX-containing composition or PPIX-containing liquid that provides at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the recommended daily dose of vitamin A or at least about 20 μ g, 50 μ g, 100 μ g, 200 μ g, 300 μ g, 400 μ g, 500 μ g, 600 μ g, 700 μ g, 800 μ g, 900 μ g or 1000 μ g of vitamin A Retinol Activity Equivalent (RAE). In some embodiments, the PPIX-containing composition is a PPIX-containing liquid, and/or a fractionated PPIX-containing composition or PPIX-containing liquid that provides a vitamin a Retinol Activity Equivalent (RAE) of no more than about 2000 μ g, 2500 μ g, or 3000 μ g. Alternatively and/or additionally, the PPIX-containing composition is a PPIX-containing liquid, and/or a fractionated PPIX-containing liquid. Alternatively and/or additionally, the PPIX-containing composition is a PPIX-containing liquid, and/or a fractionated PPIX-containing composition or PPIX-containing liquid providing about 0.25mg, 0.5mg, 1mg, 1.5mg, 2mg, 2.5mg, 3mg, 4mg, 5mg, 6mg, 9mg, 10mg, 12mg or 15mg of beta-carotene.

Alternatively and/or additionally, the PPIX-containing composition is a PPIX-containing liquid and/or provides a fraction or less of the daily recommended limit for saturated fat of an edible product, e.g., no more than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the daily recommended saturated fat of a fractionated PPIX-containing composition or PPIX-containing liquid. Alternatively and/or additionally, the PPIX-containing composition is a PPIX-containing liquid and/or a fractionated PPIX-containing composition or PPIX-containing liquid that provides no more than 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.5%, 2%, 5% or 10% of the total saturated fat in the edible composition or finished product made from the edible composition.

In some embodiments, the PPIX-containing composition is combined with additional ingredients to produce a meat-like product (meal-like product). These meat-like products may include clean meat or cultured meat (made from animal cells grown in the laboratory or outside the animal), vegetable-based and non-animal-based meat (made from vegetable components and/or non-animal derived components). In some embodiments, the PPIX-containing composition made from overproduced algae is combined with additional ingredients to produce a meat-like product, whereby the addition of the PPIX-containing composition imparts a red or red-like color, a meat-like flavor, a meat-like taste, and/or a meat-like texture to the meat-like product. In some embodiments, the meat-like character imparted by the PPIX-containing composition imparts a raw or uncooked product. In some embodiments, the meat-like characteristics imparted by the PPIX-containing composition are imparted to the culinary product. Alternatively, at least one characteristic of meat or meat-like flavor or aroma, meat or meat-like texture, blood sample appearance, meat or meat-like color is derived from the algal preparation.

In some embodiments, the entire algae or the fractionated algae is combined with an additional protein source in the edible composition. For example, the protein source may be a wheat protein (such as wheat protein, texturized wheat protein), pea protein, texturized pea protein, soy protein, texturized soy protein, potato protein, whey protein, yeast extract, fungal protein (such as bayone), or other plant-based protein source or any combination thereof. In some embodiments, the entire algae or the fractionated algae is combined with an oil or fat source in the edible composition. For example, the oil or fat source may be coconut oil, rapeseed oil, sunflower oil, safflower oil, corn oil, olive oil, avocado oil, nut oil, or other vegetable based oil or fat source, or any combination thereof. In some embodiments, the whole or fractionated algae is combined with a starch or other carbohydrate source, such as from potato, chickpeas, wheat, soy (soy), beans (beans), corn or other plant-based starches or carbohydrates, or any combination thereof. In some embodiments, the whole algae or the graded algae are combined with a thickener in the edible composition. For example, starches (such as arrowroot, corn starch, catakul starch, potato starch, sago, tapioca starch) and starch derivatives thereof can be used as thickeners; the microorganisms and vegetable gums used as food thickeners include algin, guar gum, locust bean gum, konjac, and xanthan gum; proteins such as collagen and egg white may be used as thickeners; sugar polymers used as thickeners include agar, methylcellulose, carboxymethylcellulose, pectin, and carrageenan. In some embodiments, the whole algae or algae fraction may be combined with vitamins and minerals in an edible composition, such as vitamin E, vitamin C, thiamine (vitamin B1), zinc, niacin, vitamin B6, riboflavin (vitamin B2), and vitamin B12.

In some embodiments, the entire algae or algae fractions may be combined with additional ingredients such that the edible composition and/or finished product is vegetarian, strictly vegetarian, or gluten-free and thus may conform to the dietary guidelines of jewish and halal. Thus, in some embodiments, the edible composition and/or finished product may be suitable for consumption by vegetarians, vegans, gluten-free people, koshers, and halans. In some embodiments, the whole algae or algae fraction may be combined with additional ingredients such that the edible composition and/or finished product is GMO free and/or free of any ingredients derived from genetically engineered organisms or cells.

Exemplary numbering embodiments

The following embodiments set forth non-limiting permutations of the combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is deemed to be dependent upon or related to each previously or subsequently numbered embodiment regardless of the order in which they are listed.