EP-4735618-A1 - CELL FREE PRODUCTION OF ANTHOCYANINS

EP4735618A1EP 4735618 A1EP4735618 A1EP 4735618A1EP-4735618-A1

Abstract

The invention provides methods and. compositions for production of anthocyanins. The invention provides cell-free methods and compositions for bioproduction of anthocyanins for a. substrate.

Inventors

KIM, NEILL
EMILEH, Ali
AMAYA, Jose
LI, JINGYI
BRIDEAU, Nicholas

Assignees

Debut Biotechnology, Inc.

Dates

Publication Date: 20260506
Application Date: 20240628

Claims (1)

Patent Application Claims: 1. A method for cell-free production of anthocyanins, wherein the method comprises: providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of one or more substrates to anthocyanins. 2. The method of claim 1, wherein the substrate is a catechin. 3. The method of claim 2, wherein catechin is (+)-catechin. 4. The method of claim 3, wherein the anthocyanin is cyanidin-3-glucoside. 5. The method of claim 1, wherein the one or more enzymes are selected from a group consisting of: anthocyanidin synthase enzyme (ANS) and anthocyanidin 3-O- glucosyltransferase (3GT). 6. The method of claim 5, wherein the anthocyanidin synthase enzyme (ANS) catalyzes the conversion of catechin to cyanidin. 7. The method of claim 5, wherein the anthocyanidin 3-O-glucosyltransferase (3GT) catalyzes the conversion of cyanidin to cyanidin-3-glucoside. 8. The method of claim 1, wherein the cell-free medium comprises an activated sugar. 9. The method of claim 8, wherein the activated sugar is UDP-glucose. 10. The method of claim 9, wherein the UDP-glucose is added to the cell-free medium. 11. The method of claim 9, wherein the UDP-glucose is synthesized from one or more ingredients selected from the group consisting of: sucrose, glucose, UTP, UDP, ATP, glucose-6-phosophate, glucose-1-phosphate, and/or polyphosphate. 12. The method of claim 11, wherein the one or more enzymes is selected from the group consisting of sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), Patent Application phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and nucleoside diphosphate kinase (NDK). 13. The method of claim 11, wherein the one or more enzymes is sucrose synthase (SuSy). 14. The method of claim 1, wherein the cell-free medium is a cell lysate. 15. The method of claim 14, wherein the cell lysate is a cell lysate from cells from a host organism expressing the one or more enzymes. 16. The method of claim 15, wherein the host organism is selected from a group consisting of: bacteria, yeast, and/or mammalian cells. 17. The method of claim 16, wherein the one or more enzymes are introduced in the host organism by integration into genome of the host organism or on a plasmid. 18. The method of claim 16, wherein the host organisms expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes. 19. The method of claim 18, further comprising lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of anthocyanins. 20. The method of any of claims 1-19, wherein ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and/or NDK are present in the cell-free medium for cell-free production of anthocyanins. 21. The method of claim 20, wherein the method does not include separating and/or purifying ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and/or NDK, for cell-free production of anthocyanins. Patent Application 22. The method of claim 20, wherein the method comprises separating and/or purifying at least one of a ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and/or NDK, for cell-free production of anthocyanins. 23. The method of any of claims 1-22, wherein the cell-free medium further comprises: buffer, catechin, an activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and/or water. 24. The method of claim 23, wherein the buffer is a phosphate buffer. 25. The method of claim 23, wherein the activated sugar is UDP-glucose. 26. The method of claim 23, wherein the cell-free medium further comprises sodium ascorbate, ascorbic acid, ammonium iron sulphate, and 2-oxoglutarate. 27. The method of claim 23, wherein the catechin is (+)-catechin. 28. The methods of any of claims 1 – 27, wherein the method results in titer value of produced anthocyanins is from about 2 times to about 5000 times higher than methods for cell-based production of anthocyanins. 29. The methods of any of claims 1 – 27, wherein the method results in titer value of produced anthocyanins is from about 5 times to about 1000 times higher than methods for cell-based production of anthocyanins. 30. The method of claim 23, wherein pH of cell-free medium is from about 5 to about 10. 31. The method of claim 23, wherein pH of cell-free medium is from about 6 to about 8. 32. The method of claim 26, wherein the buffer is in the concentration of from about 1 mM to about 200 mM. Patent Application 33. The method of claim 26, wherein ammonium iron sulfate concentration is from about 0.25 mM to about 10 mM. 34. The method of claim 26, wherein the cell-free medium further comprises α-ketoglutaric acid in a concentration of from about 5 mM to about 50 mM. 35. The method of claim 26, wherein the ascorbic acid is in the concentration of from about 1 mM to about 100 mM. 36. The method of claim 26, wherein catechin is the concentration of from about 0.5 mM to about 50 mM. 37. The method of claim of 26, wherein magnesium chloride is present in the range of about 1 mM to about 20 mM. 38. The method of claim of 26, wherein sucrose is present in the range of about 10 mM to about 600 mM. 39. The method of claim 26, wherein the cell lysate including the one or more enzymes is present at a concentration of about 5% (v/v) to about 40% (v/v). 40. The method of claim 26, wherein reaction for cell-free production is carried out for a duration of about 0.5 hours to about 48 hours. 41. The method of claim 26, wherein reaction for cell-free production is carried out for a duration of about 0.5 hours to about 20 hours. 42. The method of claim 26, wherein temperature of the cell-free medium is from about 20 ℃ to about 40 ℃. 43. The method of claim 1, wherein reaction is conducted in a bubble column reactor, wherein the one or more enzymes are in a solution. Patent Application 44. The method of claim 1, wherein reaction is conducted in a packed bed reactor, wherein the one or more enzymes are immobilized. 45. The method of claim 5, wherein the anthocyanidin synthase (ANS) is an enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of: Accession # Organism XP 0227367581 D i ib hi 46. The method of claim 5, wherein the anthocyanidin 3-O-glucosyltransferase (3GT) is an enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of: Accession # Organism Patent Application AFJ52972.1 Linum usitatissimum ABR24135.1 Vitis labrusca 47. T he method of claim 13, wherein the sucrose synthase (SuSy) is an enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of: Accession # Organism Patent Application 48. The methods of any of claims 5 or 13, wherein the ANS, 3GT, and/or SuSy enzymes are modified and optimized for the cell-free production of anthocyanins. 49. A composition for cell-free production of anthocyanins, wherein the composition comprises: one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of one or more substrates to an anthocyanin. 50. The composition of claim 49, wherein the substrate is a catechin. 51. The composition of claim 50, wherein catechin is (+)-catechin. 52. The composition of claim 51, wherein the anthocyanin is cyanidin-3-glucoside. 53. The composition of claim 49, wherein the one or more enzymes are selected from a group consisting of: anthocyanidin synthase enzyme (ANS) and anthocyanidin 3-O- glucosyltransferase (3GT). 54. The composition of claim 53, wherein the anthocyanidin synthase enzyme (ANS) catalyzes the conversion of catechin to cyanidin. 55. The composition of claim 53, wherein the anthocyanidin 3-O-glucosyltransferase (3GT) catalyzes the conversion of cyanidin to cyanidin-3-glucoside. 56. The composition of claim 49, wherein the cell-free medium comprises an activated sugar. 57. The composition of claim 56, wherein the activated sugar is UDP-glucose. 58. The composition of claim 57, wherein the UDP-glucose is added to the cell-free medium. 59. The composition of claim 58, wherein the UPD-glucose is synthesized in the cell-free medium by the one or more enzymes. Patent Application 60. The composition of claim 57, wherein the UDP-glucose is synthesized from one or more ingredients selected from the group consisting of: sucrose, glucose, UTP, UDP, ATP, glucose-6-phosophate, glucose-1-phosphate, and/or polyphosphate. 61. The composition of claim 60, wherein the one or more enzymes is sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and nucleoside diphosphate kinase (NDK). 62. The composition of claim 49, wherein the cell-free medium is a cell lysate. 63. The composition of claim 50, wherein the cell lysate is a cell lysate from cells from a host organism expressing the one or more enzymes. 64. The composition of claim 63, wherein the host organism is selected from a group consisting of: bacteria, yeast, and/or mammalian cells. 65. The composition of claim 64, wherein the one or more enzymes are introduced in the host organism by integration into genome of the host organism or on a plasmid. 66. The composition of claim 65, wherein the host organisms expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes. 67. The composition of claim 66, further comprising lysing of cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of anthocyanins. 68. The composition of any of claims 49-67, wherein ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and/or NDK are present in the cell-free medium for cell-free production of anthocyanins. Patent Application 69. The composition of claim 68, wherein the ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and/or NDK for cell-free production of anthocyanins are not separated and/or purified. 70. The composition of claim 68, wherein the ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and/or NDK for cell-free production of anthocyanins are separated and/or purified. 71. The composition of any of claims 49-70, wherein the cell-free medium further comprises: buffer, catechin, an activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and/or water. 72. The composition of claim 71, wherein the buffer is a phosphate buffer. 73. The composition of claim 71, wherein the activated sugar is UDP-glucose. 74. The composition of claim 71, wherein the cell-free medium further comprises sodium ascorbate, ascorbic acid, ammonium iron sulphate, and 2-oxoglutarate. 75. The composition of claim 71, wherein the catechin is (+)-catechin. 76. The compositions of any of claims 49 – 75, wherein the composition is used for producing anthocyanins with titer value of produced anthocyanins is from about 2 times to about 100 times higher than methods for cell-based production of anthocyanins. 77. The compositions of any of claims 49 – 75, wherein the composition is used for producing anthocyanins with titer value of produced anthocyanins is from about 5 times to about 10 times higher than methods for cell-based production of anthocyanins. 78. The composition of claim 71, wherein pH of cell-free medium is from about 5 to about 10. 79. The composition of claim 71, wherein pH of cell-free medium is from about 6 to about 8. Patent Application 80. The composition of claim 72, wherein the buffer is in the concentration of from about 1 mM to about 200 mM. 81. The composition of claim 74, wherein ammonium iron sulfate is in the range of from about 0.25 mM to about 10 mM. 82. The composition of claim 74, wherein the cell-free medium further comprises α- ketoglutaric acid in a concentration of from about 5 mM to about 50 mM. 83. The composition of claim 74, wherein the ascorbic acid is in the concentration of from about 1 mM to about 100 mM. 84. The composition of claim 74, wherein catechin is the concentration of from about 0.5 mM to about 50 mM. 85. The composition of claim 74, wherein magnesium chloride is present in the range of from about 1 mM to about 20 mM. 86. The composition of claim 74, wherein sucrose is present in the range of from about 10 mM to about 600 mM. 87. The composition of claim 74, wherein the cell lysate including the one or more enzymes is present at a concentration of about 5% (v/v) to about 40% (v/v). 88. The composition of claim 74, wherein reaction for cell-free production is carried out for a duration of about 0.5 hours to about 48 hours. 89. The composition of claim 74, wherein reaction for cell-free production is carried out for a duration of about 0.5 hours to about 20 hours. 90. The composition of claim 74, wherein temperature of the cell-free medium is from about 20 ℃ to about 40 ℃. Patent Application 91. The composition of claim 49, wherein the composition is in a bubble column reactor, wherein the one or more enzymes are in a solution. 92. The composition of claim 90, wherein the composition is in a packed bed reactor, wherein the one or more enzymes are immobilized. 93. The composition of claim 53, wherein the anthocyanidin synthase (ANS) is an enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of: Accession # Organism 94. The composition of claim 53, wherein the anthocyanidin 3-O-glucosyltransferase (3GT) is an enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of: Patent Application Accession # Organism AFJ52972.1 Linum usitatissimum 95. T he composition of claim 61, wherein the sucrose synthase (SuSy) is an enzyme with an amino acid sequence at least 95% identical to any one of enzymes selected from the group consisting of: Accession # Organism Patent Application 96. The composition of any of claims 53 or 61, wherein the ANS, 3GT, and/or SuSy enzymes are modified and optimized for the cell-free production of anthocyanins.

Description

Patent Application Cell-Free Productions of Anthocyanins: I. Field of Invention: The invention is related to materials and methods for production of anthocyanins. The invention provides methods and materials for cell-free production of one or more anthocyanins. II. Reference to Sequence Listing The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named DEBU-021-01WO.xml, created on June 28, 2024, which is 69 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. III. Background In the food coloring and pigment industry, there is a need for natural pigments and Anthocyanins are currently produced by chopping or crushing the fruit or vegetable and subsequent infusion of water acidified with a common food acid. This extract is then concentrated by non-chemical separation techniques. Pigment extracts from plant sources generally contain mixtures of different anthocyanin molecules, which vary by their level of hydroxylation, methylation and acylation. This increases the cost of production because of the purification required from these mixtures. Moreover, these factors can vary in the source plant from year to Patent Application year, and are influenced by weather and environmental factors. Thus, another challenge for the commercial production of anthocyanin pigments from plants is that harvest is often limited to once a year. This means a large volume of extract has to be prepared and stored for an extended period of time to supply the needs of the food industry throughout the year. Special storage conditions often have to be available due to the instability of anthocyanins. Thus, new methods for production of anthocyanins are required. The invention provides methods for cell-free production of anthocyanins. The methods of the invention comprise: providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of a substrate to anthocyanins. In certain aspects of the invention, the methods of the invention may provide for production of one or more intermediates during the transformation of one or more substrates to anthocyanin. In certain aspects of the invention, the anthocyanin is cyanidin-3-glucoside. In certain embodiments, the substrate is a catechin. In certain embodiments, the catechin is (+)-catechin. In certain embodiments, the one or more enzymes are selected from a group consisting of: anthocyanidin synthase enzyme (ANS) and anthocyanidin 3-O- glucosyltransferase (3GT). In certain embodiments of the invention, the anthocyanidin synthase (ANS) enzyme catalyzes the conversion of catechin to cyanidin. In certain embodiments, the anthocyanidin 3-O- glucosyltransferase (3GT) enzyme catalyzes the conversion of cyanidin to cyanidin-3-glucoside. In certain preferred embodiments, the cyanidin produced by transformation of catechin by anthocyanidin synthase (ANS) enzyme is further transformed to cyanidin-3-glucoside by anthocyanidin 3-O-glucosyltransferase (3GT) enzyme. An overview of the conversion of catechin to cyanidin-3-glucoside is provided in FIG.1. In certain embodiments, the cell-free production of anthocyanins involves a cell-free medium. In certain embodiments, the cell-free medium comprises a cell lysate. In certain embodiments, the cell-free medium comprises an activated sugar. In certain embodiments, the activated sugar is UDP-glucose. In certain embodiments, the UDP-glucose is added to the cell-free medium. In certain other embodiments, the UDP-glucose is synthesized in the cell-free medium by the one or more enzymes. In certain embodiments, UDP-glucose is synthesized from sucrose. In certain embodiments, sucrose synthase (SuSy) enzyme transforms sucrose to UDP-glucose. An Patent Application overview of the conversion of cyanidin and cyanidin-3-glucoside, wherein the UDP-glucose is synthesized from sucrose is provided in FIG.2. In certain beneficial aspects, the invention provides that the UDP-glucose used in the reaction is generated from other substrates in course of the reaction. In certain embodiments, the UDP moiety in UDP-glucose is recycled in the cell-free medium. The recycling of UDP provides economic efficiency of the processes of the invention. Accordingly, in certain aspects, the production of cyandin-3-glucoside from cyanidin catalyzed by 3GT is conducted in conjunction with other methods for UDP-glucose production or recycling of UDP. In certain embodiments, the overview of the synthetic scheme involving the UDP-glucose production and/or recycling is provided in FIG.3. In certain embodiments, UDP-glucose is generated by the reaction of uridine triphosphate (UTP) with glucose-1-phosphate. In certain preferred embodiments, the reaction of UTP and glucose-1-phosphate is catalyzed by UTP-glucose-1-phosphate uridylyltransferase (UGP). In certain preferred embodiments, the U