Bioproduction of anthocyanins in a cell-free system

Abstract

A cell-free system is provided for producing anthocyanin from anthocyanidin. Anthocyanidin is added to a reaction mixture that includes glycosyltransferase enzyme and UDP-sugar. The reaction mixture can be an aqueous solution and can include a co-solvent (e.g., an organic co-solvent, e.g., DMF). It is an object of the present invention to provide a cell-free method that allows the production of anthocyanidin (cyanidin glycoside, e.g., cyanidin-3-glycoside, cyanidin-5-glycoside, etc.) starting from anthocyanidin by a cell-free biosynthesis platform.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention features methods for producing anthocyanins by enzymatic modification of anthocyanidins. In particular, the present invention features cell-free production methods. [Background technology]

[0002] BACKGROUND OF THEINVENTION Anthocyanins (glycosides of anthocyanidins) are the major secondary metabolites found in plants that are responsible for the color variations seen in fruits and flowers. The building blocks of anthocyanins are anthocyanidins, such as one of the six major anthocyanidins cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin, which are glycosylated by uridine-5'-diphosphate (UDP) carbohydrate-dependent glycosyltransferase (UGT) enzymes. The addition of various nucleotide activated sugars (i.e., arabinose, galactose, glucose, xylose, rhamnose) in various combinations of type, number, and position contributes to the diversity of anthocyanins found in nature.

[0003] The industrial use of anthocyanins has focused on their color properties as well as potential health benefits. With the increasing shift from artificial colorants to natural equivalents in food, textile, and cosmetic products, anthocyanins are becoming a popular class of molecules. Furthermore, years of research have led to investigating the antioxidant, anti-inflammatory, anti-cancer, anti-obesity, cardioprotective, and neuroprotective properties of various anthocyanins.

[0004] Producing anthocyanins by cultivation, chemical synthesis, or in cells suffers from many problems that limit the commercial viability of high-value chemical production. First, cultivation is often economically unfeasible, requiring large amounts of land / energy / water, and the plant can only produce small amounts of high-value substances. Second, chemical synthesis requires extensive, elaborate, expensive, toxic, and inefficient multi-step chemical reactions to generate natural products that are often too complex to make in the laboratory. Finally, biofoundry (the use of whole cells) suffers from product toxicity, carbon flux redirection, diffusion problems through cell walls, and the generation of toxic by-products. To circumvent these above problems, cell-free manufacturing emerges as a viable option.

[0005] In cell-free systems, the key components of cells (i.e., cofactors and enzymes) are used in the cell-free chemical reactions. The same enzymes found in plants are produced in vivo (typically through protein overexpression in a host such as bacteria), isolated by chromatography, and then added to a bioreactor containing the substrate (starting material). The enzymes convert the substrate in the same manner as occurs in plants, but without the complexity of an organism. In this manner, natural products can begin to be produced without plants, cells, or chemical synthesis. Summary of the Invention [Means for solving the problem]

[0006] BRIEF SUMMARY OF THEINVENTION It is an object of the present invention to provide a cell-free method that allows the production of anthocyanidins (cyanidin glycosides, such as cyanidin-3-glycoside, cyanidin-5-glycoside, etc.) starting from anthocyanidins by a cell-free biosynthetic platform. The embodiments of the present invention can be freely combined with each other if they are not incompatible.

[0007] The present invention uses glycotransferase enzymes to convert anthocyanidins into several possible anthocyanidin glycosides (eg, cyanidin-3-glycosides), depending on the glycosyl donor.

[0008] The present process uses a controlled enzyme step to increase product titer in a short reaction time. In some embodiments, the controlled enzyme step can increase product titer by at least 5 times. In addition, enzyme immobilization can be used to enhance the conversion of anthocyanidins to corresponding anthocyanins. For example, enzyme immobilization has been used to improve the conversion of cyanidin to cyanidin-3-glucoside (C3G) by avoiding enzyme precipitation, inactivation, and the unreliability of non-immobilized systems.

[0009] The unique and inventive technical feature of the present invention is the use of a cell-free system for the production of anthocyanins (anthocyanidin glycosides) (e.g., cyanidin-3-glycoside). Without wishing to limit the present invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides a higher reaction concentration of starting materials, reagents, and enzymes, resulting in a higher concentration of the end product. Furthermore, the present invention eliminates the complexity of cell walls, thereby eliminating the barrier to diffusion of products and substrates. Furthermore, the present invention eliminates competition for carbon flux, which limits the efficiency of cell-based synthesis methods, thus greatly reducing the formation of by-products. Also, since there are no cells present, the present invention is not vulnerable to degradation due to cell death resulting from the formation of toxic compounds. The suitability of the system to accommodate a variety of starting anthocyanidins and glycosylated UDPs (UDP-sugars) gives the system the flexibility to produce many products and the ability to use a variety of solvents (e.g., organic solvents) to allow for higher concentrations of solutes without worrying about killing cells.

[0010] The present invention is compatible with a variety of products. In this approach, one simply alters the anthocyanidins, UDP-sugars, and UGT enzymes in the pathway to synthesize the desired anthocyanin product.

[0011] Moreover, prior literature teaches away from the present invention. The maximum titers achievable for current cell-based anthocyanin production are limited. Previously published work, involving extensive cell reprogramming and metabolic engineering, produces anthocyanins (e.g., cyanidin-3-glucoside) at a scale of 0-200 mg / L. Although there have been reports of microbial hosts producing titers approaching 300-400 mg / L, these experiments are either technically not reproducible (see, e.g., Shrestha, 2019) or contain data that do not fit the standards set forth in the art (Yan, 2008). The present invention provides C3G production titers of at least 1.5 g / L, which represents up to a 50-fold increase from reported values.

[0012] Moreover, the inventive technical features of the present invention contributed to surprising results that were not predicted from the literature of cell-based synthesis. For example, the stability of the starting material cyanidin depends on the pH of the solution in which it is contained, with the highest stability at pH<5.0. These low pH values ​​are not compatible with cell-based production, since the cell has an optimum pH of 7. The present invention works perfectly well over a range of pH values ​​(4.0-9.0). In some embodiments, to maximize the stability of anthocyanidins, it is preferred to carry out the reaction at or near the pH of maximum stability of the anthocyanidins, for example, at or near pH 5 (e.g., 4.0-6.0, 4.2-5.8, 4.3-5.7, 4.4-5.6, 4.5-5.5, 4.6-5.4, 4.7-5.3, 4.8-5.2, or 4.9-5.1).

[0013] Furthermore, the solubility of the starting anthocyanidin can be enhanced by using a solvent (e.g., organic solvent) that the present invention allows. For example, the solubility of cyanidin is only 49mg / L in water, and cyanidin decomposes within a few minutes at this concentration. Adding a co-solvent (e.g., dimethylformamide (DMF), dimethylsulfoxide (DMSO), methanol (MeOH), or ethanol (EtOH) (or a mixture of two or more of them) greatly improves the solubility and stability of cyanidin for the reaction. Some methods according to the present invention can operate at a co-solvent level of more than 60% (at least 12 times higher than cell-based anthocyanin production, since living cells are sensitive to most co-solvent concentrations above 5%).

[0014] The inventive technical features of the present invention contributed to the further unexpected result of tolerating high concentrations of UDP-sugars in the reaction mixture. For example, the conversion of cyanidin to C3G requires a UDP-conjugated sugar donor molecule (e.g., UDP-glucose). UDP-glucotransferase enzyme has low affinity for UDP-glucose, and the reaction depends on a large molar excess of this molecule. The level of UDP-glucose in the present invention is controllable and can vary from 1 mM to 30 mM (0.5-17 g / L), compared to cell-based production, which requires significant cell engineering to reach UDP-glucose levels approaching 2 g / L (Feng et al., 2020).

[0015] Any feature or combination of features described herein is included within the scope of the present invention, as long as the features included in any such combination are not mutually contradictory, as is clear from the context, the present specification, and the knowledge of a person skilled in the art.For example, although a specific anthocyanin is shown as the product of a specific anthocyanidin, a person skilled in the art will recognize that the method described herein can be generalized to produce a wide range of anthocyanins from a range of anthocyanidins.Further advantages and aspects of the present invention are apparent in the following detailed description and claims.

[0016] (Brief description of some figures of the drawing) The features and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0017] [Figure 1A] Figure 1A shows a general anthocyanidin structure. The structure on the left is the anthocyanidin core structure, and the table on the right shows the "Rn" substituents at the indicated positions of the anthocyanidin core structure.

[0018] [Figure 1B] FIG. 1B shows the chemical conversion pathway that produces anthocyanins (e.g., cyanidin-3-O-glucoside, also known as C3G) from anthocyanidins (e.g., cyanidin) and UDP-sugars (e.g., UDP-glucose).

[0019] [Diagram 2] Figures 2A-C show HPLC traces for cyanidin, cyanidin-3-glycoside standard (top panel, Figure 2A), non-immobilized enzyme reaction (middle panel, Figure 2B), and immobilized enzyme reaction (bottom panel, Figure 2C). Retention times of 6.95 min (C3G) and 8.9 min (cyanidin) are recorded at 530 nm.

[0020] [Diagram 3]Figures 3A-3B show a schematic diagram of the continuous reactor (top panel, Figure 3A) and an HPLC trace of molecular standards and cyanidin-3-glycoside produced in the continuous reactor (bottom panel, Figure 3B). Retention times at 530 nm of 6.95 min (C3G) and 8.9 min (cyanidin) are recorded. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention Before the present compounds, compositions, and / or methods are disclosed and described, it is to be understood that unless expressly stated, the present invention is not limited to specific synthetic methods or specific compositions, as these may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0022] As used herein, "reaction solution" may refer to all components necessary for the enzyme-based chemical conversion of anthocyanidins to anthocyanins, typically, but not limited to, buffers, salts, cosolvents, cofactors, and substrates (starting materials).

[0023] As used herein, a "reaction mixture" may refer to all components from a "reaction solution" plus the enzyme(s) and / or products from the reaction. In some embodiments, the "reaction mixture" may refer to only the reaction solution, without any enzymes or reaction products.

[0024] In some embodiments, the terms "reaction solution" and "reaction mixture" may be used interchangeably.

[0025] As used herein, "buffer" may refer to a chemical added to a water-based solution that resists pH changes through the action of acid-base conjugate components.

[0026] As used herein, "supernatant" may refer to the soluble liquid fraction of a sample.

[0027] As used herein, a "batch reaction" may refer to a chemical or biochemical reaction carried out in a closed system, such as a fermenter or a typical reaction flask.

[0028] As used herein, "cofactor" may refer to a non-protein chemical compound that can bind to a protein and assist in a biochemical reaction. A non-limiting example of a cofactor may include, but is not limited to, UTP.

[0029] 1A-3B, the present invention is illustrated by reference to an exemplary method for producing glycosylated cyanidin from cyanidin.

[0030] The methods shown in Figures 1A-3B can be generalized to incorporate any suitable combination of anthocyanidins, UTP-glycosides, and enzymes to produce the desired product anthocyanin. For example, the anthocyanidin can be an anthocyanidin shown in Table 1.

[0031] [Table 1]

[0032] In some embodiments, the UDP-conjugated sugar can vary, hi some embodiments, the sugar can be glucose, galactose, xylose, arabinose, or rhamnose.

[0033] In some embodiments, the product can be cyanidin-3-glucoside (C3G). In some embodiments, the product can be any product listed in Table 2 or Table 3 below.

[0034] In some embodiments, the temperature of the reaction may range from about 20° C. to about 50° C. In some embodiments, the temperature of the reaction is about 30° C.

[0035] In some embodiments, the pH of the reaction can range from about 4 to about 9.0. In some embodiments, the pH of the reaction is about 5.0 (e.g., 4.0-6.0, 4.2-5.8, 4.3-5.7, 4.4-5.6, 4.5-5.5, 4.6-5.4, 4.7-5.3, 4.8-5.2, or 4.9-5.1).

[0036] The reaction time can be varied to optimize yield or balance yield against efficient use of resources. The reaction time can be varied from 10 minutes to 48 hours, for example, from 15 minutes to about 36 hours, or from about 30 minutes to 24 hours. In some embodiments, the time to carry out the reaction can range from about 30 minutes to about 1 hour.

[0037] In some embodiments, the enzyme may be immobilized. In some embodiments, the immobilized enzyme is immobilized on a solid support. Non-limiting examples of solid supports may include, but are not limited to, epoxy methacrylate, amino C6 methacrylate, or microporous polymethacrylate. In further embodiments, various surface chemistries may be used to bind the immobilized enzyme to the solid surface, including, but not limited to, covalent bonding, adsorption, ionic bonding, affinity, encapsulation, or entrapment. In other embodiments, the enzyme is not immobilized. Either immobilized or non-immobilized enzymes may be used in batch or continuous synthesis. For example, immobilized enzymes on solid supports may be used in a continuous flow cell through which the reaction mixture passes, whereby the immobilized enzyme may catalyze the modification of a substrate to produce a product at high titers. Alternatively, a continuous method may include micromixing an enzyme solution with a substrate to produce a product at high titers while continuously removing the product, removing the substrate, or both.

[0038] The anthocyanins in Table 2 can be prepared from the corresponding anthocyanidins using 3-O-glycotransferase or 5-O-glycotransferase as the glycosylating enzyme.

[0039] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0040] The starting materials and reactants for preparing anthocyanins from anthocyanidins can be obtained from commercial sources or by synthetic processes readily available from available starting materials. For example, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin are commercially available, for example, from ChromaDex, Inc.

[0041] The methods disclosed herein can be used to prepare mono-, di-, tri-, tetra-, penta-, hexa- and poly-substituted anthocyanins. To produce di-, tri-, tetra-, penta-, hexa- and more substituted anthocyanins, multiple synthesis steps can be carried out in the provided cell-free system. The starting material for n-glycosylated anthocyanins can be n-1 glycosylated anthocyanins. For example, to produce cyanidin-3,5-O-diglucoside, the anthocyanidin is first converted to cyanidin-3-O-glucoside (C3G) by 3-glycotransferase (3GT) enzyme, and the C3G molecule is then converted to R3 by 5-glycotransferase (5GT) enzyme. 5 The second glucosyl moiety is added at the 3GT position to produce the final cyanidin-3,5-O-glucoside. The addition of glycosyl groups by 3GT enzymes is generally the first step toward n-glycosylated anthocyanins, since C3G anthocyanins are the most basic anthocyanin building blocks. Subsequent modifications of the C3G molecule with non-glucosyl donors may include, but are not limited to, acylation, malonylation, coumaroylation, caffeoylation, and feruloylation.

[0042] Table 3 shows the anthocyanin products and their potential corresponding anthocyanidins, as well as the glycosyl donor starting materials, along with the predicted enzymes required to carry out the corresponding reactions.

[0043] [Table 3-1] [Table 3-2] [Table 3-3]

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

[0044] (Co-solvent) The reaction mixture and reaction solution may include a co-solvent (i.e., a solvent with water). Various co-solvents may be used in the reaction solution and reaction mixture to improve solubility. In some embodiments, the co-solvent may comprise about 1 to about 75% (v / v) of the reaction solution or reaction mixture (e.g., about 5 to about 50% (v / v) or 10 to about 40% (v / v)). The co-solvent may be, for example, dimethylformamide (DMF), ethanol (EtOH), methanol (MeOH), dimethylsulfoxide (DMSO).

[0045] (Anthocyanin Purity) The anthocyanin product can be produced with excellent purity. For example, the anthocyanin product can be produced with a purity of more than 70%, more than 80%, more than 90%, more than 95%, more than 97%, more than 98%, more than 99%, more than 99.5%, or more than 99.9%. Thus, purities in the range of 70-99.9%, 80-99.9%, 90-99.9%, and 95-99.9% can be achieved. EXAMPLES

[0046] (Example) The following are non-limiting examples of the present invention. It should be understood that the examples are not intended to limit the present invention in any manner. Equivalents or substitutes are within the scope of the present invention.

[0047] (Enzyme expression and purification) All genes were synthesized, cloned into expression plasmids, and then transformed into E. coli for expression. Cells were grown at 37° C. and 200 rpm in TB medium supplemented with 50 μg / mL kanamycin sulfate. 600=0.6. Cells were cooled to 18°C, expression was induced, and grown for a further 18 hours. Cell pellets were collected by centrifugation, frozen, and then resuspended in 5 mL lysis buffer (50 mM sodium phosphate pH 7.5, 300 mM NaCl, 5 mM imidazole) per gram of cell paste. Cell lysates were prepared by sonication, and cell debris was removed by centrifugation. The clarified lysate was loaded onto a GE XK series column containing IMAC-Nickel resin. Proteins were eluted using a 15 CV gradient from Buffer A (50 mM sodium phosphate pH 7.5, 300 mM NaCl, 10% glycerol (w / v)) to 25% Buffer B (1 M imidazole, 50 mM sodium phosphate pH 7.5, 300 mM NaCl, 10% glycerol (w / v)). Fractions containing the protein of interest were pooled and exchanged into 50 mM sodium phosphate pH 7.5, 10% glycerol (w / v), and 0.1 mM EDTA using a GE HiPrep 26 / 10 desalting column, after which the enzyme was concentrated to a value of 5 mg / mL using an Amicon centrifugal filtration unit, mixed with 15% glycerol (w / v), and flash frozen.

[0048] Those skilled in the art will understand that E. coli cells are exemplary to demonstrate the concept of the present invention. Other suitable cells, including bacteria (e.g., bacillus subtillis) or fungi (e.g., trichoderma or aspergillus terrus), or any other host cells suitable for expressing these genes, are within the scope of the present invention.

[0049] (Analysis method) For sampling, the reaction was acidified with 2M HCl (1:10 v / v) followed by high speed centrifugation and filtration through a 0.45 μm filter. Samples were run on an HPLC system to determine the amount of cyanidin and glycosylated cyanidin present in the reaction mixture. The HPLC method was as follows: an Agilent 1200 HPLC was fitted with an Ascentis C18 HPLC column 150 mm×4.6 mm, 3 μm. The column was heated to 25° C. and the sample block was maintained at 15° C. For each sample, 10 μL was injected and the product was eluted at a flow rate of 1.0 ml / min using the following gradient of 0.1% phosphoric acid in water (solvent A) and acetonitrile (solvent B): 90% A to 50% A for 6 min, 90% A for 0.1 min, and 90% A for 2.9 min for column equilibration. The total run time was 9 min, with cyanidin-3-glucoside eluting at 3.8 min and cyanidin eluting at 4.6 min. A diode array detector (DAD) was used for detection of the molecules of interest at 530 nm.

[0050] (Optimization of C3G production and reaction using 3GT enzyme) As described herein, cyanidin is glycosylated by 3-O-glycotransferase enzymes (3GT, 2.4.2.51) to form cyanidin-3-glucoside (C3G). This 3GT enzyme family is found in plants and has been shown to be active in microbial hosts, but significant progress is needed to produce industrially relevant quantities of C3G. Enzymes from Table 4 were expressed, purified, and screened for activity to produce C3G from cyanidin. The enzymes were first screened for optimum values ​​for substrate concentration, pH, temperature, buffer, and time. The stability and solubility of the substrate cyanidin was found to be limited. Therefore, further optimization of the type and amount of co-solvent in these reactions was also required. The reaction solution (100 mM sodium acetate, pH 5.0, 20 mM UDP-glucose, 5 mM cyanidin, 5 mM MgCl2, 20% DMF (v / v)) was mixed with 20 μM enzyme for 30 minutes at 30°C. The yield increased to 1.6 g / L.

[0051] (Immobilized 3-O-glycotransferase for the production of cyanidin-3-glucoside) After demonstrating and optimizing the production of C3G from cyanidin using free enzyme, the next step was to immobilize the 3GT enzyme on a solid support to increase stability, longevity, and catalysis. Various commercially available support materials were screened for product and substrate retention, enzyme retention, and activity of the immobilized enzyme. The support assembly included various surface chemistries for the following types of binding: covalent, adsorption, ionic, affinity, encapsulation, and entrapment. Typically, 50 mg of resin was mixed with 4.0 mg of enzyme in buffer at room temperature for 16-24 hours. The amount of immobilized enzyme was quantified by measuring the protein concentration in the solution before and after immobilization by either BCA or Bradford assay. The amount of starting material and product retained on the resin was quantified by HPLC.

[0052] After initial resin screening, the best enzyme-support combination was selected for further optimization. The immobilized enzyme was again subjected to different reaction conditions (changes in substrate concentration, changes in pH, changes in temperature, changes in buffer, changes in solvent, changes in time) to ensure optimal activity.

[0053] 3GT was mixed with epoxy methacrylate resin. The enzyme-immobilized resin was then incubated with 10 mg / ml polyethyleneimine (PEI) for at least 1 h. The immobilized enzyme was used to convert cyanidin to C3G. The reaction solution (100 mM sodium acetate, pH 5.0, 15 mM UDP-glucose, 4 mM cyanidin, 40% DMF (v / v)) was mixed with 2.5 mg of immobilized enzyme at 30 °C for 30 min. The immobilized 3GT from Vitis labrusca was able to convert 2.9 mM cyanidin to a yield of 2.1 mM C3G (51.4%, 0.924 g / L) (Figure 2C).

[0054] (The use of fixed 3GT produces C3G in a continuous reactor) A 2.75 inch long and 0.125 inch outer diameter and 0.055 inch inner diameter reactor containing immobilized Vl3GT enzyme (2.7 mg enzyme on 50 mg resin) was heated to 30° C. and equilibrated with equilibration buffer (50 mM sodium acetate, pH 5.0) for 30 minutes by pumping the equilibration buffer through the reactor. After this time, substrate solution was run through the reactor at a flow rate of 2.3 μL / min. Prior to entering the reactor, the substrate solution was generated by mixing 400 μL of 4.0 mM cyanidin at a flow rate of 0.93 μL / min with 600 μL of 15 mM UDP-glucose, 100 mM sodium acetate, pH 5.0 at a flow rate of 1.38 μL / min, resulting in a total flow rate of 2.3 μL / min (30 min retention time) through the reactor. After the solution was run through the reactor, the liquid was collected and sampled by HPLC for the presence of cyanidin and C3G formation. The immobilized Vl3GT enzyme produced 0.795 g / L of C3G in this continuous reactor.

[0055] As used herein, the term "about" refers to the referenced number ±10%.

[0056] [Table 4-1] [Table 4-2]

[0057] Although some preferred embodiments of the present invention have been shown and described, it is readily apparent to one skilled in the art that modifications thereto may be made without departing from the scope of the appended claims. Thus, the scope of the present invention should be limited only by the appended claims. The drawings are merely illustrative, and the claims are not limited by the dimensions of the drawings. In some embodiments, the description of the invention described herein using the phrase "comprising" includes embodiments that could be described as "consisting essentially of" or "consisting of", and thus the description requirement for claiming one or more embodiments of the invention using the phrase "consisting essentially of" or "consisting of" is satisfied.

[0058] (Bibliography) Feng et al., “Advances in engineering UDP-sugar supply for recombinant biosynthesis of glycosides in microbes,” Biotechnology Advances, https: / / doi.org / 10.1016 / j.biotechadv. 2020.107538, (2020).

[0059] Shresthaら、「Combinatorial approach for improved cyanidin 3-O-glucoside production in Escherichia coli」、Microb.Cell Fact. 18(7)(2019)。

[0060] Yanら、「High-Yield Anthocyanin Biosynthesis in Engineered Escherichia coli、Biotech.and Bioeng. 100(1)(2008)。

Claims

1. 1. A method for producing anthocyanins from anthocyanidins, the method comprising: a) adding to a mixture containing a suitable solvent, (i) Formula I: 【Chemistry 1】 In the formula I, R ３ , R ５ , R ６ , R ７ , R ３’ , R ４’ , and R ５’ are H, OH, and OCH ３ R is independently selected from the group consisting of ３ , R ５ , R ６ , R ７ , R ３’ , R ４’ , and R ５’ At least one of is OH or CH ３ and R ３ , R ５ , R ６ , R ７ , R ３’ , R ４’ , and R ５’ at least one of which is OH; and (ii) Formula II: 【Chemistry 2】 wherein in said formula II, G is a glycosyl residue selected from the group consisting of five-membered ring monosaccharides and six-membered ring monosaccharides; (iii) a glycosyltransferase enzyme; to form a reaction mixture; b) removing the supernatant from the reaction mixture from (a); and c) isolating the anthocyanins A method comprising:

2. 1. A method for producing anthocyanins from anthocyanidins, the method comprising: a) providing a glycosyltransferase enzyme in a cell-free container; b) adding an anthocyanidin and glycosylated uridine diphosphate to the cell-free vessel to form the anthocyanin; and c) removing the anthocyanin from the cell-free container. A method comprising:

3. 1. A method for producing anthocyanins from anthocyanidins, the method comprising: a) a compound of formula I: 【Chemistry 1】 In the anthocyanidin or anthocyanin of formula I, R ３ , R ５ , R ６ , R ７ , R ３’ , R ４’ , and R ５’ Each of ３ or a sugar; R ３ , R ５ , R ６ , R ７ , R ３’ , R ４’ and at least one R5' is OH; Formula II: 【Chemistry 4】 wherein in said formula II, G is a glycosyl residue selected from the group consisting of five-membered ring monosaccharides and six-membered ring monosaccharides; Glycosyltransferase enzymes and to form a reaction mixture; b) removing the supernatant from the reaction mixture from (a); and c) isolating the anthocyanins A method comprising:

4. 2. The method of claim 1, wherein the anthocyanidin is selected from the group consisting of cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, glycosylated cyanidin, glycosylated delphinidin, glycosylated malvidin, glycosylated pelargonidin, and glycosylated peonidin.

5. The method of claim 1, wherein the anthocyanidin is selected from the group consisting of cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin.

6. The method of claim 1, wherein G is selected from the group consisting of glucose, galactose, xylose, arabinose, and rhamnose.

7. The method of either claim 2 or claim 3, wherein the anthocyanidin is selected from the group consisting of cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, glycosylated cyanidin, glycosylated delphinidin, glycosylated malvidin, glycosylated pelargonidin, and glycosylated peonidin.

8. The anthocyanidin is selected from the group consisting of monoglycosylated cyanidin, diglycosylated cyanidin, triglycosylated cyanidin, tetraglycosylated cyanidin or pentaglycosylated cyanidin, monoglycosylated delphinidin, diglycosylated delphinidin, triglycosylated delphinidin, tetraglycosylated delphinidin or pentaglycosylated delphinidin, monoglycosylated malvidin, diglycosylated malvidin, triglycosylated malvidin 8. The method of claim 7, wherein the glycosylated malvidin is selected from the group consisting of tetraglycosylated malvidin, pentaglycosylated malvidin, monoglycosylated pelargonidin, diglycosylated pelargonidin, triglycosylated pelargonidin, tetraglycosylated pelargonidin or pentaglycosylated pelargonidin, and monoglycosylated peonidin, diglycosylated peonidin, triglycosylated peonidin, tetraglycosylated peonidin or pentaglycosylated peonidin.

9. 2. The method of claim 1, wherein the glycosylated uridine diphosphate has an initial concentration of about 1 mM to about 30 mM in the reaction mixture.

10. 2. The method of claim 1, wherein the glycosyltransferase enzyme is a 3-O-glycosyltransferase or a 5-O-glycosyltransferase.

11. 2. The method of claim 1, wherein the glycosyltransferase enzyme is a member of the group consisting of 3-O-glucoside-6"-O-rhamnosyltransferase (3RT), 3-O-glucoside-2"-O-glucotransferase (3GGT), 3'-O-glycosyltransferase, or 7-O-glycosyltransferase.

12. 2. The method of claim 1, wherein the sequence of the glycosyltransferase enzyme is one of SEQ ID NOs:1-5 or an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 97.5%, at least 99%, at least 99.5%, or at least 99.9% similarity to SEQ ID NOs:1-5.

13. 2. The method of claim 1, wherein the reaction mixture has an initial pH, the initial pH being from about 4 to about 6.

14. The method of claim 1 , wherein the glycosyltransferase enzyme is not immobilized.

15. The method of claim 1 , wherein the glycosyltransferase enzyme is immobilized.

16. 10. The method of claim 1, wherein the glycosyltransferase enzyme is contained in a continuous reactor system.

17. R ３ 2. The method of claim 1, wherein is OH and the glycosyltransferase enzyme is 3-O-glycotransferase (3GT).

18. R ５ 2. The method of claim 1, wherein is OH and the glycosyltransferase enzyme is 5-O-glycotransferase (5GT).

19. 2. The method of claim 1, wherein the glycosylated uridine-5'-diphosphate is selected from the group consisting of UDP-arabinose, UDP-galactose, UDP-glucose, UDP-rhamnose, and UDP-xylose.

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