Cell-free production of carminic acid

Abstract

In some other conventional carminic acid production methods, carminic acid production may be carried out within a host organism and / or cells. The present invention relates to materials and methods for the production of carminic acid from substrates. The present invention provides methods and materials for cell-free bioproduction of carminic acid. The methods provided in the present invention relate to cell-free production of carminic acid that is more economical and reliable compared to other conventional carminic acid production methods. Furthermore, the methods provided in the present invention provide carminic acid from these processes that offers higher potency compared to carminic acid produced by other conventional methods.

Description

Technical Field

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

[0002] II. Reference to the Sequence Listing This application is filed with a sequence listing in electronic form. The sequence listing is provided as a file named DEBU-020-01WO.xml (created on June 26, 2024, size 56 kilobytes). The electronic form information of the sequence listing is hereby incorporated by reference in its entirety into this specification.

Background Art

[0003] III. Background Carminic acid is used as a food additive. Carminic acid is one of the most frequently used dyes in foods, pharmaceuticals, cosmetics, and textiles. Carminic acid is added to foods such as ketchup, strawberry milk, and candy. Carminic acid is also added to cosmetics such as eyeshadow, nail polish, and lipstick.

[0004] Carminic acid is a coloring agent, which can be extracted from the bodies of female insects of Dactylopius coccus costa (also known as Coccus cacti L.). This insect feeds on, for example, Nopalea coccinellifera, Opuntia fidus indica, and other plants of the Cactaceae family cultivated in the desert areas of Mexico, Central and South America, and the Canary Islands. This coloring agent can have colors in the spectrum from orange to purple via red depending on the pH and is generally known as cochineal or cochineal dye. The carminic acid coloring agent is widely used in foods and beverages.

[0005] In relation to current industrial production, carminic acid is collected by extracting it from dried insect bodies using water or alcohol. The insect (Dactylopius coccus) is reared on cacti. [Overview of the Initiative]

[0006] IV. Outline of the Invention Conventional industrial methods for the production of carminic acid involve the extraction of carminic acid from insect bodies. In some other conventional carminic acid production methods, the production of carminic acid may be carried out within the host organism and / or cells. Consequently, the supply can be relatively expensive and may result in undesirable differences and price fluctuations. This invention provides methods and compositions for the cell-free production of carminic acid. The methods provided in this invention relate to the cell-free production of carminic acid, which is more economical and reliable compared to other conventional carminic acid production methods. Furthermore, the methods provided in this invention provide carminic acid from these processes that offers a higher potency compared to carminic acid produced by other conventional methods.

[0007] In one embodiment, the present invention provides cell-free production of carminic acid, the method comprising providing one or more enzymes in a cell-free medium, the one or more enzymes resulting in the conversion of one or more substrates to carminic acid. In a particular embodiment, the substrate to be converted to carminic acid is kermesic acid. In a particular embodiment, the enzyme that converts kermesic acid to carminic acid is C-glucosyltransferase (CGT). In a particular embodiment, the reaction medium for the C-glucosyltransferase (CGT)-mediated enzymatic conversion of kermesic acid to carminic acid further comprises an activated sugar. In a particular embodiment, the activated sugar is uridine diphosphate glucose (UDP-glucose). In a particular embodiment, UDP-glucose is an essential cofactor for the conversion of kermesic acid to carminic acid. Figure 1 provides an overview of the conversion of kermesic acid to carminic acid.

[0008] In certain embodiments, an activated sugar and / or UDP-glucose is added to the reaction medium. In certain embodiments, the activated sugar in the reaction medium for cell-free production of carminic acid is UDP-glucose. In certain embodiments, UDP-glucose is produced from sucrose in the reaction medium. In certain embodiments, UDP-glucose is produced in the reaction medium by one or more enzymes. In certain embodiments, the production of UDP-sugar is mediated by sucrose synthase (SuSy). In certain embodiments, sucrose synthase (SuSy) mediates the conversion of sucrose to UDP-glucose. Thus, in certain embodiments, the reaction medium contains sucrose which is subsequently converted to UDP-glucose during the course of the reaction. An overview of the process in which kermesic acid is converted to carminic acid and UDP-glucose is produced from sucrose is shown in Figure 2.

[0009] In certain beneficial embodiments, the present invention provides that the UDP-glucose used in the reaction is produced from other substrates during the reaction. In certain embodiments, the UDP moiety in UDP-glucose is reused during the process. By reusing UDP, the economic efficiency of the process of the present invention is provided. Thus, in certain embodiments, the production of carminic acid from kermesic acid catalyzed by CGT is carried out in combination with other methods for UDP-glucose production or UDP reuse. In certain embodiments, an outline of a synthetic scheme including UDP-glucose production and / or reuse is provided in Figure 3. In certain embodiments, UDP-glucose is produced by the reaction of uridine triphosphate (UTP) with glucose-1-phosphate. In certain preferred embodiments, the reaction of UTP with glucose-1-phosphate is catalyzed by UTP-glucose-1-phosphate uridilyltransferase (UGP). In certain preferred embodiments, UGP may be galU.

[0010] In certain embodiments, glucose-1-phosphate is a source of UDP-glucose. Therefore, 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 added to the reaction for the preparation of carminic acid.

[0011] In certain embodiments, glucose-1-phosphate is produced during the course of the reaction. In certain embodiments, glucose-1-phosphate is produced enzymatically during the course of the reaction. Thus, in certain embodiments, glucose is converted to glucose-6-phosphate by the reaction of glucose with ATP (adenosine triphosphate). In certain embodiments, glucose-6-phosphate and adenosine diphosphate (ADP) are produced by the reaction between glucose and ATP. In certain embodiments, the conversion of glucose to glucose-6-phosphate is catalyzed by glucokinase (GLK). In certain embodiments, the conversion of 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 of glucose-6-phosphate to glucose-1-phosphate is catalyzed by phosphoglucumutase (PGM).

[0012] In certain embodiments, the present invention further provides that the nucleoside triphosphate species used in the reaction are reused. In certain embodiments, the reused 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 phosphate or polyphosphate. In certain embodiments, the conversion of ADP to ATP is catalyzed by polyphosphate kinase (PPK).

[0013] In certain beneficial embodiments, the present invention provides cost-effectiveness because the chemical intermediates involved in the reaction are reused. In certain embodiments, the present invention provides reuse of chemical intermediates such as UDP-glucose, UDP, and glucose-1-phosphate. In fact, in certain embodiments, the only reagents required non-catalytically for the production of carminic acid by the conversion of kermesic acid are polyphosphate / phosphate and glucose. The process provided in the present invention is carried out in a cell-free medium and the raw materials (required non-catalytically) are economical, making the process of the present invention cost-effective.

[0014] In certain embodiments, one or more enzymes required for cell-free production of carminic acid are expressed in a host organism. In certain embodiments, the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells. In certain embodiments, one or more enzymes are introduced into the host organism by integration into the host organism's genome or plasmid. In certain embodiments, the plasmid contains extrachromosomal DNA that can be expressed by the host organism. In certain embodiments, the host organism expressing one or more enzymes is cultured until a predetermined biomass is achieved to produce the required amount of one or more enzymes. The predetermined biomass is calculated based on the required amount of one or more enzymes required for cell-free production of carminic acid. In certain embodiments, the culture containing the host organism expressing one or more enzymes is lysed and used as a reaction medium for the method provided in the present invention. In certain other embodiments, the reaction medium for the method of the present invention is prepared by lysing the culture containing the host organism and then removing the cell debris from the lysate.

[0015] In certain embodiments, one or more enzymes expressed in the host cell are C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy). In certain embodiments, C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy) are present in a cell-free medium for cell-free production of carminic acid.

[0016] In certain embodiments, one or more enzymes expressed in the host cell are C-glucosyltransferase (CGT), 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), phosphoglucocomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are present in a cell-free medium for cell-free production of carminic acid.

[0017] In certain embodiments of the present invention, the reaction mixture comprises purified enzymes for cell-free production of carminic acid. In certain embodiments, the purified enzymes are C-glucosyltransferase (CGT), 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), phosphoglucocomutase (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 C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy).

[0018] In certain embodiments, one or more enzymes for producing carminic acid are glucosyltransferase (GT) and / or C-glucosyltransferase (CGT). In certain embodiments, the GT and / or CGT used for cell-free production of carminic acid are selected from enzymes having at least 80% amino acid sequence identity with the enzymes provided in Table 1 below. In certain embodiments, the GT and / or CGT used for cell-free production of carminic acid are selected from enzymes having at least 95% amino acid sequence identity with the enzymes provided in Table 1 below. [Table 1-1]

[0019] In certain embodiments, one or more enzymes involved in the production of UDP-glucose, the raw material for cell-free production of carminic acid, are sucrose synthases (SuSy). In certain embodiments, the SuSy used for UDP-glucose production is selected from enzymes having at least 80% amino acid sequence identity with the enzymes provided in Table 2 below. In certain embodiments, the SuSy used for UDP-glucose production is selected from enzymes having at least 95% amino acid sequence identity with the enzymes provided in Table 2 below. [Table 2-1]

[0020] In certain embodiments, one or more enzymes involved in the production of UDP-glucose and / or the reuse of one or more reaction intermediates are provided below. In certain embodiments, the enzymes used for the production of UDP-glucose (by reaction with UTP) and / or the reuse of one or more intermediates are selected from enzymes having at least 95% amino acid sequence identity with the enzymes provided in Table 3 below. [Table 3-1]

[0021] In certain embodiments, the present invention provides GT / CGT / SuSy enzymes engineered for cell-free production of carminic acid. In certain embodiments, the engineered GT / CGT / SuSy enzymes are optimized for cell-free production of carminic acid. In certain embodiments, the engineered GT / CGT / SuSy enzymes 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 these enzymes result in efficient and optimal cell-free production of carminic acid. In certain embodiments, the GT / CGT / SuSy enzymes used in cell-free production of carminic acid are any enzymes disclosed in this application.

[0022] In certain embodiments of the invention, the cell-free production method does not require purification of one or more enzymes from a lysed host organism. In certain embodiments, the methods of the invention do not require purification of one or more enzymes from lysed biomass comprising host cells that express one or more enzymes for cell-free production of calymmin. In certain embodiments, the methods of the invention do not require purification of 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). In certain embodiments, the methods of the invention do not require purification of C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy) from lysed cells for cell-free production of calymmin. This is advantageous for increasing the efficiency of the method and reducing the costs associated with purification of these enzymes.

[0023] In certain embodiments, the cell-free medium may further contain any other additional ingredients required for the cell-free production of calminic acid. In certain embodiments, the cell-free medium contains a buffer, kermesic acid, activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphoric acid, and / or water. In certain embodiments, the cell-free medium contains a buffer, kermesic acid, 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 kermesic acid to calminic acid. 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 range of 6 to 8 in the reaction mixture. 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.

[0024] In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of about 0.001 mM to about 50 mM. In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of about 0.01 mM to about 10 mM. In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of about 0.01 mM to about 5 mM. In certain embodiments, the activated sugar is UDP-glucose.

[0025] In certain embodiments, the cell-free reaction medium contains magnesium chloride at a concentration of about 0.5 mM to about 40 mM. In certain embodiments, the cell-free reaction medium contains magnesium chloride at a concentration of about 1 mM to about 20 mM.

[0026] 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 cell-free production of carminic acid. 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.

[0027] In certain embodiments, the amount of one or more enzymes for cell-free production of carminic acid depends on the target amount of carminic acid to be produced and / or the concentrations of other raw materials present in the reaction mixture. In certain embodiments, one or more enzymes are present at a concentration of about 1% to about 50% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 2.5% to about 45% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 5% to about 40% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 7.5% to about 30% (v / v).

[0028] In certain embodiments, the reaction for the production of carminic acid is carried out over a certain duration until a desired amount of carminic acid is obtained. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 10 minutes to about 48 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 10 minutes to about 36 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 20 minutes to about 24 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 30 minutes to about 20 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 1 hour to about 15 hours.

[0029] In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is varied to obtain optimal results for carminic acid production. In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is approximately 15°C to approximately 45°C. In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is approximately 20°C to approximately 40°C. In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is approximately 22.5°C to approximately 37.5°C. The temperature of the reaction mixture for cell-free production of carminic acid can affect the rate of carminic acid production. Therefore, the reaction duration can be adjusted according to the temperature of the reaction mixture to obtain an optimal yield of carminic acid in cell-free production of carminic acid.

[0030] In certain embodiments, the method provided in the present invention may be carried out in any reactor suitable for cell-free production of carminic acid. In certain embodiments, the reaction for cell-free production of carminic acid is carried out in a bubble tower reactor / bioreactor. In certain embodiments, in the bubble tower reactor / bioreactor, one or more enzymes involved in cell-free production of carminic acid are in solution. In certain embodiments, the reaction for cell-free production of carminic acid is carried out in a bubble tower reactor / bioreactor containing a lysate from a host organism. In certain embodiments, the bubble tower reactor / bioreactor is advantageous to use for cell-free production of carminic acid when the reaction mixture contains a lysate (or a lysate from which cell debris has been removed) from a host cell organism that utilizes one or more enzymes responsible for cell-free production of carminic acid. In certain embodiments, the reaction for cell-free production of carminic acid is carried out in a packed-bed reactor / bioreactor. In certain embodiments, one or more enzymes are immobilized in the packed-bed reactor / bioreactor. A packed-bed reactor / bioreactor is preferred for purified enzymes that play a role in the cell-free production of carminic acid. In certain embodiments, one or more enzymes may be immobilized in a single reactor / bioreactor. In certain other embodiments, one or more enzymes may be immobilized in different reactors / bioreactors, which are connected in sequence.

[0031] The method provided in the present invention is advantageous over other conventional methods for producing carminic acid. In certain embodiments, the method of the present invention provides cell-free production of carminic acid. Because the method of the present invention is carried out in a cell-free medium, it offers significant economic efficiency by reducing the cost of producing carminic acid in other conventional methods. In certain embodiments, the method of the present invention is cost-effective because the reaction for producing carminic acid is carried out in a lysate from a host organism expressing one or more enzymes involved in the reaction. In particular, in certain embodiments, the method of the present invention does not involve the purification of one or more enzymes. Because the method of the present invention does not require the purification of individual enzymes, it results in further economic efficiency by reducing the cost that would be required to purify individual enzymes in other methods.

[0032] Advantageously, the cell-free production of carminic acid provided herein results in a significantly higher potency compared to conventional methods. Since higher potency carminic acid allows for more efficient purification and / or concentration of the carminic acid from the reaction mixture, the higher potency of carminic acid provides a further cost advantage in carminic acid production. In certain embodiments, the method of the present invention provides a carminic acid potency at least 5 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency at least 10 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency at least 50 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency at least 100 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency at least 500 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency at least 1000 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency at least 5000 times higher than conventional methods.

[0033] In certain embodiments, the present invention provides compositions for the cell-free production of carminic acid. The compositions of the present invention are used for the cell-free production of carminic acid according to the method described above.

[0034] In certain embodiments, the composition of the present invention comprises one or more enzymes in a cell-free medium, the one or more enzymes resulting in the conversion of a substrate to carminic acid. In certain embodiments, the substrate converted to carminic acid is kermesic acid. In certain embodiments, the one or more enzymes are C-glucosyltransferases (CGTs). In certain embodiments, the composition further comprises 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.

[0035] In certain embodiments of the present invention, UDP-glucose in the composition is synthesized by one or more enzymes in a cell-free medium. In certain embodiments, UDP-glucose is synthesized from sucrose. In certain embodiments, one or more enzymes are sucrose synthases (SuSy) that synthesize UDP-glucose from sucrose. In certain embodiments, the cell-free medium is a cell lysate. In certain embodiments, the cell lysate is a cell lysate from cells of a host organism expressing one or more enzymes. In certain embodiments, the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells. In certain embodiments, one or more enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism. In certain embodiments, the host organism expressing one or more enzymes is cultured until a predetermined biomass is achieved to produce the required amount of one or more enzymes. In certain embodiments, the composition further comprises a cell lysate for use in a cell-free medium for cell-free production of carminic acid, which is produced by removing cell debris after lysing the cells.

[0036] In certain embodiments, the CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK enzymes are present in a cell-free medium for cell-free production of carminic acid. In certain embodiments, the compositions of the present invention provide CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK that have not been isolated or separated.

[0037] In certain embodiments, CGT and SuSy enzymes are present in a cell-free medium for cell-free production of carminic acid. In certain embodiments, the composition of the present invention provides CGT and SuSy that have not been isolated or separated.

[0038] In certain embodiments, the cell-free medium may further include any other additional raw materials required for the cell-free production of carminic acid. For example, in certain embodiments, the cell-free medium includes a buffer, kermesic acid, 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 kermesic acid to carminic acid. 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.

[0039] In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of approximately 0.001 mM to approximately 50 mM. In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of approximately 0.01 mM to approximately 5 mM. In certain embodiments, the activated sugar is UDP-glucose.

[0040] In certain embodiments, the cell-free reaction medium contains magnesium chloride at a concentration of about 0.5 mM to about 40 mM. In certain embodiments, the cell-free reaction medium contains magnesium chloride at a concentration of about 1 mM to about 20 mM.

[0041] In certain embodiments, the cell-free reaction medium further contains 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 cell-free production of carminic acid. 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.

[0042] In certain embodiments, the amount of one or more enzymes for cell-free production of carminic acid depends on the target amount of carminic acid to be produced and / or the concentrations of other raw materials present in the reaction mixture. In certain embodiments, one or more enzymes are present at a concentration of about 1% to about 50% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 2.5% to about 45% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 5% to about 40% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 7.5% to about 30% (v / v).

[0043] In certain embodiments of the present invention, the composition of the present invention is in a bubble column reactor, and one or more enzymes are in solution. In certain embodiments of the present invention, the composition of the present invention is in a packed bed reactor, and one or more enzymes are immobilized. [Brief explanation of the drawing]

[0044] V. Brief Description of the Drawings [Figure 1]Figure 1 provides a schematic diagram of the conversion of kermesic acid to carminic acid.

[0045] [Figure 2] Figure 2 provides a schematic diagram of the reaction scheme showing the conversion of kermesic acid to carminic acid and the conversion of sucrose to UDP-glucose.

[0046] [Figure 3] Figure 3 provides a schematic diagram of the reaction scheme showing the conversion of kermesic acid to carminic acid in which UDP is reused.

[0047] [Figure 4] Figure 4 provides an HPLC chromatogram showing the conversion of kermesic acid to carminic acid in a cell-free medium when CGT and UDP-glucose are added to the reaction mixture. [Modes for carrying out the invention]

[0048] VI. Detailed explanation This application provides a composition and method for the production of carminic acid in a cell-free medium, comprising one or more enzymes in the cell-free medium, wherein the conversion of an organic substance to carminic acid is brought about by one or more enzymes. One or more enzymes may be engineered. The engineered enzymes may not exist in nature.

[0049] When used in reference to enzymes, the term “not naturally occurring” is intended to mean that a nucleic acid or polypeptide contains at least one genetic modification not typically 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 modified,” or “recombinant.”

[0050] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the terms "including," "includes," "having," "has," "with," or their variations are intended to be as comprehensive as the term "comprising," to the extent that they are used in either the detailed description and / or the claims.

[0051] 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).

[0052] As used herein, “reaction mixture” may refer to all components derived from “reaction solution” plus the enzyme(s) and / or products from the reaction. In some embodiments, “reaction mixture” may refer only to the reaction solution without enzymes or reaction products. In some embodiments, “reaction solution” and “reaction mixture” may be used interchangeably.

[0053] As used herein, "buffering agent" may refer to a chemical substance added to an aqueous solution that suppresses pH changes through the action of acid-base conjugated components.

[0054] As used herein, “cofactor” may refer to a non-protein compound that can bind to a protein and assist in a biological or chemical reaction. Non-exclusive examples of cofactors include, but are not limited to, NADPH and NADH.

[0055] In the cell-free systems described herein, essential cellular components, namely cofactors and enzymes, are used in chemical reactions that do not contain cellular components that may directly or indirectly inhibit the desired biochemical reaction. Enzymes identical to those found in plants and other organisms may be produced in vivo (typically by overexpression of proteins in a host such as bacteria), isolated by chromatography and / or any other method, and then added to a bioreactor along with the substrate (starting material). Enzymes may also be used directly from plants without isolation. The enzymes transform the substrate in the same manner as they occur in the original organism, without the complexity of the organism. Furthermore, biochemical reactions can be enhanced by adding cosolvents, surfactants, or both, which are unacceptable or simply cannot function in whole-cell-based production methods. In this way, natural products can be produced without the use of plants, cells, or chemosynthesis. Carminic acid:

[0056] Carminic acid is used as a food coloring agent in edible foods. Carminic acid is an anthraquinone compound containing glucose molecules linked via glycosidic bonds. The unsaturated ring in the anthraquinone moiety strongly absorbs light, contributing to its very deep purple color. The coloring agent can exhibit a spectrum of colors ranging from orange through red to violet depending on the pH, and is commonly known as cochineal or cochineal dye. Carmine is the most commonly used form of carminic acid. Carminic acid and its derivatives are found in a wide range of foods, such as ice cream, confectionery, dishes, desserts, beverages, and dairy products. Carmine is also widely used in cosmetics. Carmine is a highly stable pigment that can withstand heat treatment and poor storage conditions. Carmine, the most commonly used form of carminic acid, is obtained by adding aluminum sulfate to a cochineal extract under basic conditions. This produces a complex of carminic acid with the metal, resulting in a deep color shift, and after sol-gel polymerization, an insoluble red lake formulation is obtained.

[0057] Carminic acid derivatives are widely used in the food and cosmetics industries, which can be partly explained by the fact that they are one of the few rare and exceptional naturally occurring pigments that exhibit stability comparable to artificial dyes. In fact, processing conditions such as heating or storage under light and oxygen have little effect on their degradation.

[0058] The chemical structure of carminic acid is provided below. [ka] Synthesis of carminic acid:

[0059] Carminic acid is collected by extracting it from the dried insect bodies using water or alcohol. During the aqueous-based carminic acid extraction from the insects, a certain amount of insect protein is also released from the insects and becomes included in the pigment extract. The level of insect protein is typically less than 0.5%. The aqueous-based cochineal extract contains primarily carminic acid, in addition to some cochineal protein and other trace substances that may be extracted from the insects. In the following text, this extract will be referred to as the cochineal extract solution.

[0060] The collection and processing of insects is not an easy task, as it determines the final quality of the pigment. South American countries such as Peru have ideal climatic conditions for the growth of cacti, which are an important food source for these insects, and therefore, carmine is supplied industrially in the largest quantities here. The orange crude extract of carminic acid is thus purified before being used in the production of pigment formulations. Approximately 100,000 insects need to be processed to obtain 1 kg of extract for the coloring agent.

[0061] Due to low yields and the fact that artificial colorants were not a major concern in industry until a few years ago, the local economy favored more profitable businesses such as vegetable cultivation (bell peppers, asparagus, etc.). However, in recent years, natural colorants have become a trend in the food industry, leading to a significant increase in carmine consumption between 2004 and 2010, and causing unprecedented price surges due to shortages. Because carmine is of animal origin, it is often at the center of religious debate, with both halal and kosher choices prohibiting the use of this colorant. Therefore, alternative methods for synthesizing carmine are needed.

[0062] WO2006 / 056585 describes a process in which, during aqueous-based carminic acid extraction from insects, a certain amount of insect protein is also released from the insects and contained in the pigment extract, and it has been reported that cochineal insect protein may cause some allergy-related problems. WO2006 / 056585 describes a special process for reducing the amount of insect protein from the described insect extract solution - however, the final pigment composition / product produced by WO2006 / 056585 still contains some amount of Dactylopius coccus costa insect protein.

[0063] WO2022 / 013659 provides a method for producing carminic acid from insects of the genus Dactylopius coccus. The method includes extracting blood cells from the insect hemolymph and recovering purified blood cells; culturing the purified blood cells using a culture medium for culturing insect cell lines; stimulating and activating the insect cell lines; and growing the insect cell lines by producing carminic acid. However, these methods are unpredictable and complex, given that they involve biological production in cell lines.

[0064] This invention provides methods and compositions for the cell-free production of carminic acid. Because the methods of this invention encompass the cell-free production of carminic acid, the methods provided herein relate to a more economical and reliable cell-free production of carminic acid compared to other conventional carminic acid production methods. Furthermore, the methods provided herein offer a higher potency of carminic acid from these processes compared to carminic acid produced by other conventional methods. The methods of this invention are also advantageous because cell-free production of carminic acid reduces the risk of post-production degradation of carminic acid. Furthermore, cell-free synthesis methods allow for the production of higher concentrations of carminic acid without killing cells. Additionally, cell-free production facilitates the purification of carminic acid after the reaction is complete. In certain embodiments, cell-free production of carminic acid allows for the reuse of enzymes used in the reaction to prepare carminic acid. Therefore, enzymes from the same batch may be used to produce multiple batches of carminic acid. In certain embodiments, the enzymes can be purified in downstream processes by methods such as filtration and centrifugation.

[0065] In one embodiment, the present invention provides cell-free production of carminic acid, the method comprising providing one or more enzymes in a cell-free medium, the one or more enzymes resulting in the conversion of a substrate to carminic acid. In a particular embodiment, the substrate to be converted to carminic acid is kermesic acid. In a particular embodiment, the enzyme that converts kermesic acid to carminic acid is C-glucosyltransferase (CGT). Figure 1 provides the conversion of kermesic acid to carminic acid mediated by CGT. In a particular embodiment, the reaction medium for the enzymatic conversion of kermesic acid to carminic acid mediated by C-glucosyltransferase (CGT) further comprises an activated sugar. In a particular embodiment, the activated sugar is UDP-glucose. In a particular embodiment, UDP-glucose is an essential cofactor for the conversion of kermesic acid to carminic acid.

[0066] In certain embodiments, an activated sugar and / or UDP-glucose is added to the reaction medium. In certain embodiments, the activated sugar in the reaction medium for cell-free production of carminic acid is UDP-glucose. In certain embodiments, UDP-glucose for the reaction medium is produced from sucrose. In certain embodiments, UDP-glucose is produced in the reaction medium by one or more enzymes. In certain embodiments, the production of UDP-sugar is mediated by sucrose synthase (SuSy). In certain embodiments, sucrose synthase (SuSy) mediates the conversion of sucrose to UDP-glucose. Figure 2 shows a schematic of the conversion of sucrose to UDP-glucose mediated by SuSy. As shown in Figure 2, SuSy is involved in the conversion of sucrose to UDP-glucose, and the produced UDP-glucose is involved in the conversion of kermesic acid to carminic acid. Thus, in certain embodiments, the reaction medium contains sucrose, which is subsequently converted to UDP-glucose during the course of the reaction. In certain embodiments, the conversion of sucrose to UDP-glucose may be carried out in the same reactor as the conversion of kermesic acid to carminic acid. In other embodiments, the conversion of sucrose to UDP-glucose may be carried out in a different reactor than the conversion of kermesic acid to carminic acid.

[0067] In certain beneficial embodiments, the present invention provides that the UDP-glucose used in the reaction is produced from other substrates during the reaction. In certain embodiments, the UDP moiety in UDP-glucose is reused during the process. The reuse of UDP provides economic efficiency to the process of the present invention. Thus, in certain embodiments, the production of carminic acid from kermesic acid catalyzed by CGT is carried out in combination with other methods for UDP-glucose production or UDP reuse. In certain embodiments, an outline of a synthetic scheme including UDP-glucose production and / or reuse is provided in Figure 3. In certain embodiments, UDP-glucose is produced by the reaction of uridine triphosphate (UTP) with glucose-1-phosphate. In certain preferred embodiments, the reaction of UTP with glucose-1-phosphate is catalyzed by UTP-glucose-1-phosphate uridilyltransferase (UGP). In certain preferred embodiments, UGP may be galU.

[0068] In certain embodiments, glucose-1-phosphate is a source of UDP-glucose. Therefore, 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 added to the reaction for the preparation of carminic acid.

[0069] In certain embodiments, glucose-1-phosphate is produced during the course of the reaction. In certain embodiments, glucose-1-phosphate is produced enzymatically during the course of the reaction. Thus, in certain embodiments, glucose is converted to glucose-6-phosphate by the reaction of glucose with ATP (adenosine triphosphate). In certain embodiments, glucose-6-phosphate and adenosine diphosphate (ADP) are produced by the reaction between glucose and ATP. In certain embodiments, the conversion of glucose to glucose-6-phosphate is catalyzed by glucokinase (GLK). In certain embodiments, the conversion of 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 of glucose-6-phosphate to glucose-1-phosphate is catalyzed by phosphoglucumutase (PGM).

[0070] In certain embodiments, the present invention further provides that the nucleoside triphosphate species used in the reaction are reused. In certain embodiments, the reused 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 phosphate or polyphosphate. In certain embodiments, the conversion of ADP to ATP is catalyzed by polyphosphate kinase (PPK).

[0071] In certain beneficial embodiments, the present invention provides cost-effectiveness because the chemical intermediates involved in the reaction are reused. In certain embodiments, the present invention provides reuse of chemical intermediates such as UDP-glucose, UDP, and glucose-1-phosphate. In fact, in certain embodiments, the only reagents required non-catalytically for the production of carminic acid by the conversion of kermesic acid are polyphosphate / phosphate and glucose. The process provided in the present invention is carried out in a cell-free medium and the raw materials (required non-catalytically) are economical, making the process of the present invention cost-effective.

[0072] In certain embodiments, one or more enzymes required for cell-free production of carminic acid are expressed in a host organism. In certain embodiments, the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells. In certain embodiments, one or more enzymes are introduced into the host organism by integration into the host organism's genome or plasmid. In certain embodiments, the plasmid contains extrachromosomal DNA that can be expressed by the host organism. In certain embodiments, the host organism expressing one or more enzymes is cultured until a predetermined biomass is achieved to produce the required amount of one or more enzymes. The predetermined biomass is calculated based on the required amount of one or more enzymes required for cell-free production of carminic acid. In certain embodiments, the culture containing the host organism expressing one or more enzymes is lysed and used as a reaction medium for the method provided in the present invention. In certain other embodiments, the reaction medium for the method of the present invention is prepared by lysing the culture containing the host organism and then removing the cell debris from the lysate. In certain embodiments, one or more enzymes expressed in the host cell are C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy). In certain embodiments, C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy) are present in a cell-free medium for cell-free production of carminic acid.

[0073] In certain embodiments of the present invention, the reaction mixture comprises purified enzymes for cell-free production of carminic acid. In certain embodiments, the purified enzymes are C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy).

[0074] In certain embodiments, one or more enzymes expressed in the host cell are C-glucosyltransferase (CGT), 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), phosphoglucocomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are present in a cell-free medium for cell-free production of carminic acid.

[0075] In certain embodiments of the present invention, the reaction mixture comprises purified enzymes for cell-free production of carminic acid. In certain embodiments, the purified enzymes are C-glucosyltransferase (CGT), 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, the purified enzymes are C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy).

[0076] In certain embodiments, one or more enzymes for producing carminic acid are glucosyltransferase (GT) and / or C-glucosyltransferase (CGT). C-glycosyltransferase (CGT) catalyzes the formation of C-glycosidic bonds for the biosynthesis of C-glycosides. Glycosylation occurs on O-, C-, N-, and S- atoms to produce O-glycosides, C-glycosides, N-glycosides, and S-glycosides, respectively. C-glycosides are more stable to acid hydrolysis and glycosidases than others. Chemical synthesis of C-glycosides is always difficult due to stereoselectivity and harsh reaction conditions. The difficulty in preparing sugar precursors also limits the synthesis of C-glycosides. In contrast, the biosynthesis of glycosides involves much milder conditions and exhibits high stereospecificity. Therefore, research on C-glycosyltransferase (CGT) is beneficial for exploring the biosynthesis of C-glycosides, thereby promoting the development and utilization of C-glycoside drugs.

[0077] The enzymes previously known for this process of carminic acid production were those derived from the cochineal insect Dactylopius coccus (DcUGT) and an engineered variant derived from the plant Gentiana trifloral (GtCGT). In certain embodiments, the present invention provides an improved GT enzyme engineered to alter its product specificity as a CGT rather than as an OGT.

[0078] In certain embodiments, GTs and / or CGTs used for cell-free production of carminic acid are provided in WO2022 / 164226, WO2022 / 013659, and / or US10,724,012, which are incorporated herein by reference in their entirety.

[0079] In certain embodiments, the GT and / or CGT used for cell-free production of carminic acid are selected from enzymes having at least 80% amino acid sequence identity with the enzymes provided in Table 1 below. In certain embodiments, the GT and / or CGT used for cell-free production of carminic acid are selected from enzymes having at least 95% amino acid sequence identity with the enzymes provided in Table 1 (reproduced below). [Table 1-2]

[0080] In certain embodiments, one or more enzymes involved in the production of UDP-glucose, the raw material for cell-free production of carminic acid, are sucrose synthases (SuSy). Sucrose synthases (SuSy) convert sucrose and uridine 5-bisphosphate (UDP) to UDP-glucose. By linking the SuSy reaction and the GT reaction in a one-pot cascade transformation, a UDP cycle is created, and this cycle continuously regenerates UDP-glucose, making it a suitable donor for glucoside production. In certain embodiments, the SuSy used for UDP-glucose production is selected from enzymes having at least 80% amino acid sequence identity with the enzymes provided in Table 2 below. In certain embodiments, the SuSy used for UDP-glucose production is selected from enzymes having at least 95% amino acid sequence identity with the enzymes provided in Table 2 (reproduced below). [Table 2-2]

[0081] In certain embodiments, one or more enzymes involved in the production of UDP-glucose and / or the reuse of one or more reaction intermediates are provided below. In certain embodiments, the enzymes used for the production of UDP-glucose (by reaction with UTP) and / or the reuse of one or more intermediates are selected from enzymes having at least 95% amino acid sequence identity with the enzymes provided in Table 3 below. [Table 3-2]

[0082] In certain embodiments, the present invention provides GT / CGT / SuSy enzymes engineered for cell-free production of carminic acid. In certain embodiments, the engineered GT / CGT / SuSy enzymes are optimized for cell-free production of carminic acid. In certain embodiments, the engineered GT / CGT / SuSy enzymes 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 these enzymes result in efficient and optimal cell-free production of carminic acid. In certain embodiments, the GT / CGT / SuSy enzymes used in cell-free production of carminic acid are any enzymes disclosed in this application.

[0083] In certain embodiments, the present invention provides GLK / PGM / UGP / NDK / PPK enzymes that have been manipulated and optimized for the reuse of reaction intermediates involved in UDP-glucose production and / or cell-free production of carminic acid. In certain embodiments, the manipulated and / or modified GLK / PGM / UGP / NDK / PPK enzymes 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 these enzymes result in efficient and optimal cell-free production of carminic acid.

[0084] In certain embodiments of the present invention, the cell-free production method does not require the purification of one or more enzymes 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 lysed biomass containing host cells expressing one or more 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 lysed cells for cell-free production of carminic acid. This is advantageous in increasing the efficiency of the method and reducing the costs associated with the purification of these enzymes.

[0085] In certain embodiments, the cell-free medium may further include any other additional raw materials required for the cell-free production of carminic acid. For example, in certain embodiments, the cell-free medium includes a buffer, kermesic acid, activated sugar, magnesium chloride, cell lysate, sucrose, and / or water. In certain embodiments, the cell-free reaction mixture further includes uridine diphosphate (UDP). In certain embodiments, the cell-free reaction mixture further includes polyphosphate and / or glucose. In certain embodiments, the cell-free reaction mixture contains UDP-glucose and / or UDP in catalytic amounts. In certain embodiments, UDP-glucose is produced enzymatically in the reaction. In certain embodiments, the reaction mixture contains 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 includes glucose and polyphosphate.

[0086] In certain preferred embodiments, the process of the present invention utilizes kermesic acid, glucose, and polyphosphate as starting materials for the production of carminic acid. In these embodiments, the present invention provides that other intermediates in the process for the production of carminic acid can be reused.

[0087] In a particular embodiment, the process of the present invention utilizes kermesic acid and UDP-glucose as starting materials for the production of carminic acid.

[0088] In certain embodiments, the process of the present invention utilizes kermesic acid and sucrose as starting materials for the production of carminic acid. In these embodiments, the present invention provides that other intermediates can be reused in the process for the production of carminic acid.

[0089] In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for the enzymatic conversion of kermesic acid to carminic acid. 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.

[0090] In certain embodiments, the buffer is selected from the group consisting of Tris, HEPES, phosphoric acid, carbonic acid / bicarbonate, acetic acid, sodium hydroxide-glycine, and / or citric acid. The concentration of the buffer for the reaction mixture depends on the concentrations of the starting materials and other raw materials in the reaction mixture. 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 250 mM. In certain embodiments, the buffer is present at a concentration of about 5 mM to about 100 mM. In certain embodiments, the phosphate buffer is present at a concentration of about 1 mM to about 250 mM. In certain embodiments, the phosphate buffer is present at a concentration of about 5 mM to about 100 mM.

[0091] In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of approximately 0.001 mM to approximately 50 mM. In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of approximately 0.01 mM to approximately 5 mM. In certain embodiments, the activated sugar is UDP-glucose.

[0092] In certain embodiments, the cell-free reaction medium includes a source of magnesium ions. In certain embodiments, the source of magnesium ions is magnesium chloride at a concentration of about 0.5 mM to about 40 mM. In certain embodiments, the cell-free reaction medium contains magnesium chloride at a concentration of about 1 mM to about 20 mM.

[0093] In certain embodiments, the cell-free reaction medium further contains 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 cell-free production of carminic acid. 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.

[0094] In certain embodiments, the cell-free reaction medium further contains glucose. In certain embodiments, glucose is a starting material for providing UDP-glucose for the conversion of kermesic acid to carminic acid. 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 carminic acid. 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.

[0095] In certain embodiments, the cell-free reaction medium further comprises polyphosphate. In certain embodiments, polyphosphate is also a starting material for providing UDP-glucose for the conversion of kermesic acid to carminic acid. 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 carminic acid. In certain embodiments, polyphosphate is present in the cell-free reaction medium at a concentration of about 10 mM to about 1000 mM. In certain embodiments, polyphosphate is present in the cell-free reaction medium at a concentration of about 20 mM to about 800 mM. In certain embodiments, polyphosphate is present in the cell-free reaction medium at a concentration of about 50 mM to about 600 mM.

[0096] In certain embodiments, the amount of one or more enzymes for cell-free production of carminic acid depends on the target amount of carminic acid to be produced and / or the concentrations of other raw materials present in the reaction mixture. In certain embodiments, one or more enzymes are present at a concentration of about 1% to about 50% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 2.5% to about 45% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 5% to about 40% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 7.5% to about 30% (v / v).

[0097] In certain embodiments, the reaction for the production of carminic acid is carried out over a certain duration until a desired amount of carminic acid is obtained. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 10 minutes to about 48 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 10 minutes to about 36 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 20 minutes to about 24 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 30 minutes to about 20 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out for about 1 hour to about 15 hours.

[0098] In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is varied to obtain optimal results for carminic acid production. In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is approximately 15°C to approximately 45°C. In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is approximately 20°C to approximately 40°C. In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is approximately 22.5°C to approximately 37.5°C. The temperature of the reaction mixture for cell-free production of carminic acid can affect the rate of carminic acid production. Therefore, the reaction duration can be adjusted according to the temperature of the reaction mixture to obtain an optimal yield of carminic acid in cell-free production of carminic acid.

[0099] In certain embodiments, the method provided in the present invention may be carried out in any reactor suitable for cell-free production of carminic acid. In certain embodiments, the reaction for cell-free production of carminic acid is carried out in a bubble tower reactor / bioreactor. In certain embodiments, in the bubble tower reactor / bioreactor, one or more enzymes involved in cell-free production of carminic acid are in solution. In certain embodiments, the reaction for cell-free production of carminic acid is carried out in a bubble tower reactor / bioreactor containing a lysate from a host organism. In certain embodiments, the bubble tower reactor / bioreactor is advantageous to use for cell-free production of carminic acid when the reaction mixture contains a lysate (or a lysate from which cell debris has been removed) from a host cell organism that utilizes one or more enzymes responsible for cell-free production of carminic acid. In certain embodiments, the reaction for cell-free production of carminic acid is carried out in a packed-bed reactor / bioreactor. In certain embodiments, one or more enzymes are immobilized in the packed-bed reactor / bioreactor. A packed-bed reactor / bioreactor is preferred for purified enzymes that play a role in the cell-free production of carminic acid. In certain embodiments, one or more enzymes may be immobilized in a single reactor / bioreactor. In certain other embodiments, one or more enzymes may be immobilized in different reactors / bioreactors, in which case these reactors / bioreactors are connected in sequence. In certain embodiments, the bioreactor system is provided in PCT / US2021 / 064049, which is incorporated in its entirety by reference.

[0100] The method provided in the present invention is advantageous over other conventional methods for producing carminic acid. In certain embodiments, the method of the present invention provides cell-free production of carminic acid. Because the method of the present invention is carried out in a cell-free medium, it offers significant economic efficiency by reducing the cost of producing carminic acid in other conventional methods. In certain embodiments, the method of the present invention is cost-effective because the reaction for producing carminic acid is carried out with a lysate from a host organism expressing one or more enzymes involved in the reaction. In particular, in certain embodiments, the method of the present invention does not involve the purification of one or more enzymes. Because the method of the present invention does not require the purification of individual enzymes, it offers further economic efficiency by reducing the cost that would be required to purify individual enzymes in other methods.

[0101] Advantageously, the cell-free production of carminic acid provided herein offers a significantly higher potency value for carminic acid compared to conventional methods. A higher potency carminic acid provides a further cost advantage for carminic acid production, as the higher potency carminic acid provides efficiency in the purification and / or concentration of 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 carminic acid 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 carminic acid potency value at least 500 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency value at least 1000 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency value at least 5000 times higher than conventional methods.

[0102] In some embodiments, the isolated carminic acid 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%.

[0103] In other embodiments, the isolated carminic acid 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%. Composition for cell-free production of carminic acid

[0104] In certain embodiments, the present invention provides compositions for the cell-free production of carminic acid. The compositions of the present invention are used for the cell-free production of carminic acid according to the method described above.

[0105] In certain embodiments, the composition of the present invention comprises one or more enzymes in a cell-free medium, the one or more enzymes resulting in the conversion of a substrate to carminic acid. In certain embodiments, the substrate converted to carminic acid is kermesic acid. In certain embodiments, the one or more enzymes are C-glucosyltransferases (CGTs). In certain embodiments, the composition further comprises 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.

[0106] In certain embodiments of the present invention, UDP-glucose in the composition is synthesized by one or more enzymes in a cell-free medium. In certain embodiments, UDP-glucose is synthesized from sucrose. In certain embodiments, one or more enzymes are sucrose synthases (SuSy) that synthesize UDP-glucose from sucrose. In certain embodiments, the cell-free medium is a cell lysate. In certain embodiments, the cell lysate is a cell lysate from cells of a host organism expressing one or more enzymes. In certain embodiments, the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells. In certain embodiments, one or more enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism. In certain embodiments, the host organism expressing one or more enzymes is cultured until a predetermined biomass is achieved to produce the required amount of one or more enzymes. In certain embodiments, the composition further comprises a cell lysate for use in a cell-free medium for cell-free production of carminic acid, which is produced by removing cell debris after lysing the cells. In certain embodiments, the CGT and SuSy enzymes are present in a cell-free medium for cell-free production of carminic acid. In certain embodiments, the composition of the present invention provides CGT and SuSy that have not been purified or separated.

[0107] In certain embodiments, the composition further comprises glucose-1-phosphate. In certain embodiments, glucose-1-phosphate is a source of UDP-glucose. Therefore, 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 added to the reaction for the preparation of carminic acid.

[0108] In certain embodiments, glucose-1-phosphate is produced during the course of the reaction in the composition of the present invention. In certain embodiments, glucose-1-phosphate is produced enzymatically during the course of the reaction. Therefore, in certain embodiments, the composition of the present invention comprises glucose and / or ATP. In certain embodiments, glucose is converted to glucose-6-phosphate by a reaction between glucose and ATP. 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 of glucose to glucose-6-phosphate is catalyzed by glucokinase (GLK). In certain embodiments, the conversion of 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 of glucose-6-phosphate to glucose-1-phosphate is catalyzed by phosphoglucumutase (PGM). Therefore, in certain embodiments, the composition comprises GLK, HK, and / or PGM.

[0109] In certain embodiments, the present invention further provides that the nucleoside triphosphate species used in the reaction are reused. In certain embodiments, the reused 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 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.

[0110] In certain embodiments, the composition further comprises C-glucosyltransferase (CGT), 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), phosphoglucocomutase (PGM), polyphosphate kinase (PPK), UTP-glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK) are present in a cell-free medium for cell-free production of carminic acid.

[0111] In certain embodiments, the cell-free medium may further include any other additional raw materials required for the cell-free production of carminic acid. For example, in certain embodiments, the cell-free medium includes a buffer, kermesic acid, 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 kermesic acid to carminic acid. 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.

[0112] In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of approximately 0.001 mM to approximately 50 mM. In certain embodiments, the cell-free reaction medium contains activated sugar at a concentration of approximately 0.01 mM to approximately 5 mM. In certain embodiments, the activated sugar is UDP-glucose.

[0113] In certain embodiments, the cell-free reaction medium contains magnesium chloride at a concentration of about 0.5 mM to about 40 mM. In certain embodiments, the cell-free reaction medium contains magnesium chloride at a concentration of about 1 mM to about 20 mM.

[0114] In certain embodiments, the cell-free reaction medium further contains 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 cell-free production of carminic acid. 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.

[0115] In certain embodiments, the cell-free reaction medium further contains glucose. In certain embodiments, the concentration of glucose in the reaction mixture is determined by the amount of UDP-glucose required for the cell-free production of carminic acid. 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.

[0116] In certain embodiments, the cell-free reaction medium further comprises phosphoric acid / polyphosphate. In certain embodiments, the cell-free reaction medium further comprises polyphosphate. In certain embodiments, 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 carminic acid. In certain embodiments, 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, 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, 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, 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, polyphosphate is present at a concentration of about 25 g / L.

[0117] 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.

[0118] In certain embodiments, the cell-free reaction medium further contains 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.

[0119] In certain embodiments, the cell-free reaction medium contains glucose-6-phosphate and / or glucose-1-phosphate. In certain embodiments, glucose-6-phosphate and / or glucose-1-phosphate are generated and / or reused in the reaction medium. Therefore, 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 may 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.

[0120] In certain embodiments, the amount of one or more enzymes for cell-free production of carminic acid depends on the target amount of carminic acid to be produced and / or the concentrations of other raw materials present in the reaction mixture. In certain embodiments, one or more enzymes are present at a concentration of about 1% to about 50% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 2.5% to about 45% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 5% to about 40% (v / v). In certain embodiments, one or more enzymes are present at a concentration of about 7.5% to about 30% (v / v).

[0121] In certain embodiments of the present invention, the composition of the present invention is in a bubble column reactor containing one or more enzymes in solution. In certain embodiments of the present invention, the composition of the present invention is in a packed bed reactor containing one or more immobilized enzymes.

[0122] In some embodiments, the enzyme may be immobilized. In some embodiments, the immobilized enzyme may 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, but are not limited to, various surface chemistrys including covalent bonding, adsorption, ionic bonding, affinity, encapsulation, or incorporation may be used to link the immobilized enzyme to a solid surface. In other embodiments, the immobilized enzyme may be immobilized on a crosslinking enzyme aggregate. In other embodiments, the enzyme is unimmobilized. Either immobilized or unimmobilized enzymes can be used in batch or serial synthesis. For example, an immobilized enzyme on a solid support may be used in a cartridge through which the reaction mixture passes, thereby allowing the immobilized enzyme to catalyze substrate modification at a high titer to produce the product. Alternatively, a continuous method may involve continuously removing either the product, the substrate (e.g., recovery), or both by micro-mixing the enzyme solution and the substrate to produce a product with a high titer. In some embodiments, the removed (e.g., recovered) substrate can be reused to increase process efficiency and overall yield.

[0123] In some 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 immobilized.

[0124] In some 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 immobilized.

[0125] In some embodiments, the enzyme is reused 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 from the reaction mixture for continuous purification. In a particular embodiment, a bioreactor system is provided in PCT / US2021 / 064049, which is incorporated in its entirety by reference.

[0126] Advantageously, the cell-free production of carminic acid provided herein offers a significantly higher potency value of carminic acid compared to conventional methods. A higher potency carminic acid provides a further cost advantage to carminic acid production, as the higher potency of carminic acid provides efficiency in the purification and / or concentration of 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 carminic acid 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 carminic acid potency value at least 500 times higher than conventional methods. In certain embodiments, the method of the present invention provides a carminic acid potency value at least 1000 times higher than conventional methods.

[0127] In some embodiments, the isolated carminic acid 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%.

[0128] In other embodiments, the isolated carminic acid 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]

[0129] VII. Examples (Example 1) Biological production of carminic acid The reaction conditions are provided in Table 4 below. The exemplary reaction conditions provided below are for the reactions provided for the conversion of kermesic acid to carminic acid and / or sucrose to UDP-glucose. [Table 4]

[0130] Figure 4 shows the results of the reaction. As shown in Figure 4, when CGT is added to a mixture containing kermesic acid, the kermesic acid is converted to carminic acid. (Example 2) Reactions for the production of UDP-glucose and / or the reuse of intermediates

[0131] The reaction conditions for the reaction to produce UDP-glucose and / or reuse of the intermediate are provided in Table 5 below. [Table 5]

[0132] In a particular embodiment, the enzymes in Table 5 above are CGT, SuSy, NDK, UGP, PPK, PGM, GLK, and / or HK.

[0133] Table 6 provides exemplary sequences of glycosyltransferase enzymes according to the method of the present invention. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

[0134] Table 7 provides exemplary sequences of sucrose synthase enzymes according to the method of the present invention. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7]

[0135] Table 8 provides exemplary sequences of enzymes involved in the production and / or reuse of UDP-glucose, a reaction intermediate according to the method of the present invention. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0136] Embedding by reference

[0137] Throughout this disclosure, references and citations are made to other documents, including patents, patent applications, patent publications, journals, books, articles, web content, and publicly accessible databases. All such documents are incorporated herein in their entirety by reference for any purpose. Equal portions

[0138] In addition to those shown and described herein, various modifications of the invention and many further embodiments will be apparent to those skilled in the art from the entirety of this document, including references to the scientific and patent literature cited herein. The subject matter of this specification includes important information, examples, and guidance applicable to the practice of various embodiments of the invention and their equivalents.

Claims

1. A method for cell-free production of carminic acid, the method comprising 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 carminic acid.

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

3. The method according to claim 2, wherein the enzyme is C-glucosyltransferase (CGT).

4. The method according to claim 2, further comprising the cell-free medium containing an activated sugar.

5. The method according to claim 4, wherein the activated sugar is UDP-glucose.

6. The method according to claim 5, wherein the UDP-glucose is added to the cell-free medium.

7. The method according to claim 5, wherein the UDP-glucose is synthesized in the cell-free medium by one or more enzymes.

8. The method according to claim 7, wherein the UDP-glucose is synthesized from one or more raw materials selected from the group consisting of sucrose, glucose, UTP, UDP, ATP, glucose-6-phosphate, glucose-1-phosphate, and / or polyphosphate.

9. The method according to claim 8, wherein one 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).

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

11. The method according to claim 10, wherein the cell lysate is a cell lysate from a host organism that expresses one or more enzymes.

12. The method according to claim 11, wherein the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells.

13. The method according to claim 11, wherein one or more enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism.

14. The method according to claim 13, wherein the host organism expressing the one or more enzymes is cultured until a predetermined biomass is achieved to produce the required amount of the one or more enzymes.

15. The method according to claim 14, 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 carminic acid.

16. The method according to any one of claims 1 to 15, wherein the one or more enzymes are selected from the group consisting of CGT, SuSy, GLK, HK, PGM, PPK, UGP, and NDK.

17. The method according to claim 16, wherein one or more enzymes are present in the cell-free medium for the cell-free production of carminic acid.

18. The method according to claim 16, wherein the method does not involve separating and / or purifying one or more enzymes for cell-free production of carminic acid.

19. The method according to any one of claims 1 to 18, wherein the cell-free medium further comprises a buffer, kermesic acid, activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and / or water.

20. The method according to claim 19, wherein the buffer is a phosphate buffer.

21. The method according to claim 19, wherein the activated sugar is UDP-glucose.

22. The method according to any one of claims 1 to 18, wherein the method yields a potency of produced carminic acid that is about 10 to 5,000 times higher than that of a cell-based method for producing carminic acid.

23. The method according to claim 19, wherein the pH of the cell-free medium is approximately 5 to approximately 9.

24. The method according to claim 19, wherein the buffer is present at a concentration of about 5 mM to about 250 mM.

25. The method according to claim 19, wherein the activated sugar is present at a concentration of about 0.01 mM to about 10 mM.

26. The method according to claim 19, wherein magnesium chloride is present at a concentration of about 1 mM to about 20 mM.

27. The method according to claim 19, wherein sucrose is present at a concentration of approximately 10 mM to approximately 1000 mM.

28. The method according to claim 19, wherein sucrose is present at a concentration of approximately 50 mM to approximately 600 mM.

29. The method according to claim 19, wherein the cell lysate containing one or more of the enzymes is present at a concentration of about 5% (v / v) to about 50% (v / v).

30. The method according to claim 19, wherein the reaction for cell-free production is carried out over a duration of approximately 0.5 hours to approximately 48 hours.

31. The method according to claim 19, wherein the reaction for cell-free production is carried out over a duration of approximately 0.5 hours to approximately 20 hours.

32. The method according to claim 19, wherein the temperature of the cell-free medium is approximately 20°C to approximately 40°C.

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

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

35. A composition for cell-free production of carminic acid, wherein the composition comprises one or more enzymes in a cell-free medium, the one or more enzymes causing the conversion of one or more substrates to carminic acid.

36. The composition according to claim 35, wherein the substrate is kermesic acid.

37. The composition according to claim 36, wherein the enzyme is C-glucosyltransferase (CGT).

38. The composition according to claim 36, further comprising the cell-free medium containing an activated sugar.

39. The composition according to claim 38, wherein the activated sugar is UDP-glucose.

40. The composition according to claim 39, wherein the UDP-glucose is added to the cell-free medium.

41. The composition according to claim 39, wherein the UDP-glucose is synthesized in the cell-free medium by one or more enzymes.

42. The composition according to claim 41, wherein the UDP-glucose is synthesized from one or more raw materials selected from the group consisting of sucrose, glucose, UTP, UDP, ATP, glucose-6-phosphate, glucose-1-phosphate, and / or polyphosphate.

43. The composition according to claim 42, wherein one 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).

44. The composition according to claim 35, wherein the cell-free medium is a cell lysate.

45. The composition according to claim 44, wherein the cell lysate is a cell lysate from a host organism that expresses one or more enzymes.

46. The composition according to claim 45, wherein the host organism is selected from the group consisting of bacteria, yeast, and / or mammalian cells.

47. The composition according to claim 46, wherein one or more enzymes are introduced into the host organism by integration into the genome or plasmid of the host organism.

48. The composition according to claim 47, wherein the host organism expressing the one or more enzymes is cultured until a predetermined biomass is achieved to produce the required amount of the one or more enzymes.

49. The composition according to claim 48, 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 carminic acid.

50. The composition according to any one of claims 35 to 40, wherein the one or more enzymes are selected from the group consisting of CGT, SuSy, GLK, HK, PGM, PPK, UGP, and NDK.

51. The composition according to claim 50, wherein one or more enzymes are present in the cell-free medium for the cell-free production of carminic acid.

52. The composition according to claim 51, wherein the method does not involve separating and / or purifying one or more enzymes for cell-free production of carminic acid.

53. The composition according to any one of claims 35 to 52, wherein the cell-free medium further comprises a buffer, kermesic acid, activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and / or water.

54. The composition according to claim 53, wherein the buffer is a phosphate buffer.

55. The composition according to claim 53, wherein the activated sugar is UDP-glucose.

56. The composition according to any one of claims 35 to 52, wherein the method yields a potency of produced carminic acid that is about 10 to about 5000 times higher than that of a cell-based method for producing carminic acid.

57. The composition according to claim 53, wherein the pH of the cell-free medium is approximately 5 to approximately 9.

58. The composition according to claim 53, wherein the buffer is present at a concentration of about 5 mM to about 100 mM.

59. The composition according to claim 53, wherein the activated sugar is present at a concentration of about 0.01 mM to about 5 mM.

60. The composition according to claim 53, wherein magnesium chloride is present at a concentration of about 1 mM to about 20 mM.

61. The composition according to claim 53, wherein sucrose is present at a concentration of about 10 mM to about 1000 mM.

62. The composition according to claim 53, wherein sucrose is present at a concentration of about 50 mM to about 600 mM.

63. The composition according to claim 53, wherein a cell lysate containing one or more of the enzymes is present at a concentration of about 5% (v / v) to about 50% (v / v).

64. The composition according to claim 53, wherein the reaction for cell-free production is carried out over a duration of approximately 0.5 hours to approximately 48 hours.

65. The composition according to claim 53, wherein the reaction for cell-free production is carried out for a duration of about 0.5 hours to about 20 hours.

66. The composition according to claim 53, wherein the temperature of the cell-free medium is about 20°C to about 40°C.

67. The composition according to claim 35, wherein the reaction is carried out in a bubble column reactor and the one or more enzymes are in solution.

68. The composition according to claim 35, wherein the reaction is carried out in a packed-bed reactor and the one or more enzymes are immobilized.

69. An engineered host cell wherein the engineered host cell comprises one or more gene modifications for producing CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK.

70. The engineered host cell according to claim 69, wherein the one or more gene modifications are for enhancing the production of CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK.

71. The manipulated host cell according to claim 69, wherein the manipulated host cell is selected from the group consisting of bacteria, yeast, and / or mammalian cells.

72. The engineered host cell according to claim 69, wherein one or more gene modifications for producing CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK are introduced into the genome of the engineered host cell.

73. The engineered host cell according to claim 69, wherein one or more gene modifications for producing CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK are introduced into a plasmid.

74. The engineered host cell according to claim 69, wherein the produced CGT, SuSy, GLK, HK, PGM, PPK, UGP, and / or NDK are used for the production of carminic acid in a cell-free medium.

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