Cell-free production of anthocyanins

Abstract

The present invention provides methods and compositions for the production of anthocyanins. The present invention provides cell-free methods and compositions for the bioproduction of anthocyanins for substrates. In certain embodiments of the present 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.

Description

Technical Field

[0001] I. Field of the Invention: The present invention relates to materials and methods for the production of anthocyanins. The present invention provides methods and materials for the cell-free production of one or more anthocyanins.

[0002] II. Reference to the Sequence Listing This application is filed with a sequence listing in electronic format. The sequence listing is provided as the file name DEBU-021-01WO.xml (creation date: June 28, 2024. Size: 69 kilobytes). The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

Background Art

[0003] III. Background In the food coloring and pigment industries, there is a need for natural pigments, and anthocyanins are highly sought-after compounds. These pigments have a range of colors that span from red to purple to blue depending on the functional groups of the molecule and the pH, and there is also a demand for them not only with respect to their colors but also with respect to their health-promoting properties. These molecules are responsible for the colors of various plants and have many beneficial attributes that range from cancer treatment to supporting the development of solar energy technologies.

[0004] Common sources of anthocyanins commercially used as natural food colorants are grape skin extracts, purple cabbage, purple carrots, and elderberries. They are natural pH indicators, being red under acidic conditions where they are most stable and becoming bluish and more unstable as the pH increases. Thus, a stable natural blue source that can be used in the food and beverage industries has not yet been identified.

Summary of the Invention

Means for Solving the Problems

[0005] IV. Summary of the Invention Anthocyanins are currently produced by chopping or crushing fruits or vegetables and subsequently infusing them with water acidified with a common food-grade acid. This extract is then concentrated using non-chemical separation techniques. Pigment extracts derived from plant sources generally contain a mixture of different anthocyanin molecules, varying in their levels of hydroxylation, methylation, and acylation. This increases production costs due to the purification required from these mixtures. Furthermore, these factors can fluctuate year to year in the raw material plants and are influenced by weather and environmental factors. Therefore, another challenge for the commercial production of plant-derived anthocyanin pigments is that harvesting is limited to once a year. This means that large quantities of extract must be prepared and stored for extended periods to meet the needs of the food industry throughout the year. Due to the instability of anthocyanins, special storage conditions often need to be available.

[0006] Therefore, a new method for the production of anthocyanins is needed. The present invention provides a method for cell-free production of anthocyanins. The method of the present invention comprises the step of providing one or more enzymes in a cell-free medium, wherein the one or more enzymes comprise the step of resulting in the conversion of a substrate to anthocyanin. In a particular aspect of the present invention, the method of the present invention may provide the production of one or more intermediates during the conversion of one or more substrates to anthocyanin. In a particular aspect of the present invention, the anthocyanin is cyanidin-3-glucoside. In a particular embodiment, the substrate is catechin. In a particular embodiment, the catechin is (+)-catechin. In a particular embodiment, the one or more enzymes are selected from the group consisting of anthocyanidin synthase enzymes (ANS) and anthocyanidin 3-O-glucosyltransferases (3GT).

[0007] In certain embodiments of the present 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 the conversion of catechin by the anthocyanidin synthase (ANS) enzyme is further converted to cyanidin-3-glucoside by the anthocyanidin 3-O-glucosyltransferase (3GT) enzyme. An overview of the conversion from catechin to cyanidin-3-glucoside is provided in Figure 1.

[0008] In certain embodiments, the cell-free production of the anthocyanins requires 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 one or more enzymes. In certain embodiments, the UDP-glucose is synthesized from sucrose. In certain embodiments, the sucrose synthase (SuSy) enzyme converts sucrose to UDP-glucose. An overview of the conversion of cyanidin and cyanidin-3-glucoside, from which UDP-glucose is synthesized from sucrose, is provided in Figure 2.

[0009] In a particular beneficial aspect, the present invention provides a method in which the UDP-glucose used in the above reaction is generated from other substrates during the course of the reaction. In a particular embodiment, the UDP portion of the UDP-glucose is recycled in the cell-free medium. The recycling of UDP provides economic efficiency for the process of the present invention. Thus, in a particular aspect, the production of cyanidin-3-glucoside from cyanidin catalyzed by 3GT is carried out in conjunction with other methods for UDP-glucose production or UDP recycling. In a particular embodiment, an outline of a synthetic scheme requiring the above UDP-glucose production and / or recycling is provided in Figure 3. In a particular embodiment, UDP-glucose is produced by the reaction of uridine triphosphate (UTP) with glucose-1-phosphate. In a particular preferred embodiment, the reaction of UTP and glucose-1-phosphate is catalyzed by UTP-glucose-1-phosphate uridilyltransferase (UGP). In a particular preferred embodiment, the UGP may be galU.

[0010] In certain contexts, glucose-1-phosphate is a source for the formation of UDP-glucose. Therefore, in certain embodiments, glucose-1-phosphate can be added to a reaction for the production of cyanidin-3-glucoside. In certain embodiments, the glucose-1-phosphate can be supplied to a reaction for the preparation of cyanidin-3-glucoside.

[0011] In certain embodiments, glucose-1-phosphate is produced during the course of the above reaction. In certain embodiments, glucose-1-phosphate is produced by an enzyme during the course of the above reaction. Thus, in certain embodiments, glucose is converted to glucose-6-phosphate by the reaction of ATP (adenosine triphosphate) and glucose. In certain embodiments, the reaction between glucose and ATP results in the production of glucose-6-phosphate and adenosine diphosphate (ADP). In certain embodiments, the conversion from glucose to glucose-6-phosphate is catalyzed by glucokinase (GLK). In certain embodiments, the conversion from glucose to glucose-6-phosphate is catalyzed by hexokinase (HK). In certain embodiments, glucose-6-phosphate is converted to glucose-1-phosphate. In certain embodiments, the conversion from glucose-6-phosphate to glucose-1-phosphate is catalyzed by phosphoglucumutase (PGM).

[0012] In certain aspects, the present invention further provides that the nucleoside triphosphate species used in the above reaction are recycled. In certain embodiments, the recycled nucleoside triphosphates are ATP and UTP. In certain embodiments, UTP is produced by the reaction of UDP and ATP. In certain embodiments, the production of UTP from UDP and ATP is catalyzed by nucleoside diphosphate kinase (NDK). In certain embodiments, ATP may be produced from ADP and a phosphate or polyphosphate. In certain embodiments, the conversion from ADP to ATP is catalyzed by polyphosphate kinase (PPK).

[0013] In certain beneficial aspects, the present invention provides that the process is cost-effective due to the recycling of chemical intermediates involved in the reaction. In certain embodiments, the present invention provides that chemical intermediates such as UDP-glucose, UDP, and glucose-1-phosphate are recycled. In fact, in certain embodiments, the only reagents required in non-catalytic amounts for the production of cyanidin-3-glucoside by the conversion of catechin and / or cyanidin are polyphosphate / phosphate and glucose. The process provided in the present invention is cost-effective because it is carried out in a cell-free medium and its components (required in non-catalytic amounts) are economical.

[0014] In a particular embodiment, one or more of the enzymes used in the conversion of a substrate to anthocyanin are expressed in a host organism. In a particular embodiment, the cell lysate in the cell-free medium is the cell lysate of the host organism expressing one or more of the enzymes for the conversion of a substrate to anthocyanin. In a particular embodiment, the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells. In a particular embodiment, one or more of the enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism. In a particular embodiment, the host organism expressing one or more of the enzymes is cultured until a predetermined biomass is achieved that produces the essential amount of one or more of the enzymes for the conversion of a substrate to anthocyanin. In a particular embodiment, the method comprises the step of lysing cells and subsequently removing cell debris to produce cell lysates for use in the cell-free medium for cell-free production of anthocyanins.

[0015] In certain embodiments, anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and / or sucrose synthase (SuSy) are present in the cell-free medium for cell-free production of anthocyanins. In certain embodiments, the method does not include the step of separating and / or purifying anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and sucrose synthase (SuSy) for cell-free production of anthocyanins. It is beneficial for the method to have a system that does not require enzyme separation because it eliminates an extra step and results in a reduction of production costs. In certain embodiments, the method includes the step of separating and / or purifying at least one of anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and sucrose synthase (SuSy) for cell-free production of anthocyanins.

[0016] In a particular embodiment, the one or more enzymes expressed in the host cell are anthocyanidin synthase (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucocomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). In certain embodiments, anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are present in the cell-free medium for cell-free generation of cyanidin-3-glucoside.

[0017] In certain embodiments of the present invention, the reaction mixture comprises a purified enzyme for cell-free production of cyanidin-3-glucoside. In certain embodiments, the purified enzyme is anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). In certain embodiments, the purified enzyme is anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and / or sucrose synthase (SuSy). In certain embodiments, the purified enzyme is anthocyanidin synthase enzyme (ANS) and / or anthocyanidin 3-O-glucosyltransferase (3GT).

[0018] In certain embodiments, the CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK enzymes are present in the cell-free medium for cell-free production of carminic acid. In certain embodiments, the composition of the present invention provides CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK, which are neither purified nor separated.

[0019] In certain embodiments, the cell-free medium may further comprise any other additional components required for the cell-free production of cyanidin-3-glucoside. In certain embodiments, the cell-free medium comprises a buffer, catechin, cyanidin, activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and / or water. In certain embodiments, the cell-free medium comprises a buffer, catechin, cyanidin, activated sugar, magnesium chloride, cell lysate, sucrose, and / or water. In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for the enzymatic conversion of catechin and / or cyanidin to cyanidin-3-glucoside. In certain embodiments, the buffer maintains a pH of about 5 to about 9 in the reaction mixture. In certain embodiments, the buffer maintains a pH of about 6 to about 8 in the reaction mixture. In a particular embodiment, the buffer maintains the pH in the reaction mixture in the range of 6 to 8. In a particular embodiment, the buffer is a phosphate buffer. In a particular embodiment, the buffer is present at a concentration of about 1 mM to about 200 mM. In a particular embodiment, the buffer is present at a concentration of about 5 mM to about 100 mM.

[0020] In certain embodiments, the cell-free medium further comprises a buffer, catechin, activated sugar, magnesium chloride, a cell lysate having an enzyme, sucrose, and / or water. In certain embodiments, the cell-free medium further comprises sodium ascorbate, ascorbic acid, ammonium iron sulfate, and α-ketoglutaric acid / 2-oxoglutarate.

[0021] In certain embodiments, the buffer in the cell-free medium maintains the pH of the reaction mixture within an optimal range. In certain embodiments, the pH of the reaction medium is about 5.5 to about 10. In certain embodiments, the pH of the cell-free medium is about 6 to about 9. In certain embodiments, the pH of the cell-free medium is about 6 to about 8. In certain embodiments, the concentration of the buffer in the cell-free medium is about 1 mM to about 1000 mM. In certain embodiments, the concentration of the buffer is about 2.5 mM to about 750 mM. In certain embodiments, the concentration of the buffer is about 5 mM to about 100 mM. In certain embodiments, the concentration of the buffer is about 20 mM to about 100 mM. In certain embodiments, the buffer is a phosphate buffer.

[0022] In a particular embodiment, the activated sugar in the cell-free medium is UDP-glucose. In a particular embodiment, the UDP-glucose is added to the cell-free medium. In a particular embodiment, the UDP-glucose in the cell-free medium is synthesized from sucrose. In a particular embodiment, the cell-free medium contains UDP. In a particular embodiment, the concentration of the activated sugar in the cell-free medium is about 0.01 mM to about 10 mM. In a particular embodiment, the concentration of the activated sugar in the cell-free medium is about 0.05 mM to about 5 mM. In a particular embodiment, the concentration of the activated sugar in the cell-free medium is about 0.1 mM to about 2.5 mM.

[0023] In a particular embodiment, the substrate is catechin. In a particular embodiment, the concentration of catechin in the cell-free medium is about 0.1 mM to about 50 mM. In a particular embodiment, the concentration of catechin in the cell-free medium is about 0.5 mM to about 40 mM. In a particular embodiment, the concentration of catechin in the cell-free medium is about 1 mM to about 20 mM.

[0024] In certain embodiments, the cell-free medium contains magnesium chloride. The magnesium chloride can be present in the range of about 1 mM to about 50 mM. In certain embodiments, the cell-free medium contains magnesium chloride in the range of about 1 mM to about 25 mM. In certain embodiments, the cell-free medium contains magnesium chloride in the range of about 1 mM to about 20 mM.

[0025] In certain embodiments, the cell-free medium contains a lysate of a host microorganism having the one or more enzymes for the cell-free reaction medium. In certain embodiments, the cell-free medium contains a lysate having the one or more enzymes at a concentration of about 1% (v / v) to about 50% (v / v). In certain embodiments, the cell-free medium contains a lysate having the one or more enzymes at a concentration of about 5% (v / v) to about 40% (v / v). In certain embodiments, the cell-free medium contains a lysate having the one or more enzymes at a concentration of about 10% (v / v) to about 30% (v / v).

[0026] In certain embodiments, the cell-free medium contains sucrose. In certain embodiments, sucrose is present at a concentration of about 10 mM to about 1000 mM. In certain embodiments, sucrose is present at a concentration of about 20 mM to about 800 mM. In certain embodiments, sucrose is present at a concentration of about 50 mM to about 600 mM.

[0027] In certain embodiments, the cell-free medium further contains sodium ascorbate / ascorbic acid, ferric ammonium sulfate, and α-ketoglutaric acid.

[0028] In certain embodiments, the cell-free medium contains sodium ascorbate / ascorbic acid. In certain embodiments, the cell-free medium contains from about 1 mM to about 100 mM. In certain embodiments, the cell-free medium contains from about 2 mM to about 50 mM. In certain embodiments, the cell-free medium contains from about 5 mM to about 30 mM.

[0029] In certain embodiments, the cell-free medium contains ferric ammonium sulfate. In certain embodiments, the cell-free medium contains ferric ammonium sulfate at a concentration of from about 0.1 mM to about 10 mM. In certain embodiments, the cell-free medium contains ferric ammonium sulfate at a concentration of from about 0.5 mM to about 5 mM. In certain embodiments, the cell-free medium contains ferric ammonium sulfate at a concentration of from about 1 mM to about 3 mM.

[0030] In certain embodiments, the cell-free medium contains α-ketoglutaric acid. In certain embodiments, the cell-free medium contains α-ketoglutaric acid at a concentration of from about 1 mM to about 100 mM. In certain embodiments, the cell-free medium contains α-ketoglutaric acid at a concentration of from about 2.5 mM to about 50 mM. In certain embodiments, the cell-free medium contains α-ketoglutaric acid at a concentration of from about 10 mM to about 30 mM.

[0031] In certain embodiments, the cell-free reaction for anthocyanin production is carried out for an essential duration until the desired amount of the anthocyanin is produced in the reaction. In certain embodiments, the reaction for cell-free production of anthocyanin is carried out for a duration of from about 0.1 hour to about 20 hours. In certain embodiments, the reaction for cell-free production of anthocyanin is carried out for a duration of from about 0.5 hour to about 20 hours. In certain embodiments, the reaction for cell-free production of anthocyanin is carried out for a duration of from about 1 hour to about 15 hours.

[0032] In certain embodiments, the reaction in the cell-free medium is adjusted for an optimal yield of anthocyanins to be produced. In certain embodiments, the temperature of the cell-free medium is approximately 20°C to approximately 40°C. In certain embodiments, the reaction is carried out in a bubble column reactor, where the enzymes (1 or more) are in solution. In certain embodiments, the reaction is carried out in a packed bed reactor, where the enzymes (1 or more) are immobilized.

[0033] In certain embodiments, the cell-free medium comprises an anthocyanidin synthase (ANS) enzyme. In certain embodiments, the anthocyanidin synthase (ANS) used in the method of the present invention comprises the following: [Table 5] An enzyme having an amino acid sequence that is at least 95% identical to one of the more selected enzymes.

[0034] In certain embodiments, the anthocyanidin synthase (ANS) used in the method of the present invention may include further modifications designed to optimize the yield of the product produced by the method of the present invention.

[0035] In certain embodiments, the cell-free medium comprises anthocyanidin 3-O-glucosyltransferase (3GT) enzyme. In certain embodiments, the anthocyanidin 3-O-glucosyltransferase (3GT) used in the method of the present invention is comprised of the following: [Table 6-1] [Table 6-2] An enzyme having an amino acid sequence that is at least 95% identical to one of the more selected enzymes.

[0036] In certain embodiments, the anthocyanidin 3-O-glucosyltransferase (3GT) enzyme used in the method of the present invention may include further modifications designed to optimize the yield of the product produced by the method of the present invention.

[0037] In a particular embodiment, the cell-free medium comprises the enzyme sucrose synthase (SuSy). In a particular embodiment, the sucrose synthase (SuSy) comprises the group consisting of: [Table 7] An enzyme having an amino acid sequence that is at least 95% identical to one of the more selected enzymes.

[0038] In certain embodiments, the sucrose synthase (SuSy) enzyme used in the method of the present invention may include further modifications designed to optimize the yield of the product and / or intermediate produced by the method of the present invention.

[0039] In a particular embodiment, the one or more enzymes involved in the production of UDP-glucose and / or the recycling of one or more reaction intermediates are provided below. In a particular embodiment, the enzymes used for the production of UDP-glucose (by reaction with UTP) and / or the recycling of one or more reaction intermediates are the enzymes provided in the table below: [Table 8] The enzymes are selected from those having at least 95% amino acid sequence identity.

[0040] In certain embodiments, the present invention provides an ANS / 3GT / SuSy enzyme engineered for cell-free synthesis of cyanidin-3-glucoside. In certain embodiments, the engineered ANS / 3GT / SuSy enzyme is optimized for cell-free synthesis of cyanidin-3-glucoside. In certain embodiments, the engineered ANS / 3GT / SuSy enzyme may include genetic modifications. In certain embodiments, the genetic modifications may be selected from the group consisting of point mutations, insertions, deletions, and / or any other modifications, such that the enzymes result in efficient and optimal cell-free synthesis of cyanidin-3-glucoside. In certain embodiments, the ANS / 3GT / SuSy enzyme used in cell-free synthesis of cyanidin-3-glucoside is any enzyme disclosed herein.

[0041] In certain embodiments, the present invention provides GLK / PGM / UGP / NDK / PPK enzymes for the recycling of reaction intermediates involved in UDP-glucose production and / or cell-free production of cyanidin-3-glucoside. In certain embodiments, the above-mentioned GLK / PGM / UGP / NDK / PPK enzymes may include genetic modifications. In certain embodiments, the above-mentioned genetic modifications may be selected from the group consisting of point mutations, insertions, deletions, and / or any other modifications such that the enzymes result in efficient and optimal cell-free production of cyanidin-3-glucoside.

[0042] In certain aspects of the present invention, the cell-free production method described above does not require the purification of one or more enzymes derived from the lysed host organism. In certain embodiments, the method of the present invention does not require the purification of one or more enzymes from the lysed biomass containing host cells expressing one or more of the above enzymes for cell-free production of carminic acid. In certain embodiments, the method of the present invention does not require the purification of CGT / SuSy / GLK / PGM / UGP / NDK / PPK from the lysed cells for cell-free production of cyanidin-3-glucoside. This is advantageous because it increases the efficiency of the method and reduces the costs associated with the purification of these enzymes.

[0043] In certain embodiments, the cell-free medium may further comprise any other additional components required for the cell-free production of cyanidin-3-glucoside. For example, in certain embodiments, the cell-free medium comprises a buffer, catechin, cyanidin, activated sugar, magnesium chloride, cell lysate, sucrose, and / or water. In certain embodiments, the cell-free reaction mixture further comprises uridine diphosphate (UDP). In certain embodiments, the cell-free reaction mixture further comprises a polyphosphate and / or glucose. In certain embodiments, the cell-free reaction mixture comprises UDP-glucose and / or UDP in catalytic amounts. In certain embodiments, UDP-glucose is produced by an enzyme in the reaction. In certain embodiments, the reaction mixture comprises glucose-1-phosphate. Glucose-1-phosphate can be produced in the cell-free reaction. Therefore, in certain embodiments, glucose-1-phosphate is also present in catalytic amounts. In certain embodiments, the reaction medium comprises glucose and polyphosphate.

[0044] In certain preferred embodiments, the process of the present invention utilizes catechin, glucose, and polyphosphate as starting materials for the production of cyanidin-3-glucoside. In certain embodiments, the process of the present invention utilizes cyanidin, glucose, and polyphosphate as starting materials for the production of cyanidin-3-glucoside. In these embodiments, the present invention provides that other intermediates in the process for the production of cyanidin-3-glucoside can be recycled.

[0045] Conventional methods for the production of anthocyanins require either the extraction of the product from plants (which is expensive and unpredictable) or certain cell-based approaches (e.g., as described in U.S. Patent No. 8,962,327, which is incorporated by reference). However, these cell-based approaches have complexities such as processes that result in low yields, numerous byproducts, and expensive purification and isolation protocols. The method of the present invention is beneficial in that it provides a cell-free approach for the bioproduction of anthocyanins. In particular, the product titer of the anthocyanins produced by the method of the present invention is higher than the product titer of the anthocyanins produced by any other known method for the production of anthocyanins. In certain embodiments, the product titer of the product produced by the method of the present invention is at least twice as high as that of other known methods for the production of anthocyanins. In certain embodiments, the product titer of the product produced by the method of the present invention is at least five times higher than that of other known methods for the production of anthocyanins. In certain embodiments, the product titer of the product produced by the method of the present invention is at least ten times higher than that of other known methods for the production of anthocyanins. In a particular embodiment, the titer of the product produced by the method of the present invention is at least 100 times higher than that of other known methods for the production of anthocyanins. In a particular embodiment, the titer of the product produced by the method of the present invention is at least 500 times higher than that of other known methods for the production of anthocyanins. In a particular embodiment, the titer of the product produced by the method of the present invention is at least 1000 times higher than that of other known methods for the production of anthocyanins. In a particular embodiment, the titer of the product produced by the method of the present invention is at least 5000 times higher than that of other known methods for the production of anthocyanins.

[0046] In another beneficial aspect of the method of the present invention, the method of the present invention provides an economically efficient method for the production of anthocyanins. Since the method of the present invention provides cell-free production of anthocyanins, it results in a reduction in the cost of purifying the reaction products. Furthermore, in certain aspects of the present invention, the above 1 or more enzymes are contained in the same reaction mixture. That is, the enzymes do not need to be isolated after being expressed in the host organism. The above 1 or more enzymes can be expressed simultaneously in the host organism without the need for any additional purification and / or separation steps and can be utilized for the cell-free production.

[0047] In certain other beneficial aspects of the present invention, the use of one or more of the above enzymes for cell-free production of the anthocyanins results in a high yield of the product to be produced. One or more of the above enzymes may be further modified to optimize the yield of the product while it is being produced in the cell-free reaction. [Brief explanation of the drawing]

[0048] Brief explanation of the drawing [Figure 1] Figure 1 shows schematic pathways for the conversion of catechins to cyanidins and cyanidin-3-glucosides mediated by ANS and 3GT, respectively.

[0049] [Figure 2] Figure 2 provides a schematic pathway for the conversion of catechin to cyanidin-3-glucoside mediated by ANS and 3GT, where UDP-glucose is provided by the SuSy-mediated conversion of sucrose to UDP-glucose.

[0050] [Figure 3]Figure 3 provides a schematic pathway for the conversion of cyanidin to cyanidin-3-glucoside mediated by 3GT and drugs, where UDP-glucose is produced during the reaction by the recycling and / or generation of UDP-glucose.

[0051] [Figure 4-1] Figure 4 provides HPLC plots showing the conversion of cyanidin to cyanidin-3-glucoside. The top two panels provide HPLC plots for cyanidin-3-glucoside and cyanidin, respectively, while the bottom two panels provide HPLC plots monitoring the reaction in systems with and without SuSy-mediated conversion from sucrose to UDP-glucose, respectively. [Figure 4-2] Same as above. [Modes for carrying out the invention]

[0052] VI. Detailed explanation This application provides compositions and methods for producing anthocyanins in a cell-free medium, wherein one or more enzymes are present in the cell-free medium, and the one or more enzymes result in the conversion of organic matter to anthocyanins. The one or more enzymes may be manipulated. The manipulated enzymes may not exist in nature.

[0053] The term “not naturally occurring,” when used in reference to enzymes, is intended to mean that a nucleic acid or polypeptide contains at least one genetic modification not normally found in naturally occurring polypeptides or nucleic acid sequences. Naturally occurring nucleic acids and polypeptides may be referred to as “wild-type” or “original.” A host cell, organism, or microorganism containing at least one genetic modification produced by human intervention may also be referred to as “not naturally occurring,” “engineered,” “genetically engineered,” or “recombinant.”

[0054] As used herein, the singular forms “a” (one, a), “an” (one, a), and “the” (the above, this, that) are intended to also include the plural form unless the context otherwise clearly indicates. Furthermore, the terms “include, encompass, cite,” “include, encompass, cite,” “have,” “possess,” “accompany,” or variations thereof, to the extent that they are used in either the detailed description and / or the claims, are intended to be inclusive in a manner similar to that of the terms “include, encompass.”

[0055] As used herein, “reaction solution” may refer to all components necessary for an enzyme-based chemical transformation. These typically include, but are not limited to, buffers, salts, cofactors, and substrates (starting materials).

[0056] As used herein, “reaction mixture” may refer to the “reaction solution” plus all components from the enzyme and / or the product from the reaction. In some embodiments, the “reaction mixture” may refer only to the reaction solution without any enzyme or reaction product. In some embodiments, the “reaction solution” and the “reaction mixture” may be used interchangeably.

[0057] As used herein, “buffering agent” may refer to a chemical substance added to a water-based solution that is resistant to changes in pH through the action of acid-base conjugated components.

[0058] As used herein, “cofactor” may refer to a non-protein compound that can bind to a protein and assists in a biological or chemical reaction. Non-limiting examples of cofactors include, but are not limited to, NADPH and NADH.

[0059] As used herein, the term used with respect to the enzyme, anthocyanin synthase (ANS), may also refer to enzymes known in the art as leucoanthocyanidin dioxygenase. The above enzyme, anthocyanin synthase (ANS), may also be known as leucoanthocyanidin dioxygenase.

[0060] In the cell-free systems described herein, the most important components of the cells, namely cofactors and enzymes, are used in chemical reactions without the cellular components that could directly or indirectly inhibit the desired biochemical reaction. The same enzymes found in plants and other organisms can be produced in vivo (typically through protein overexpression in a host such as bacteria), isolated via chromatography and / or any other method, and then added to a bioreactor along with a substrate (starting material). The enzymes can also be used directly from plants without any isolation. The enzymes transform the substrate in the same way that occurs in the original organism without the complexity of the organism. In addition, the biochemical reaction can be enhanced by the addition of cosolvents, detergents, or both that are not permissible or simply do not function by whole-cell-based production methods. In this way, natural products can be produced without plants, cells, or chemosynthesis.

[0061] Anthocyanins: Anthocyanins are natural plant pigments that have beneficial effects on plants, as well as on humans and animals. Dietary sources of anthocyanins are generally easy to identify because they are red, blue, or purple. Examples include berries and red-skinned grapes, apples, as well as pears and various vegetables (e.g., radishes and red / purple cabbage). Anthocyanins can also be ingested through their use as food additives and dietary supplements, and are available as anthocyanin-rich fruit extracts, powders, and refined compounds. Dietary consumption of anthocyanins is high compared to the consumption of other flavonoids due to their wide distribution in plant foods. Numerous studies have shown the potential effects that this flavonoid family may have in reducing the incidence of cardiovascular disease, cancer, hyperlipidemia, and other insulin resistance-related diseases through the consumption of anthocyanin-rich foods. Anthocyanins are also found in abundant concentrations in tea, wine, fruits, vegetables, nuts, olive oil, cocoa, and cereals, and they appear as vivid red to blue.

[0062] Anthocyanins are glycosides composed of an anthocyanidin aglycone + one or more glycosidically linked monosaccharide or oligosaccharide units. The structures of commonly known anthocyanidins are provided below: [ka]

[0063] As is evident from the structures of anthocyanidins provided above, all anthocyanidins have hydroxyl groups at positions 3, 5, and 7, but each structure may have its own characteristic hydroxyl or methoxyl group on the so-called B ring: (I) cyanidin, (II) delphinidin, (III) malvidin, (IV) peonidin, (V) pelargonidin, and (VI) petunidine.

[0064] The glycosylation of anthocyanidins that form each anthocyanin can occur at different hydroxyl groups of molecules that have 3-OH as the most abundant glycosylation site in nature to produce 3-O-β-glucosides. The reactions provided below demonstrate the conversion from cyanidin (anthocyanidin) to cyanidin-3-glucoside (anthocyanidin). [ka]

[0065] Anthocyanins and anthocyanidins, like other polyphenols and flavonoids, have the ability to act as free radical scavengers against harmful oxidants such as reactive oxygen species and reactive nitrogen species (ROS and RNS). The central role of antioxidant activity is the oxidation of the phenolic hydroxyl group of anthocyanins; in particular, the para and ortho phenolic groups are important for semiquinone formation and stabilization of the one-electron oxidation product.

[0066] Preparation of anthocyanins: Regarding the extraction of anthocyanins and anthocyanidins from biological matrices, various strategies exist based on the selective search for specific molecules with complexity and particular chemical characteristics. For example, the extraction of total anthocyanidins is generally performed by using an aqueous / organic mixture in which the contribution of the organic part is prominent. Alternatively, the anthocyanins are often extracted in a more hydrophilic solvent or a more prominent aqueous-based mixture. In all these cases, it is very useful to maintain the ionization state of the compounds in their flavilium form, and this objective can be achieved by adding an inorganic or organic acid to the aqueous phase. Plant, food, and agricultural samples are usually extracted with an ethanol / methanol:water mixture (70-95:30-5) acidified with HCl, formic acid or other organic acids (e.g., citric acid).

[0067] Following the above extraction, the first step in the purification procedure, depending on the matrix (fruit, leaf, or liquid sample), is to distinguish between extracts rich in green and red pigments. The first case indicates a strong chlorophyll component that must be pre-purified, while the second case indicates a matrix (fruit, flower, or liquid biological sample) richer in anthocyanins. After the removal of chlorophyll, the above purification is generally carried out by different chromatographic steps requiring differential steady-state phases based on the purpose.

[0068] The anthocyanin biosynthetic pathway is well-characterized and appears to be strongly conserved in both the Arabidopsis thaliana model plant and various crops. The anthocyanin biosynthetic pathway constitutes an important branch of the phenylpropanoid pathway, sharing several biosynthetic enzymes with those for other flavonoids (e.g., flavones and flavanones) in its initial steps. It begins with phenylalanine, which is converted to cinnamic acid by phenylalanine ammonia lyase (PAL). The cinnamic acid is then converted to coumaric acid by cinnamate-4-hydroxylase (C4H), and subsequently to 4-coumaroyl-CoA by 4-coumaroyl-CoA ligase (4Cl). Following the condensation of 4-coumaroyl-CoA and malonyl-CoA, naringenin chalcone is produced by chalcone synthase (CHS), subsequently converted to naringenin by chalcone isomerase (CHI), and then converted to dihydroflavonols, such as dihydrokaempferol and dihydroquercetin, by flavanone 3-hydroxylase (F3H) and flavonoid 3'-hydroxylase (F3'H), respectively. The final step in the above biosynthetic pathway results in the production of leucocyanidins, cyanidins, and anthocyanins by dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid-3-O-glycosyltransferase (UFGT), respectively. Anthocyanin biosynthesis occurs in the cytosol, but they are stored in vacuoles by specific transporters. In Arabidopsis, for example, the TT12 and AHA10 genes have been associated with the transport and vacuolar accumulation of anthocyanins in seeds. In particular, TT12 encodes a membrane protein belonging to the "multidrug and toxic elution antiporter" family and has been shown to be involved at the vacuolar level in the accumulation of glycosylated flavan-3-ols and protoanthocyanidins. AHA10 encodes a plasma membrane H+-ATPase, which, in turn, is responsible for vacuolar acidification. A detailed description of the above biosynthetic pathways is provided in Mattioli et al., Anthocyanins: A Comprehensive Review of Their Chemical Properties and Health Effects on Cardiovascular and Neurodegenerative Diseases, Molecules. 2020 Sep; 25(17): 3809 (which is incorporated herein by reference in its entirety).

[0069] While biotechnological approaches to increase anthocyanin levels in food are being explored, none of the previous approaches have offered cell-free synthesis of anthocyanins. Anthocyanins are currently produced by chopping or crushing fruits or vegetables and subsequently injecting them with water acidified with a common food-grade acid. This extract is then concentrated by non-chemical separation techniques. Pigment extracts derived from plant sources generally contain a mixture of different anthocyanin molecules, which vary depending on their levels of hydroxylation, methylation, and acylation. This increases production costs due to the purification required from these mixtures. Furthermore, these factors can fluctuate year to year in the raw material plants and are influenced by weather and environmental factors. Thus, another challenge for the commercial production of plant-derived anthocyanin pigments is that harvesting is limited to once a year. This means that large quantities of extracts must be prepared and stored for extended periods to meet the needs of the food industry throughout the year. Due to the instability of anthocyanins, special storage conditions often have to be available.

[0070] Therefore, a new method for the production of anthocyanins is needed. The present invention provides a method for the cell-free production of anthocyanins. The method of the present invention comprises the step of providing one or more enzymes into a cell-free medium, wherein the one or more enzymes comprise the step of bringing about the conversion of a substrate to anthocyanin. In a particular aspect of the present invention, the method of the present invention may provide the production of one or more intermediates during the conversion of the substrate to anthocyanin. In a particular aspect of the present invention, the substrate may be anthocyanidin or its precursor. In a particular embodiment, the anthocyanidin is selected from the group consisting of delphinidin, malvidin, peonidin, pelargonidin, and petunidine. In a particular embodiment, the anthocyanin may be glycosylated delphinidin, malvidin, peonidin, pelargonidin, and petunidine.

[0071] In a particular aspect of the present invention, the anthocyanin is cyanidin-3-glucoside. In a particular embodiment, the substrate is catechin, and in a particular embodiment, the catechin is (+)-catechin. In a particular embodiment, the above 1 or more enzymes are selected from the group consisting of anthocyanidin synthase enzymes (ANS) and anthocyanidin 3-O-glucosyltransferases (3GT).

[0072] In certain embodiments of the present 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. A schematic diagram of the reaction pathway for the conversion of catechin to cyanidin-3-glucoside is provided in Figure 1. In certain preferred embodiments, the cyanidin produced by the conversion of catechin by the anthocyanidin synthase (ANS) enzyme is further converted to cyanidin-3-glucoside by the anthocyanidin 3-O-glucosyltransferase (3GT) enzyme.

[0073] In certain embodiments, the cell-free production of the anthocyanins requires 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 one or more enzymes. In certain embodiments, the UDP-glucose is synthesized from sucrose. In certain embodiments, the sucrose synthase (SuSy) enzyme converts sucrose to UDP-glucose. Figure 2 provides a schematic reaction scheme for the production of UDP-glucose from sucrose mediated by SuSy, where the UDP-glucose is involved in the production of cyanidin-3-glucoside from catechin.

[0074] In a particular beneficial aspect, the present invention provides a method in which the UDP-glucose used in the above reaction is generated from other substrates during the course of the reaction. In a particular embodiment, the UDP portion of the UDP-glucose is recycled in the cell-free medium. The recycling of UDP provides economic efficiency for the process of the present invention. Thus, in a particular aspect, the production of cyanidin-3-glucoside from cyanidin catalyzed by 3GT is carried out in conjunction with other methods for UDP-glucose production or UDP recycling. In a particular embodiment, an outline of a synthetic scheme requiring the above UDP-glucose production and / or recycling is provided in Figure 3. In a particular embodiment, UDP-glucose is produced by the reaction of uridine triphosphate (UTP) with glucose-1-phosphate. In a particular preferred embodiment, the reaction of UTP and glucose-1-phosphate is catalyzed by UTP-glucose-1-phosphate uridilyltransferase (UGP). In a particular preferred embodiment, the UGP may be galU.

[0075] In certain contexts, glucose-1-phosphate is a source for the formation of UDP-glucose. Therefore, in certain embodiments, glucose-1-phosphate can be added to a reaction for the production of cyanidin-3-glucoside. In certain embodiments, the glucose-1-phosphate can be supplied to a reaction for the preparation of cyanidin-3-glucoside.

[0076] In certain embodiments, glucose-1-phosphate is produced during the course of the above reaction. In certain embodiments, glucose-1-phosphate is produced by an enzyme during the course of the above reaction. Thus, in certain embodiments, glucose is converted to glucose-6-phosphate by the reaction of ATP (adenosine triphosphate) and glucose. In certain embodiments, the reaction between glucose and ATP results in the production of glucose-6-phosphate and adenosine diphosphate (ADP). In certain embodiments, the conversion from glucose to glucose-6-phosphate is catalyzed by glucokinase (GLK). In certain embodiments, the conversion from glucose to glucose-6-phosphate is catalyzed by hexokinase (HK). In certain embodiments, glucose-6-phosphate is converted to glucose-1-phosphate. In certain embodiments, the conversion from glucose-6-phosphate to glucose-1-phosphate is catalyzed by phosphoglucumutase (PGM).

[0077] In certain aspects, the present invention further provides that the nucleoside triphosphate species used in the above reaction are recycled. In certain embodiments, the recycled nucleoside triphosphates are ATP and UTP. In certain embodiments, UTP is produced by the reaction of UDP and ATP. In certain embodiments, the production of UTP from UDP and ATP is catalyzed by nucleoside diphosphate kinase (NDK). In certain embodiments, ATP may be produced from ADP and a phosphate or polyphosphate. In certain embodiments, the conversion from ADP to ATP is catalyzed by polyphosphate kinase (PPK).

[0078] In certain beneficial aspects, the present invention provides that the process of the present invention is cost-effective due to the recycling of chemical intermediates involved in the above reaction. In certain embodiments, the present invention provides that chemical intermediates such as UDP-glucose, UDP, and glucose-1-phosphate are recycled. In fact, in certain embodiments, the only reagents required in non-catalytic amounts for the production of cyanidin-3-glucoside by the conversion of catechin are polyphosphate / phosphate and glucose. The process of the present invention is cost-effective because the process provided in the present invention is carried out in a cell-free medium and its components (required in non-catalytic amounts) are economical.

[0079] In a particular embodiment, one or more of the enzymes used in the conversion of a substrate to anthocyanin are expressed in a host organism. In a particular embodiment, the cell lysate in the cell-free medium is the cell lysate of the host organism expressing one or more of the enzymes for the conversion of a substrate to anthocyanin. In a particular embodiment, the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells. In a particular embodiment, one or more of the enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism. In a particular embodiment, the host organism expressing one or more of the enzymes is cultured until a predetermined biomass is achieved that produces the essential amount of one or more of the enzymes for the conversion of a substrate to anthocyanin. In a particular embodiment, the method comprises the step of lysing cells and subsequently removing cell debris to produce cell lysates for use in the cell-free medium for cell-free production of anthocyanins.

[0080] In certain embodiments, anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and / or sucrose synthase (SuSy) are present in the cell-free medium for cell-free production of anthocyanins. In certain embodiments, the method does not involve the step of separating and / or purifying anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and sucrose synthase (SuSy) for cell-free production of anthocyanins. Similarly, in certain embodiments, the present invention also provides that enzymes involved in the UDP-glucose recycling pathway are not manipulated. Having a system that does not require enzyme separation is beneficial because it eliminates extra steps and results in reduced production costs. In a particular embodiment, the above method includes the step of isolating and / or purifying at least one of the following: anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and sucrose synthase (SuSy) for cell-free production of anthocyanins.

[0081] In certain embodiments of the present invention, the reaction mixture comprises a purified enzyme for cell-free production of cyanidin-3-glucoside. In certain embodiments, the purified enzyme is anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and / or sucrose synthase (SuSy).

[0082] In a particular embodiment, the one or more enzymes expressed in the host cell are anthocyanidin synthase (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucocomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). In certain embodiments, C-glucosyltransferase (CGT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are present in the cell-free medium for cell-free production of cyanidin-3-glucoside.

[0083] In certain embodiments of the present invention, the reaction mixture comprises a purified enzyme for cell-free production of cyanidin-3-glucoside. In certain embodiments, the purified enzyme is anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). In certain embodiments, C-glucosyltransferase (CGT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are not purified. In certain embodiments, the purified enzymes are anthocyanidin synthase (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), and / or sucrose synthase (SuSy).

[0084] In certain embodiments, the cell-free medium further comprises a buffer, catechin, activated sugar, magnesium chloride, a cell lysate having an enzyme, sucrose, and / or water. In certain embodiments, the cell-free medium further comprises sodium ascorbate, ascorbic acid, ammonium iron sulfate, and α-ketoglutaric acid / 2-oxoglutarate.

[0085] In certain embodiments, the buffer in the cell-free medium maintains the pH of the reaction mixture within an optimal range. In certain embodiments, the pH of the reaction medium is about 5.5 to about 10. In certain embodiments, the pH of the cell-free medium is about 6 to about 9. In certain embodiments, the pH of the cell-free medium is about 6 to about 8.

[0086] In certain embodiments, the buffer is selected from the group consisting of Tris, HEPES, phosphate, carbonate / bicarbonate, acetate, sodium hydroxide-glycine, and / or citrate. The concentration of the buffer for the reaction mixture depends on the concentrations of the starting materials and other components in the reaction mixture. In certain embodiments, the buffer is a phosphate buffer. In certain embodiments, the concentration of the buffer in the cell-free medium is about 1 mM to about 1000 mM. In certain embodiments, the concentration of the buffer is about 2.5 mM to about 750 mM. In certain embodiments, the concentration of the buffer is about 5 mM to about 100 mM. In certain embodiments, the concentration of the buffer is about 20 mM to about 100 mM. In certain embodiments, the buffer is a phosphate buffer.

[0087] In a particular embodiment, the activated sugar in the cell-free medium is UDP-glucose. In a particular embodiment, the UDP-glucose is added to the cell-free medium. In a particular embodiment, the UDP-glucose in the cell-free medium is synthesized from sucrose. In a particular embodiment, the cell-free medium contains UDP. In a particular embodiment, the concentration of the activated sugar in the cell-free medium is about 0.01 mM to about 10 mM. In a particular embodiment, the concentration of the activated sugar in the cell-free medium is about 0.05 mM to about 5 mM. In a particular embodiment, the concentration of the activated sugar in the cell-free medium is about 0.1 mM to about 2.5 mM.

[0088] In a particular embodiment, the substrate is catechin. In a particular embodiment, the concentration of catechin in the cell-free medium is about 0.1 mM to about 50 mM. In a particular embodiment, the concentration of catechin in the cell-free medium is about 0.5 mM to about 40 mM. In a particular embodiment, the concentration of catechin in the cell-free medium is about 1 mM to about 20 mM.

[0089] In certain embodiments, the cell-free medium contains magnesium chloride. The magnesium chloride may be present in a concentration of about 1 mM to about 50 mM. In certain embodiments, the cell-free medium contains magnesium chloride in a concentration of about 1 mM to about 25 mM. In certain embodiments, the cell-free medium contains magnesium chloride in a concentration of about 1 mM to about 20 mM.

[0090] In a particular embodiment, the cell-free medium comprises a lysate of a host microorganism having 1 or more of the enzymes described above for the cell-free reaction medium. In a particular embodiment, the cell-free medium comprises a lysate having 1 or more of the enzymes described above at a concentration of about 1% (v / v) to about 50% (v / v). In a particular embodiment, the cell-free medium comprises a lysate having 1 or more of the enzymes described above at a concentration of about 5% (v / v) to about 40% (v / v). In a particular embodiment, the cell-free medium comprises a lysate having 1 or more of the enzymes described above at a concentration of about 10% (v / v) to about 30% (v / v).

[0091] In certain embodiments, the cell-free medium contains sucrose. In certain embodiments, the sucrose is present at a concentration of about 10 mM to about 1000 mM. In certain embodiments, the sucrose is present at a concentration of about 20 mM to about 800 mM. In certain embodiments, the sucrose is present at a concentration of about 50 mM to about 600 mM.

[0092] In certain embodiments, the cell-free reaction medium further comprises glucose. In certain embodiments, glucose is a starting material providing UDP-glucose for the conversion of catechin / cyanidin to cyanidin-3-glucoside. Thus, the concentration of glucose in the reaction mixture is determined by the amount of UDP-glucose required for the reaction for cell-free production of cyanidin-3-glucoside. In certain embodiments, glucose is present in the cell-free reaction medium at a concentration of about 10 mM to about 1000 mM. In certain embodiments, glucose is present in the cell-free reaction medium at a concentration of about 20 mM to about 800 mM. In certain embodiments, glucose is present in the cell-free reaction medium at a concentration of about 50 mM to about 600 mM.

[0093] In certain embodiments, the cell-free reaction medium further comprises a polyphosphate. In certain embodiments, the polyphosphate is also a starting material that provides UDP-glucose for the conversion of catechin / cyanidin to cyanidin-3-glucoside. Thus, the concentration of polyphosphate in the reaction mixture is determined by the amount of UDP-glucose required for the reaction for cell-free production of cyanidin-3-glucoside. In certain embodiments, the polyphosphate is present in the cell-free reaction medium at a concentration of about 10 mM to about 1000 mM. In certain embodiments, the polyphosphate is present in the cell-free reaction medium at a concentration of about 20 mM to about 800 mM. In certain embodiments, the polyphosphate is present in the cell-free reaction medium at a concentration of about 50 mM to about 600 mM.

[0094] In a particular embodiment, the cell-free medium further comprises sodium ascorbate / ascorbic acid, ammonium iron sulfate, and α-ketoglutaric acid.

[0095] In certain embodiments, the cell-free medium contains sodium ascorbate / ascorbic acid. In certain embodiments, the cell-free medium contains about 1 mM to about 100 mM. In certain embodiments, the cell-free medium contains about 2 mM to about 50 mM. In certain embodiments, the cell-free medium contains about 5 mM to about 30 mM.

[0096] In certain embodiments, the cell-free medium contains ammonium iron sulfate. In certain embodiments, the cell-free medium contains ammonium iron sulfate at a concentration of about 0.1 mM to about 10 mM. In certain embodiments, the cell-free medium contains ammonium iron sulfate at a concentration of about 0.5 mM to about 5 mM. In certain embodiments, the cell-free medium contains ammonium iron sulfate at a concentration of about 1 mM to about 3 mM.

[0097] In certain embodiments, the cell-free medium contains α-ketoglutarate. In certain embodiments, the cell-free medium contains α-ketoglutarate at a concentration of about 1 mM to about 100 mM. In certain embodiments, the cell-free medium contains α-ketoglutarate at a concentration of about 2.5 mM to about 50 mM. In certain embodiments, the cell-free medium contains α-ketoglutarate at a concentration of about 10 mM to about 30 mM.

[0098] In certain embodiments, the cell-free reaction for anthocyanin production is carried out for a required duration until a desired amount of the anthocyanin is produced in the reaction. In certain embodiments, the cell-free reaction for anthocyanin production is carried out for a duration of about 0.1 hours to about 20 hours. In certain embodiments, the cell-free reaction for anthocyanin production is carried out for a duration of about 0.5 hours to about 20 hours. In certain embodiments, the cell-free reaction for anthocyanin production is carried out for a duration of about 1 hour to about 15 hours.

[0099] In certain embodiments, the temperature of the reaction mixture for cell-free production of anthocyanins is varied to obtain optimal results for anthocyanin production. In certain embodiments, the temperature of the reaction mixture for cell-free production of anthocyanins is about 15°C to about 45°C. In certain embodiments, the temperature of the reaction mixture for cell-free production of anthocyanins is about 20°C to about 40°C. In certain embodiments, the temperature of the reaction mixture for cell-free production of anthocyanins is about 22.5°C to about 37.5°C. The temperature of the reaction mixture for cell-free production of anthocyanins may affect the rate of anthocyanin production. In conclusion, the duration of the reaction can be adjusted according to the temperature of the reaction mixture to obtain an optimal yield of anthocyanins in cell-free production of anthocyanins.

[0100] In certain circumstances, the method provided in the present invention may be carried out in any reactor suitable for cell-free production of anthocyanins. In certain embodiments, the cell-free production reaction of anthocyanins is carried out in a bubble column reactor / bioreactor. In certain embodiments, in the bubble column reactor / bioreactor, the 1 or more enzymes involved in cell-free production of anthocyanins are in solution. In certain embodiments, the reaction for cell-free production of anthocyanins is carried out in a bubble column reactor / bioreactor containing a lysate from the host organism. In certain embodiments, it is advantageous to use the bubble column reactor / bioreactor for cell-free production of anthocyanins when the reaction mixture involves a lysate from the host cell organism (or a lysate with cell debris removed from the host cell organism) utilizing the 1 or more enzymes responsible for cell-free production of anthocyanins. In certain embodiments, the reaction for cell-free production of anthocyanins is carried out in a packed bed reactor / bioreactor. In certain embodiments, the above 1 or more enzymes are immobilized in the packed bed reactor / bioreactor. The packed bed reactor / bioreactor is preferred for the purified enzymes that play a role in cell-free production of anthocyanins. In certain embodiments, the above 1 or more enzymes may be immobilized in a single reactor / bioreactor. In certain other embodiments, the above 1 or more enzymes may be immobilized in different reactors / bioreactors, where these reactors / bioreactors are linked in sequence. In certain embodiments, the bioreactor system is provided in PCT / US2021 / 064049 (which is incorporated by reference in its entirety).

[0101] The methods provided in the present invention are advantageous over other conventional methods of anthocyanin production. In certain embodiments, the methods of the present invention provide cell-free production of anthocyanins. Since the methods of the present invention are carried out in a cell-free medium, they offer significant economic efficiency by reducing the cost of anthocyanin production in other conventional methods. In certain embodiments, the methods for anthocyanin production are cost-effective because they are carried out from the lysates of the host organism expressing the above-mentioned enzyme 1 or more involved in the above reaction. In particular, in certain embodiments, the methods of the present invention do not require the purification of the above-mentioned enzyme 1 or more. Since the purification of individual enzymes is not required in the methods of the present invention, they offer further economic efficiency by reducing the costs required in purifying individual enzymes in other respects.

[0102] Advantageously, the cell-free production of anthocyanins provided herein offers significantly higher potency of anthocyanins compared to conventional methods. A higher potency of anthocyanins provides an additional cost advantage for anthocyanin production because a higher potency of anthocyanins provides greater efficiency in purifying and / or concentrating the anthocyanins from the reaction mixture. In certain embodiments, the method of the present invention provides an anthocyanin potency at least 5 times higher than conventional methods. In certain embodiments, the method of the present invention provides an anthocyanin potency at least 10 times higher than conventional methods. In certain embodiments, the method of the present invention provides an anthocyanin potency at least 50 times higher than conventional methods. In certain embodiments, the method of the present invention provides an anthocyanin potency at least 100 times higher than conventional methods. In certain embodiments, the method of the present invention provides an anthocyanin potency at least 500 times higher than conventional methods. In certain embodiments, the method of the present invention provides an anthocyanin potency at least 1000 times higher than conventional methods. In a particular embodiment, the method of the present invention provides an anthocyanin potency at least 5,000 times higher than that of conventional methods.

[0103] In some embodiments, the isolated anthocyanins have a purity of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

[0104] In other embodiments, the isolated anthocyanins have a purity of about 10% to 95%, or about 10% to 90%, or about 10% to 80%, or about 10% to 70%, or about 10% to 60%, or about 10% to 50%, or about 10% to 40%, or about 20% to 95%, or about 20% to 90%, or about 20% to 80%, or about 20% to 70%, or about 20% to 60%, or about 20% to 50%, or about 20% to 40%, or about 50% to 95%, or about 50% to 90%, or about 50% to 80%, or about 50% to 70%, or about 50% to 60%.

[0105] In certain embodiments, the cell-free medium comprises an anthocyanidin synthase (ANS) enzyme. In certain embodiments, the anthocyanidin synthase (ANS) used in the method of the present invention comprises the following: [Table 9] An enzyme having an amino acid sequence that is at least 95% identical to one of the more selected enzymes.

[0106] In certain embodiments, the anthocyanidin synthase (ANS) used in the method of the present invention may include further modifications designed to optimize the yield of the product produced by the method of the present invention.

[0107] In certain embodiments, the cell-free medium comprises anthocyanidin 3-O-glucosyltransferase (3GT) enzyme. In certain embodiments, the anthocyanidin 3-O-glucosyltransferase (3GT) used in the method of the present invention is comprised of the following: [Table 10] An enzyme having an amino acid sequence that is at least 95% identical to one of the more selected enzymes.

[0108] In certain embodiments, the anthocyanidin 3-O-glucosyltransferase (3GT) enzyme used in the method of the present invention may include further modifications designed to optimize the yield of the product produced by the method of the present invention.

[0109] In a particular embodiment, the cell-free medium comprises the enzyme sucrose synthase (SuSy). In a particular embodiment, the sucrose synthase (SuSy) comprises the group consisting of: [Table 11-1] [Table 11-2] An enzyme having an amino acid sequence that is at least 95% identical to one of the more selected enzymes.

[0110] In certain embodiments, the sucrose synthase (SuSy) enzyme used in the method of the present invention may include further modifications designed to optimize the yield of the product and / or intermediate produced by the method of the present invention.

[0111] In a particular embodiment, the one or more enzymes involved in the production of UDP-glucose and / or the recycling of one or more reaction intermediates are provided below. In a particular embodiment, the enzymes used for the production of UDP-glucose (by reaction with UTP) and / or the recycling of one or more reaction intermediates are the enzymes provided in the table below: [Table 12-1] [Table 12-2] The enzymes are selected from those having at least 95% amino acid sequence identity.

[0112] In certain embodiments, the present invention provides an ANS / 3GT / SuSy enzyme engineered for cell-free synthesis of cyanidin-3-glucoside. In certain embodiments, the engineered ANS / 3GT / SuSy enzyme is optimized for cell-free synthesis of cyanidin-3-glucoside. In certain embodiments, the engineered ANS / 3GT / SuSy enzyme may include genetic modifications. In certain embodiments, the genetic modifications may be selected from the group consisting of point mutations, insertions, deletions, and / or any other modifications, such that the enzymes result in efficient and optimal cell-free synthesis of cyanidin-3-glucoside. In certain embodiments, the ANS / 3GT / SuSy enzyme used in cell-free synthesis of cyanidin-3-glucoside is any enzyme disclosed herein.

[0113] In certain embodiments, the present invention provides GLK / PGM / UGP / NDK / PPK enzymes for the recycling of reaction intermediates involved in UDP-glucose production and / or cell-free production of cyanidin-3-glucoside. In certain embodiments, the above-mentioned GLK / PGM / UGP / NDK / PPK enzymes may include genetic modifications. In certain embodiments, the above-mentioned genetic modifications may be selected from the group consisting of point mutations, insertions, deletions, and / or any other modifications such that the enzymes result in efficient and optimal cell-free production of cyanidin-3-glucoside.

[0114] In certain aspects of the present invention, the cell-free production method described above does not require the purification of one or more enzymes derived from the lysed host organism. In certain embodiments, the method of the present invention does not require the purification of one or more enzymes from the lysed biomass containing host cells expressing one or more of the above enzymes for cell-free production of carminic acid. In certain embodiments, the method of the present invention does not require the purification of CGT / SuSy / GLK / PGM / UGP / NDK / PPK from the lysed cells for cell-free production of cyanidin-3-glucoside. This is advantageous because it increases the efficiency of the method and reduces the costs associated with the purification of these enzymes.

[0115] In certain embodiments, the cell-free medium may further comprise any other additional components required for the cell-free production of cyanidin-3-glucoside. For example, in certain embodiments, the cell-free medium comprises a buffer, catechin, cyanidin, activated sugar, magnesium chloride, cell lysate, sucrose, and / or water. In certain embodiments, the cell-free reaction mixture further comprises uridine diphosphate (UDP). In certain embodiments, the cell-free reaction mixture further comprises a polyphosphate and / or glucose. In certain embodiments, the cell-free reaction mixture comprises UDP-glucose and / or UDP in catalytic amounts. In certain embodiments, UDP-glucose is produced by an enzyme in the reaction. In certain embodiments, the reaction mixture comprises glucose-1-phosphate. Glucose-1-phosphate can be produced in the cell-free reaction. Therefore, in certain embodiments, glucose-1-phosphate is also present in catalytic amounts. In certain embodiments, the reaction medium comprises glucose and polyphosphate.

[0116] In certain preferred embodiments, the process of the present invention utilizes catechin, glucose, and polyphosphate as starting materials for the production of cyanidin-3-glucoside. In certain embodiments, the process of the present invention utilizes cyanidin, glucose, and polyphosphate as starting materials for the production of cyanidin-3-glucoside. In these embodiments, the present invention provides that other intermediates in the process for the production of cyanidin-3-glucoside can be recycled.

[0117] Conventional methods for the production of anthocyanins require either the extraction of the product from plants (which is expensive and unpredictable) or certain cell-based approaches (e.g., as described in U.S. Patent No. 8,962,327, which is incorporated by reference). However, these cell-based approaches have complexities such as processes that result in low yields, numerous byproducts, and expensive purification and isolation protocols. The method of the present invention is beneficial in that it provides a cell-free approach for the bioproduction of anthocyanins. In particular, the product titer of the anthocyanins produced by the method of the present invention is higher than the product titer of the anthocyanins produced by any other known method for the production of anthocyanins. In certain embodiments, the product titer of the product produced by the method of the present invention is at least twice as high as that of other known methods for the production of anthocyanins. In certain embodiments, the product titer of the product produced by the method of the present invention is at least five times higher than that of other known methods for the production of anthocyanins. In certain embodiments, the product titer of the product produced by the method of the present invention is at least ten times higher than that of other known methods for the production of anthocyanins. In a particular embodiment, the titer of the product produced by the method of the present invention is at least 100 times higher than that of other known methods for the production of anthocyanins. In a particular embodiment, the titer of the product produced by the method of the present invention is at least 500 times higher than that of other known methods for the production of anthocyanins. In a particular embodiment, the titer of the product produced by the method of the present invention is at least 1000 times higher than that of other known methods for the production of anthocyanins. In a particular embodiment, the titer of the product produced by the method of the present invention is at least 5000 times higher than that of other known methods for the production of anthocyanins.

[0118] In another beneficial aspect of the method of the present invention, the method of the present invention provides an economically efficient method for the production of anthocyanins. Since the method of the present invention provides cell-free production of anthocyanins, it results in a reduction in the cost of purifying the reaction products. Furthermore, in certain aspects of the present invention, the above 1 or more enzymes are contained in the same reaction mixture. That is, the enzymes do not need to be isolated after being expressed in the host organism. The above 1 or more enzymes can be expressed simultaneously in the host organism without the need for any additional purification and / or separation steps and can be utilized for the cell-free production.

[0119] In certain other beneficial aspects of the present invention, the use of one or more of the above enzymes for cell-free production of the anthocyanins results in a high yield of the product to be produced. One or more of the above enzymes may be further modified to optimize the yield of the product while it is being produced in the cell-free reaction.

[0120] Composition for cell-free production of anthocyanins In certain contexts, the present invention provides compositions for cell-free production of anthocyanins. The compositions of the present invention are used for cell-free production of anthocyanins according to the method described above. In certain embodiments, the compositions include components for cell-free production of cyanidin-3-glucoside.

[0121] In a particular context, the present invention provides a composition for the cell-free production of cyanidin-3-glucoside. The composition of the present invention is used for the cell-free production of cyanidin-3-glucoside according to the method described above.

[0122] In certain embodiments, the composition of the present invention comprises one or more enzymes in a cell-free medium, where the one or more enzymes result in the conversion of a substrate to cyanidin-3-glucoside. In certain embodiments, the substrate converted to cyanidin-3-glucoside is cyanidin. In certain embodiments, the substrate converted to cyanidin-3-glucoside is catechin (and cyanidin as an intermediate). In certain embodiments, the one or more enzymes catalyzing the conversion from catechin to anidine is ANS. In certain embodiments, the one or more enzymes is 3GT. In certain embodiments, the composition is a cell-free medium. In certain embodiments, the cell-free medium comprises an activated sugar. In certain embodiments, the activated sugar is UDP-glucose. In certain embodiments, UDP-glucose is added to the cell-free medium.

[0123] In a particular aspect of the present invention, UDP-glucose in the composition is synthesized in the cell-free medium by one or more enzymes. In a particular embodiment, UDP-glucose is synthesized from sucrose. In a particular embodiment, one or more enzymes is sucrose synthase (SuSy) that synthesizes UDP-glucose from sucrose. In a particular embodiment, the cell-free medium is a cell lysate. In a particular embodiment, the cell lysate is a cell lysate from the cells of a host organism expressing one or more enzymes. In a particular embodiment, the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells. In a particular embodiment, one or more enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism. In a particular embodiment, the host organism expressing one or more enzymes is cultured until a predetermined biomass is achieved that produces an essential amount of one or more enzymes. In certain embodiments, the composition further comprises a cell lysate produced by cell lysis and subsequent removal of cell debris for use in the cell-free medium for cell-free production of cyanidin-3-glycoside. In certain embodiments, the enzymes ANS, 3GT, and SuSy are present in the cell-free medium for cell-free production of cyanidin-3-glycoside. In certain embodiments, the composition of the present invention provides ANS, 3GT, and SuSy in their unpurified and unseparated form.

[0124] In certain embodiments, the composition further comprises glucose-1-phosphate. In certain embodiments, glucose-1-phosphate is a source for the formation of UDP-glucose. Thus, in certain embodiments, glucose-1-phosphate may be added to the reaction for the production of carminic acid. In certain embodiments, glucose-1-phosphate may be supplied to the reaction for the preparation of carminic acid.

[0125] In certain embodiments, glucose-1-phosphate is produced in the composition of the present invention during the course of the reaction. In certain embodiments, glucose-1-phosphate is produced by an enzyme during the course of the reaction. Thus, in certain embodiments, the composition of the present invention contains glucose and / or ATP. In certain embodiments, glucose is converted to glucose-6-phosphate by a reaction between ATP and glucose. In certain embodiments, the reaction between glucose and ATP results in the production of glucose-6-phosphate and adenosine diphosphate (ADP). In certain embodiments, the conversion from glucose to glucose-6-phosphate is catalyzed by glucokinase (GLK). In certain embodiments, the conversion from glucose to glucose-6-phosphate is catalyzed by hexokinase (HK). In certain embodiments, glucose-6-phosphate is converted to glucose-1-phosphate. In certain embodiments, the conversion from glucose-6-phosphate to glucose-1-phosphate is catalyzed by phosphoglucumutase (PGM). Therefore, in certain embodiments, the composition comprises GLK, HK, and / or PGM.

[0126] In certain aspects, the present invention further provides that the nucleoside triphosphate species used in the above reaction are recycled. In certain embodiments, the recycled nucleoside triphosphates are ATP and UTP. In certain embodiments, UTP is produced by the reaction of UDP and ATP. In certain embodiments, the production of UTP from UDP and ATP is catalyzed by nucleoside diphosphate kinase (NDK). In certain embodiments, ATP may be produced from ADP and a phosphate or polyphosphate. In certain embodiments, the conversion of ADP to ATP is catalyzed by polyphosphate kinase (PPK). In certain embodiments, the composition of the present invention further comprises ATP, UTP, and / or UDP. In certain embodiments, the composition of the present invention further comprises PPK.

[0127] In certain embodiments, the composition further comprises anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). In certain embodiments, C-glucosyltransferase (CGT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are present in the cell-free medium for cell-free generation of cyanidin-3-glycoside.

[0128] In certain embodiments, the cell-free medium may further contain any other additional components required for the cell-free production of cyanidin-3-glycoside. For example, in certain embodiments, the cell-free medium comprises a buffer, catechin, cyanidin, activated sugar, magnesium chloride, cell lysate, sucrose, and / or water. In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for the enzymatic conversion of catechin / cyanidin to cyanidin-3-glycoside. In certain embodiments, the buffer maintains a pH of about 5 to about 9 in the reaction mixture. In certain embodiments, the buffer maintains a pH of about 6 to about 8 in the reaction mixture. In certain embodiments, the buffer maintains a pH in the reaction mixture in the range of 6 to 8. In certain embodiments, the buffer is a phosphate buffer. In certain embodiments, the buffer is present at a concentration of about 1 mM to about 200 mM. In certain embodiments, the buffer is present at a concentration of about 5 mM to about 100 mM.

[0129] In a particular embodiment, the cell-free reaction medium contains activated sugar at a concentration of about 0.001 mM to about 50 mM. In a particular embodiment, the cell-free reaction medium contains an activated sugar at a concentration of about 0.01 mM to about 5 mM. In a particular embodiment, the activated sugar is UDP-glucose.

[0130] In a particular embodiment, the cell-free reaction medium contains magnesium chloride at a concentration of about 0.5 mM to about 40 mM. In a particular embodiment, the cell-free reaction medium contains magnesium chloride at a concentration of about 1 mM to about 20 mM.

[0131] In certain embodiments, the cell-free reaction medium further comprises sucrose. In certain embodiments, the sucrose is converted to UDP-glucose. Thus, the concentration of sucrose in the reaction mixture is determined by the amount of UDP-glucose required for the reaction for cell-free production of cyanidin-3-glycoside. In certain embodiments, sucrose is present in the cell-free reaction medium at a concentration of about 10 mM to about 1000 mM. In certain embodiments, sucrose is present in the cell-free reaction medium at a concentration of about 20 mM to about 800 mM. In certain embodiments, sucrose is present in the cell-free reaction medium at a concentration of about 50 mM to about 600 mM.

[0132] In certain embodiments, the cell-free reaction medium further comprises glucose. In certain embodiments, the concentration of glucose in the reaction mixture is determined by the amount of UDP-glucose required for the reaction for cell-free production of cyanidin-3-glycoside. In certain embodiments, glucose is present in the cell-free reaction medium at a concentration of about 1 mM to about 1000 mM. In certain embodiments, glucose is present in the cell-free reaction medium at a concentration of about 10 mM to about 1000 mM. In certain embodiments, glucose is present in the cell-free reaction medium at a concentration of about 50 mM to about 600 mM.

[0133] In certain embodiments, the cell-free reaction medium further comprises a phosphate / polyphosphate. In certain embodiments, the cell-free reaction medium further comprises a polyphosphate. In certain embodiments, the concentration of the polyphosphate in the reaction mixture is determined by the amount of UDP-glucose required for the cell-free production of carminic acid. In certain embodiments, the polyphosphate is present in the cell-free reaction medium at a concentration of about 0.1 g / L to about 500 g / L. In certain embodiments, the polyphosphate is present in the cell-free reaction medium at a concentration of about 1 g / L to about 100 g / L. In certain embodiments, the polyphosphate is present in the cell-free reaction medium at a concentration of about 1 g / L to about 60 g / L. In certain embodiments, the polyphosphate is present in the cell-free reaction medium at a concentration of about 5 g / L to about 40 g / L. In certain embodiments, the polyphosphate is present at a concentration of about 25 g / L.

[0134] In certain embodiments, the cell-free reaction medium further comprises UTP and / or UDP. In certain embodiments, the concentration of UTP and / or UDP in the reaction mixture is determined by the amount of UDP-glucose required for the reaction for cell-free production of carminic acid. In certain embodiments, the concentration of UTP and / or UDP in the cell-free medium is about 0.01 μM to about 100 μM. In certain embodiments, the concentration of UTP and / or UDP in the cell-free medium is about 0.1 μM to about 50 μM. In certain embodiments, the concentration of UTP and / or UDP in the cell-free medium is about 0.1 μM to about 10 μM. In certain embodiments, the concentration of UTP and / or UDP in the cell-free medium is about 1 μM. In certain embodiments, the concentration of UTP and / or UDP in the cell-free medium is about 5 μM.

[0135] In certain embodiments, the cell-free reaction medium further comprises ATP. In certain embodiments, the concentration of ATP in the reaction mixture is determined by the amount of UDP-glucose required for the cell-free production of carminic acid. In certain embodiments, the concentration of ATP in the cell-free medium is about 0.01 μM to about 100 μM. In certain embodiments, the concentration of ATP in the cell-free medium is about 0.1 μM to about 50 μM. In certain embodiments, the concentration of ATP in the cell-free medium is about 0.1 μM to about 10 μM. In certain embodiments, the concentration of ATP in the cell-free medium is about 1 μM. In certain embodiments, the concentration of ATP in the cell-free medium is about 5 μM.

[0136] In certain embodiments, the cell-free reaction medium comprises glucose-6-phosphate and / or glucose-1-phosphate. In certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate are generated and / or recycled in the reaction medium. Thus, in certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate are present in catalytic amounts in the reaction medium. In certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate can be added to the reaction medium. In certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate are present at concentrations ranging from about 0.01 mM to about 500 mM. In certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate are present at concentrations ranging from about 0.01 mM to about 1 mM. In certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate are present at concentrations ranging from about 1 mM to about 500 mM. In certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate are present at concentrations ranging from about 10 mM to about 500 mM. In certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate are present at concentrations ranging from about 10 mM to about 100 mM.

[0137] In a particular embodiment, the amount of enzyme 1 or more above for cell-free production of carminic acid depends on the target amount of carminic acid to be produced and / or the concentrations of other components present in the reaction mixture. In a particular embodiment, enzyme 1 or more above is present at a concentration of about 1% to about 50% (v / v). In a particular embodiment, enzyme 1 or more above is present at a concentration of about 2.5% to about 45% (v / v). In a particular embodiment, enzyme 1 or more above is present at a concentration of about 5% to about 40% (v / v). In a particular embodiment, enzyme 1 or more above is present at a concentration of about 7.5% to about 30% (v / v).

[0138] In a particular aspect of the present invention, the composition of the present invention is in a bubble column reactor, where the above 1 or more enzymes are in solution. In a particular embodiment, the composition of the present invention is in a packed bed reactor, where the above 1 or more enzymes are immobilized.

[0139] In some embodiments, the enzyme can be immobilized. In some embodiments, the immobilized enzyme can be immobilized on a solid support. Non-limiting examples of solid supports include (but are not limited to) epoxy methacrylate, carboxymethyl cellulose, starch, collagen, ion exchange resin, amino C6 methacrylate, or microporous polymethacrylate. In further embodiments, various surface chemistrys can be used to link the immobilized enzyme to a solid surface (including, but not limited to, covalent, adsorption, ionic, affinity, encapsulation, or capture). In other embodiments, the immobilized enzyme can be immobilized in the form of a crosslinked enzyme aggregate. In other embodiments, the enzyme is not immobilized. Both immobilized and unimmobilized enzymes can be used in batch or serial synthesis. For example, an immobilized enzyme on a solid support can be used in a cartridge through which a reaction mixture passes, thereby allowing the immobilized enzyme to catalyze substrate modification to produce the product at a high titer. Alternatively, a continuous method may involve micromixing of the enzyme solution and substrate to produce the above product at a high titer, while continuously withdrawing the product, withdrawing the substrate (e.g., recovering it), or both. In some embodiments, the withdrawn (e.g., recovered) substrate may be recycled to increase process efficiency and overall yield.

[0140] In some embodiments, anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucocomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are immobilized.

[0141] In some embodiments, anthocyanidin synthase enzyme (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucocomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are not immobilized.

[0142] In some embodiments, the enzyme is recycled by ultrafiltration. In some embodiments, an ion exchange resin may be used to capture carminic acid during production. For example, an amine-functionalized solid support may be added to capture carminic acid for continuous purification from the reaction mixture. In a particular embodiment, the bioreactor system described above is provided in PCT / US2021 / 064049 (which is incorporated herein by reference in its entirety).

[0143] Advantageously, the cell-free production of carminic acid provided herein offers a significantly higher potency value with respect to cyanidin-3-glycoside compared to conventional methods. A higher potency value of cyanidin-3-glycoside provides an additional cost advantage for the production of cyanidin-3-glycoside, because a higher potency of cyanidin-3-glycoside provides efficiency in purifying and / or concentrating carminic acid from the reaction mixture. In certain embodiments, the method of the present invention provides a carminic acid potency value at least 5 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency value at least 10 times higher than conventional methods. In certain embodiments, the method of the present invention provides a cyanidin-3-glycoside potency value at least 50 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency value at least 100 times higher than conventional methods. In certain embodiments, the method of the present invention provides a cyanidin-3-glycoside potency value at least 500 times higher than conventional methods. In a particular embodiment, the method of the present invention provides a carminic acid potency at least 1000 times higher than that of conventional methods.

[0144] In some embodiments, the isolated cyanidin-3-glycoside has a purity of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

[0145] In other embodiments, the isolated cyanidin-3-glycoside has a purity of about 10% to 95%, or about 10% to 90%, or about 10% to 80%, or about 10% to 70%, or about 10% to 60%, or about 10% to 50%, or about 10% to 40%, or about 20% to 95%, or about 20% to 90%, or about 20% to 80%, or about 20% to 70%, or about 20% to 60%, or about 20% to 50%, or about 20% to 40%, or about 50% to 95%, or about 50% to 90%, or about 50% to 80%, or about 50% to 70%, or about 50% to 60%. [Examples]

[0146] Examples Example 1: Bioproduction of cyanidin-3-glucoside: The reaction conditions for cell-free production of cyanidin-3-glucoside are provided below: [Table 13]

[0147] Figure 4 shows HPLC graphs relating to the formation of cyanidin and cyanidin-3-glucoside.

[0148] Example 2: Reactions relating to UDP-glucose production and / or intermediate recycling The reaction conditions for the reaction relating to the production of UDP-glucose and / or the recycling of intermediates are provided in the table below. [Table 14-1] [Table 14-2]

[0149] Table 1 provides exemplary sequences of anthocyanin synthase (ANS) enzymes according to the method of the present invention. Table 1: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0150] Table 2 provides exemplary sequences of anthocyanidin 3-O-glucosyltransferase (3GT) enzymes according to the method of the present invention. Table 2: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0151] Table 3 provides exemplary sequences of sucrose synthase (SuSy) enzymes according to the method of the present invention. Table 3: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0152] Table 4 provides exemplary sequences of enzymes involved in the production and / or recycling of UDP-glucose, which is a reaction intermediate according to the method of the present invention. Table 4: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0153] Supporting display References and citations to other documents (e.g., patents, patent applications, patent gazettes, journals, books, articles, web content, and publicly accessible databases) are made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes.

[0154] Equal parts Various modifications of the present invention and many further embodiments will be apparent to those skilled in the art from the entirety of this document, including references to scientific and patent documents cited herein, in addition to those shown and described herein. The subject matter herein includes important information, examples, and guidance that can be adapted to the practice of the present invention in its various embodiments and equivalents.

Claims

1. A method for cell-free production of anthocyanins, the method comprising the step of providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in the conversion of one or more substrates to anthocyanins.

2. The method according to claim 1, wherein the substrate is catechin.

3. The method according to claim 2, wherein the catechin is (+)-catechin.

4. The method according to claim 3, wherein the anthocyanin is cyanidin-3-glucoside.

5. The method according to claim 1, wherein the 1 or more enzymes are selected from the group consisting of anthocyanidin synthase enzymes (ANS) and anthocyanidin 3-O-glucosyltransferases (3GT).

6. The method according to claim 5, wherein the anthocyanidin synthase enzyme (ANS) catalyzes the conversion of catechin to cyanidin.

7. The method according to claim 5, wherein the anthocyanidin 3-O-glucosyltransferase (3GT) catalyzes the conversion of cyanidin to cyanidin-3-glucoside.

8. The method according to claim 1, wherein the cell-free medium comprises an activated sugar.

9. The method according to claim 8, wherein the activated sugar is UDP-glucose.

10. The method according to claim 9, wherein the UDP-glucose is added to the cell-free medium.

11. The method according to claim 9, wherein the UDP-glucose is synthesized from one or more components selected from the group consisting of sucrose, glucose, UTP, UDP, ATP, glucose-6-phosphate, glucose-1-phosphate, and / or polyphosphate.

12. The method according to claim 11, wherein the 1 or more enzymes are selected from the group consisting of sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and nucleoside diphosphate kinase (NDK).

13. The method according to claim 11, wherein the 1 or more enzymes are sucrose synthase (SuSy).

14. The method according to claim 1, wherein the cell-free medium is a cell lysate.

15. The method according to claim 14, wherein the cell lysate is a cell lysate derived from a host organism that expresses one or more of the enzymes.

16. The method according to claim 15, wherein the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells.

17. The method according to claim 16, wherein the 1 or more enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism.

18. The method according to claim 16, wherein the host organism expressing 1 or more of the enzymes is cultured until a predetermined biomass is produced in an essential amount of 1 or more of the enzymes.

19. The method according to claim 18, further comprising the step of lysing cells, subsequently removing cell debris, to produce a cell lysate for use in the cell-free medium for cell-free production of anthocyanins.

20. The method according to any one of claims 1 to 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 according to claim 20, wherein the method does not include a step of separating and / or purifying ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK for cell-free production of anthocyanins.

22. The method according to claim 20, comprising the step of separating and / or purifying at least one of ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK for cell-free production of anthocyanins.

23. The method according to any one of claims 1 to 22, wherein the cell-free medium further comprises a buffer, catechin, activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and / or water.

24. The method according to claim 23, wherein the buffer is a phosphate buffer.

25. The method according to claim 23, wherein the activated sugar is UDP-glucose.

26. The method according to claim 23, wherein the cell-free medium further comprises sodium ascorbate, ascorbic acid, ammonium iron sulfate, and 2-oxoglutarate.

27. The method according to claim 23, wherein the catechin is (+)-catechin.

28. The method according to any one of claims 1 to 27, wherein the results of the method in terms of the potency of the anthocyanins produced are about 2 to about 5,000 times higher than those of methods for cell-based production of anthocyanins.

29. The method according to any one of claims 1 to 27, wherein the results of the method in terms of the potency of the anthocyanins produced are about 5 to about 1000 times higher than those of methods for cell-based production of anthocyanins.

30. The method according to claim 23, wherein the pH of the cell-free medium is approximately 5 to approximately 10.

31. The method according to claim 23, wherein the pH of the cell-free medium is approximately 6 to approximately 8.

32. The method according to claim 26, wherein the buffer solution is at a concentration of 1 mM to about 200 mM.

33. The method according to claim 26, wherein the ammonium iron sulfate concentration is approximately 0.25 mM to approximately 10 mM.

34. The method according to claim 26, wherein the cell-free medium further comprises α-ketoglutaric acid at a concentration of about 5 mM to about 50 mM.

35. The method according to claim 26, wherein the ascorbic acid is at a concentration of about 1 mM to about 100 mM.

36. The method according to claim 26, wherein the catechin is at a concentration of approximately 0.5 mM to approximately 50 mM.

37. The method according to claim 26, wherein magnesium chloride is present in a concentration ranging from about 1 mM to about 20 mM.

38. The method according to claim 26, wherein the sucrose is present in a range of approximately 10 mM to approximately 600 mM.

39. The method according to claim 26, wherein the cell lysate containing one or more of the enzymes is present at a concentration of about 5% (v / v) to about 40% (v / v).

40. The method according to claim 26, wherein the reaction for cell-free generation is carried out over a duration of approximately 0.5 hours to approximately 48 hours.

41. The method according to claim 26, wherein the reaction for cell-free generation is carried out over a duration of approximately 0.5 hours to approximately 20 hours.

42. The method according to claim 26, wherein the temperature of the cell-free medium is approximately 20°C to approximately 40°C.

43. The method according to claim 1, wherein the reaction is carried out in a bubble column reactor, and the 1 or more enzymes are present in solution.

44. The method according to claim 1, wherein the reaction is carried out in a packed bed reactor, and the 1 or more enzymes are immobilized.

45. The aforementioned anthocyanidin synthases (ANS) consist of the following group: Table 15 The method according to claim 5, wherein the enzyme has an amino acid sequence that is at least 95% identical to any one of the enzymes selected from the more selected enzymes.

46. The anthocyanidin 3-O-glucosyltransferase (3GT) is comprised of the following group: Table 16-1 Table 16-2 The method according to claim 5, wherein the enzyme has an amino acid sequence that is at least 95% identical to any one of the enzymes selected from the more selected enzymes.

47. The sucrose synthase (SuSy) mentioned above is comprised of the following group: Table 17 The method according to claim 13, wherein the enzyme has an amino acid sequence that is at least 95% identical to any one of the enzymes selected from the more selected enzymes.

48. The method according to any one 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, the one or more enzymes resulting in the conversion of one or more substrates to anthocyanins.

50. The composition according to claim 49, wherein the substrate is catechin.

51. The composition according to claim 50, wherein the catechin is (+)-catechin.

52. The composition according to claim 51, wherein the anthocyanin is cyanidin-3-glucoside.

53. The composition according to claim 49, wherein the 1 or more enzymes are selected from the group consisting of anthocyanidin synthase enzymes (ANS) and anthocyanidin 3-O-glucosyltransferases (3GT).

54. The composition according to claim 53, wherein the anthocyanidin synthase enzyme (ANS) catalyzes the conversion of catechin to cyanidin.

55. The composition according to claim 53, wherein the anthocyanidin 3-O-glucosyltransferase (3GT) catalyzes the conversion of cyanidin to cyanidin-3-glucoside.

56. The composition according to claim 49, wherein the cell-free medium comprises an activated sugar.

57. The composition according to claim 56, wherein the activated sugar is UDP-glucose.

58. The composition according to claim 57, wherein the UDP-glucose is added to the cell-free medium.

59. The composition according to claim 58, wherein the UPD-glucose is synthesized in the cell-free medium by one or more enzymes.

60. The composition according to claim 57, wherein the UDP-glucose is synthesized from one or more components selected from the group consisting of sucrose, glucose, UTP, UDP, ATP, glucose-6-phosphate, glucose-1-phosphate, and / or polyphosphate.

61. The composition according to claim 60, wherein the one or more enzymes are 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 according to claim 49, wherein the cell-free medium is a cell lysate.

63. The composition according to claim 50, wherein the cell lysate is a cell lysate of cells derived from a host organism that expresses one or more of the enzymes.

64. The composition according to claim 63, wherein the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells.

65. The composition according to claim 64, wherein the one or more enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism.

66. The composition according to claim 65, wherein the host organism expressing one or more of the enzymes is cultured until a predetermined biomass is produced in an essential amount of one or more of the enzymes.

67. The composition according to claim 66, further comprising lysing cells and subsequently removing cell debris to produce a cell lysate for use in the cell-free medium for cell-free production of anthocyanins.

68. The composition according to any one of claims 49 to 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.

69. The composition according to claim 68, wherein for cell-free production of anthocyanins, the ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK are not separated and / or purified.

70. The composition according to claim 68, wherein the ANS, 3GT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK are separated and / or purified for cell-free production of anthocyanins.

71. The cell-free medium further comprises a buffer, catechin, activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and / or water, according to any one of claims 49 to 70.

72. The composition according to claim 71, wherein the buffer is a phosphate buffer.

73. The composition according to claim 71, wherein the activated sugar is UDP-glucose.

74. The composition according to claim 71, wherein the cell-free medium further comprises sodium ascorbate, ascorbic acid, ammonium iron sulfate, and 2-oxoglutarate.

75. The composition according to claim 71, wherein the catechin is (+)-catechin.

76. The composition according to any one of claims 49 to 75, wherein the composition is used to produce anthocyanins, and the potency of the anthocyanins produced is about 2 to about 100 times higher than that of a cell-based method for producing anthocyanins.

77. The composition according to any one of claims 49 to 75, wherein the composition is used to produce anthocyanins, and the potency of the anthocyanins produced is about 5 to 10 times higher than that of a cell-based method for producing anthocyanins.

78. The composition according to claim 71, wherein the pH of the cell-free medium is about 5 to about 10.

79. The composition according to claim 71, wherein the pH of the cell-free medium is about 6 to about 8.

80. The composition according to claim 72, wherein the buffer solution has a concentration of about 1 mM to about 200 mM.

81. The composition according to claim 74, wherein the concentration of iron ammonium sulfate is in the range of about 0.25 mM to about 10 mM.

82. The composition according to claim 74, wherein the cell-free medium further comprises α-ketoglutaric acid at a concentration of about 5 mM to about 50 mM.

83. The composition according to claim 74, wherein the ascorbic acid is at a concentration of about 1 mM to about 100 mM.

84. The composition according to claim 74, wherein the catechin is concentrated at a concentration of about 0.5 mM to about 50 mM.

85. The composition according to claim 74, wherein magnesium chloride is present in a concentration ranging from about 1 mM to about 20 mM.

86. The composition according to claim 74, wherein sucrose is present in a range of about 10 mM to about 600 mM.

87. The composition according to claim 74, wherein the cell lysate containing one or more of the enzymes is present at a concentration of about 5% (v / v) to about 40% (v / v).

88. The composition according to claim 74, wherein the reaction for cell-free generation is carried out over a duration of approximately 0.5 hours to approximately 48 hours.

89. The composition according to claim 74, wherein the reaction for cell-free generation is carried out over a duration of approximately 0.5 hours to approximately 20 hours.

90. The composition according to claim 74, wherein the temperature of the cell-free medium is about 20°C to about 40°C.

91. The composition according to claim 49, wherein the composition is in a bubble column reactor, and the one or more enzymes are in solution.

92. The composition according to claim 90, wherein the composition is in a packed bed reactor and the one or more enzymes are immobilized.

93. The aforementioned anthocyanidin synthases (ANS) consist of the following group: Table 18 The composition according to claim 53, wherein the enzyme has an amino acid sequence that is at least 95% identical to any one of the enzymes selected from the above.

94. The anthocyanidin 3-O-glucosyltransferase (3GT) is comprised of the following group: Table 19 The composition according to claim 53, wherein the enzyme has an amino acid sequence that is at least 95% identical to any one of the enzymes selected from the above.

95. The sucrose synthase (SuSy) mentioned above is comprised of the following group: Table 20 The composition according to claim 61, wherein the enzyme has an amino acid sequence that is at least 95% identical to any one of the enzymes selected from the above.

96. The composition according to claim 53 or 61, wherein the ANS, 3GT, and / or SuSy enzymes are modified and optimized for cell-free production of the anthocyanins.

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