Novel chalcone dyes and compositions and methods for their preparation

Abstract

To provide a chalcone dye compound that can be produced from living cells of Escherichia coli and has high industrial utility, and means and methods for the production of the same.SOLUTION: The present invention provides a composition for producing a chalcone dye compound which is a glycoside of isoliquiritigenin with a methylated hydroxy group at 2' position, the composition comprising cells of Escherichia coli expressing 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), isoliquiritigenin 2'-O-methyltransferase (I2'MT), and UDP glycosyltransferase (UGT). There are also provided a chalcone dye compound and a method for producing the same.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates to chalcone compounds and compositions and methods for their production. [Background technology]

[0002] Flavonoid compounds are known to have various useful physiological activities and are also industrially useful as pigments or their precursors. Naringenin chalcone and isoliquiritigenin are chalcones that are precursors of the flavonoids (more specifically, flavanones) naringenin and liquiritigenin, respectively. Chalcones are classified as a type of flavonoid, in which the central C ring of the tricyclic flavonoid skeleton is open and linear. Flavonoids, including flavanones and chalcones, are synthesized by plants in nature.

[0003] Generally, chalcones are pigments found in flowers such as safflowers, carnations, and peonies, contributing to their yellow to orange color. Naringenin chalcone and isoliquiritigenin are also yellow pigments. These chalcones are also characterized by relatively high hydrophobicity. Naringenin chalcone and isoliquiritigenin spontaneously isomerize to naringenin and liquiritigenin, respectively. The flavanones naringenin and liquiritigenin are industrially useful compounds, either as themselves or as precursors to other substances. However, they are colorless and cannot be used as pigments themselves. In other words, the spontaneous conversion of naringenin chalcone and isoliquiritigenin to naringenin and liquiritigenin, respectively, indicates discoloration.

[0004] In Patent Document 1, the present inventors have disclosed the production of flavanones such as naringenin through the synthesis of naringenin chalcone and the like by bioconversion using live Escherichia coli. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-3034 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present disclosure is to provide a dye compound that can be produced using living Escherichia coli cells and has high industrial usefulness, as well as a production means and method therefor. [Means for solving the problem]

[0007] The present inventors have discovered that a novel chalcone dye that is both resistant to fading and hydrophilic can be produced in vivo using living Escherichia coli cells, and have thereby completed the present invention. The present disclosure includes the following embodiments. [1] A composition for producing a chalcone dye compound which is a glycoside of isoliquiritigenin in which the hydroxyl group at the 2'-position is methylated, containing Escherichia coli cells expressing 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), isoliquiritigenin 2'-O-methyltransferase (I2'MT), and UDP glycosyltransferase (UGT). composition. [2] The composition described in [1], wherein the E. coli cells further express an EFP protein. [3] The composition according to [1] or [2], wherein the E. coli cells further express tyrosine ammonia lyase (TAL). [4] The composition according to any one of [1] to [3], wherein the UGT has (iso)flavone 7-O-glucosyltransferase activity. [5] A method for producing a chalcone dye, comprising adding p-coumaric acid to the composition according to any one of [1] to [4]. [6] A method for producing a chalcone dye, comprising adding tyrosine to the composition according to [3]. [7] A chalcone pigment compound that is a glycoside of isoliquiritigenin with the 2'-hydroxyl group methylated. [8] Escherichia coli cells expressing 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), isoliquiritigenin 2'-O-methyltransferase (I2'MT), and UDP glycosyltransferase (UGT). [9] [8] The Escherichia coli cell according to [8], further expressing a tyrosine ammonia lyase (TAL) and / or an EFP protein. [Effects of the Invention]

[0008] The dye compounds provided by the embodiments of the present disclosure overcome the drawbacks of, for example, fading and hydrophobicity observed in naringenin chalcone dyes, and have improved industrial usefulness. Because the embodiments of the present disclosure can produce this dye by an in vivo reaction using Escherichia coli, they can be characterized by sustainability, simplicity, low cost, and the like. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows LC-MS data showing the production and accumulation of 2′-O-methylated isoliquiritigenin (m / z 269) in transformed E. coli. [Figure 2] FIG. 2 shows LC-MS data showing the production and accumulation of the target compound, 2′-O-methylated isoliquiritigenin glycoside (m / z 431), in transformed E. coli. [Figure 3] Figure 3 shows LC-MS analysis data from an experiment examining the UV-visible light absorption of culture extracts obtained using transformed E. coli that produced the target compound, 2'-O-methylated isoliquiritigenin glycoside (MIL-G in peak P1), and other by-products in vivo. DETAILED DESCRIPTION OF THE INVENTION

[0010] In one aspect, the present disclosure provides a composition for producing a chalcone dye compound that is a glycoside of isoliquiritigenin in which the hydroxyl group at the 2'-position is methylated. In another aspect, the present disclosure provides a chalcone dye compound that is a glycoside of isoliquiritigenin in which the hydroxyl group at the 2'-position is methylated, which can be produced using such a composition.

[0011] The structural formula of isoliquiritigenin is shown below. [ka]

[0012] A specific example of isoliquiritigenin in which the 2'-hydroxyl group is methylated and the 4'-hydroxyl group is glycosylated can be represented by the following structural formula: In this formula, Glc represents glucose. [ka]

[0013] That is, "methylation of the 2'-hydroxyl group" of isoliquiritigenin means that the hydrogen atom of the 2'-hydroxyl group of isoliquiritigenin is replaced with a methyl group. This can also be described as isoliquiritigenin being 2'-O-methylated. It can also be described as isoliquiritigenin being methoxylated at the 2'-position. "Glycosidation of the 4'-hydroxyl group" means that the hydrogen atom of the 4'-hydroxyl group is replaced with a sugar (typically a monosaccharide, such as a glucose ring). This can also be described as a sugar being glycosidically linked to the 4'-hydroxyl group. In other words, the anomeric carbon of a sugar, such as glucose, is O-glycosidically linked to the 4'-position of isoliquiritigenin. It can also be described as isoliquiritigenin being glycosidized at the 4'-position. Typically, the main product is 2'-O-methylated isoliquiritigenin glycosylated at the 4' position, as shown in the structural formula above, but the possibility that it may also contain 2'-O-methylated isoliquiritigenin glycosylated at the 4-position of the other benzene ring (B ring) is not excluded.

[0014] The structural formula of liquiritigenin is shown below. From the left, the three rings correspond to the A, C, and B rings of the flavonoid skeleton. [ka]

[0015] Isoliquiritigenin is a yellow pigment, but spontaneously isomerizes to liquiritigenin, and liquiritigenin is colorless.However, by methoxylating the 2'-position of isoliquiritigenin, cyclization to liquiritigenin is prevented, and the color development as a pigment can be stabilized.The compound of this embodiment is yellowish in itself, but can also be used as a starting material for derivatization into further pigment compounds.

[0016] Isoliquiritigenin also has the disadvantage of being highly hydrophobic and poorly soluble in water, which is one of the reasons that makes its industrial application as a dye difficult. Methylation of the hydroxyl group is thought to further increase its hydrophobicity. However, the chalcone dye of this embodiment has sufficiently high water solubility due to the presence of sugars in glycosidation, and this disadvantage can be overcome.

[0017] The composition of this embodiment includes Escherichia coli cells expressing 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), isoliquiritigenin 2'-O-methyltransferase (I2'MT), and UDP glycosyltransferase (UGT). The expression of these enzymes (and additional enzymes or proteins described below) by Escherichia coli cells means that the genes encoding these enzymes or proteins have been exogenously introduced into Escherichia coli, i.e., genetically engineered, and transcription and translation of the gene DNA occurs, resulting in the production of functional polypeptides as gene products. The present inventors have discovered that by expressing these multiple exogenous gene products, designed biosynthetic steps, from the synthesis of chalcone precursors to the methylation and glycosidation of chalcone, can be effectively linked in Escherichia coli.

[0018] 4CL has 4-coumarate-CoA ligase activity, ATP + 4-coumarate + CoA → AMP + diphosphate + 4-coumaroyl-CoA 4CLs are a known family of enzymes that catalyze the reaction: 4CL3 (SEQ ID NO: 1). Various plant 4CLs, such as 4CLs derived from plant 4CL3 or 4CL4 isoforms, can be used in this embodiment. As an example, a 4CL having 90% or more sequence identity to the 4CL3 amino acid sequence of SEQ ID NO: 1 derived from soybean (Glycine max) is preferred. This sequence identity may be 95% or more, 98% or more, 99% or more, or 100%. In another example, a 4CL having 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to any of the 4CL amino acid sequences of SEQ ID NOs: 2 to 4 derived from snapdragon (Antirrhinum majus) can be used.

[0019] CHS is a known family of enzymes that has chalcone synthesis activity and can synthesize one molecule of naringenin chalcone from one molecule of 4-coumarate-CoA and three molecules of malonyl-CoA. CHSs derived from various plants can be used in this embodiment. As an example, a chalcone synthase having 90% or more sequence identity to the CHS1 amino acid sequence of SEQ ID NO: 5 derived from soybean is preferred. As another example, a chalcone synthase having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 6 derived from Antirrhinum majus is preferred. The sequence identity may be 95% or more, 98% or more, 99% or more, or 100%.

[0020] The E. coli cell may further express an EFP protein. The EFP (Enhancer of Flavonoid Production) gene is known to be involved in flavonoid production and flower color development in plants. The EFP gene encodes a chalcone isomerase (CHI)-like protein, but it lacks several amino acid residues involved in CHI enzyme activity and is considered to be a non-enzyme protein. Patent Document 1 showed that the presence of EFP can suppress by-product synthesis when producing flavanones via chalcones in E. coli. The addition of EFP can have the effect of increasing the selectivity and / or yield of the production of the 2'-O-methylated isoliquiritigenin glycoside of this embodiment. EFPs derived from various plants can be used in this embodiment. As an example, an EFP derived from soybean having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 7 is preferred. As another example, an EFP derived from snapdragon having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 8 is preferred. In yet another example, EFPs having 90% or more sequence identity to any of the amino acid sequences of SEQ ID NOS: 9 to 13 derived from various other plants can be used, and the sequence identity may be 95% or more, 98% or more, 99% or more, or 100%.

[0021] Patent Document 1 describes that for the production of naringenin in E. coli, the selection of the combination of plant species from which 4CL, CHS, and EFP are derived can be important in terms of naringenin production efficiency. However, in embodiments of the present disclosure, 2'-O-methylated isoliquiritigenin glycoside can also be produced using combinations of plant origin different from those described in Patent Document 1. Enzymes derived from different plant species may exhibit slightly different levels of activity in E. coli, and naringenin synthesis in Patent Document 1 appeared to be particularly sensitive to the activity ratio of 4CL to CHS (which is also influenced by EFP). However, it is possible that some positive feedback mechanism acts during the branching to the isoliquiritigenin synthesis route and the subsequent progression of methylation and glycosidation, reducing sensitivity to the enzyme activity ratio.

[0022] The CHR has chalcone reductase activity, and in its presence, the CHS can synthesize isoliquiritigenin instead of naringenin chalcone (although this does not necessarily mean that the production of naringenin chalcone is zero). In other words, the 6'-hydroxyl group present in naringenin chalcone is reduced. The CHR family is known. CHRs derived from various plants (e.g., legumes) can be used in this embodiment. As an example, a chalcone reductase derived from soybean having 90% or more sequence identity to the CHR1 amino acid sequence of SEQ ID NO: 14 is suitable. In another example, a chalcone reductase having 90% or more sequence identity to the CHR5 amino acid sequence of SEQ ID NO: 15 can be used. The sequence identity may be 95% or more, 98% or more, 99% or more, or 100%.

[0023] I2'MT has isoliquiritigenin 2'-O-methyltransferase activity and is a methyltransferase that binds to isoliquiritigenin in plants. S-Adenosyl-L-methionine + isoliquiritigenin → S-Adenosyl-L-homocysteine ​​+ 2'-O-methylisoliquiritigenin I2'MT is a known family of enzymes that can catalyze the reaction: I2'MT from various plants (e.g., legumes) can be used in this embodiment. As a non-limiting example, I2'MT having 90% or more sequence identity to the I2'MT amino acid sequence of SEQ ID NO: 16 from alfalfa (Medicago sativa) is suitable. This sequence identity may be 95% or more, 98% or more, 99% or more, or 100%.

[0024] UGTs are a known family of enzymes that have UDP glycosyltransferase activity and can transfer the sugar moiety of a UDP (uridine diphosphate)-based sugar nucleotide, such as UDP-glucose or UDP-galactose, to the hydroxyl group (oxygen atom) of a sugar acceptor. UGTs derived from various plants (e.g., legumes) can be used in this embodiment. As a non-limiting example, a UDP glycosyltransferase having 90% or more sequence identity to the UGT1 amino acid sequence of SEQ ID NO: 17 derived from soybean is suitable. This sequence identity may be 95% or more, 98% or more, 99% or more, or 100%.

[0025] Unexpectedly, chalcone 4'-glucosyltransferase (C4'GT), an enzyme known to naturally glycosylate chalcone (at the 4' position) in plant cells, failed to efficiently synthesize 2'-O-methylated isoliquiritigenin glycoside in E. coli cells, whereas UGTs were successfully used. In nature, UGTs typically possess isoflavone 7-O-glucosyltransferase activity. The 7-position of isoflavone corresponds to the 4'-position of chalcone. UGT1 was originally identified as an isoflavone 7-O-glucosyltransferase and named IF7GT. However, it was later discovered that flavones (and even flavanones and flavanols) could also be used as glycosyl acceptors, leading to the name UGT1 (Funaki et al., Plant Cell Physiol. 56(8): 1512-1520 (2015)). UGTs that can accept both isoflavones and flavones as glycosyl acceptors can also be called (iso)flavone 7-O-glucosyltransferases. In any case, the fact that isoflavone 7-O-glucosyltransferase can act favorably on chalcones and even 2'-O-methylated chalcones in E. coli cells was an unexpected discovery.

[0026] Preferably, all of the exogenous enzymes and proteins described above are derived from plants. Other genes may be introduced into the E. coli of this embodiment. For example, a plasmid vector typically carries an antibiotic resistance gene. The E. coli cells may also express a tyrosine ammonia-lyase enzyme (TAL). TALs are a known family of enzymes that convert L-tyrosine to p-coumaric acid (producing ammonia as a by-product). Introducing these enzymes into E. coli along with the other enzymes described above enables efficient and inexpensive pigment synthesis using the natural amino acid tyrosine as a starting substrate. For example, the TAL may be derived from bacteria, such as, but not limited to, Herpetosiphon aurantiacus. In one embodiment, the TAL is a tyrosine ammonia-lyase with 90% or more sequence identity to the amino acid sequence of TAL1 from Herpetosiphon aurantiacus, as set forth in SEQ ID NO: 19. This sequence identity may be 95% or more, 98% or more, 99% or more, or 100%.

[0027] As commonly understood by those skilled in the art, sequence identity is a percentage indicating the number of pairs of perfectly matched amino acid residues found among the total number of pairs when a query sequence (e.g., an amino acid sequence provided herein with a SEQ ID NO) and a target sequence are aligned over the entire length of the query sequence to form pairs of positionally corresponding amino acid residues (this is generally called alignment). When an insertion or deletion of an amino acid residue occurs, this may also include pairs of amino acids in the query sequence with blanks in the target sequence (or vice versa). Sequence identity can be easily calculated using software known to those skilled in the art, such as BLAST.

[0028] Those skilled in the art can appropriately select a promoter suitable for an E. coli expression system. Examples include, but are not limited to, the T7 promoter, SP6 promoter, T3 promoter, PBAD promoter, tac promoter, and cspA promoter. The T7 promoter is particularly preferred. While it is possible to create an operon in which multiple genes are transcribed from a single promoter, it is preferable to have each exogenous gene have its own promoter. The promoters possessed by each exogenous gene may be the same type of promoter (e.g., the T7 promoter). The promoter for inducing transcription is not necessarily a constitutive promoter, but may also be an inducible promoter. Therefore, "E. coli expressing" a certain gene product should be understood to mean E. coli that has the ability to express that gene product, not necessarily one that constantly expresses that gene product.

[0029] Means known to those skilled in the art to support the expression of heterologous genes in E. coli may be used as appropriate. Such means may include, for example, one or more of the following: inclusion of a ribosome binding site (RBS) sequence, codon optimization of the coding sequence for E. coli, and use of a host strain with an additional tRNA gene. Various techniques for introducing foreign genes accompanied by promoters into E. coli cells are known to those skilled in the art and can be used to generate the E. coli of this embodiment. These genes are preferably introduced into E. coli cells contained in one or more plasmid vectors. One or more of these heterologous genes may also be integrated into the E. coli genome chromosome to create a stable expression strain.

[0030] The polypeptides of the present disclosure may consist solely of the amino acid sequence shown, or may have a peptide tag attached to the N-terminus and / or C-terminus. Peptide tags are well known to those skilled in the art, and examples include, but are not limited to, His tags (HHHHHH), HA tags (YPYDVPDYA), FLAG tags (DYKDDDDK), Myc tags (EQKLISEEDL), and S tags (KETAAAKFERQHMDS). The length of a single peptide tag is preferably 25 amino acids or less, more preferably 20 amino acids or less, even more preferably 15 amino acids or less, and particularly preferably 10 amino acids or less. As known to those skilled in the art, these peptide tags are typically added to facilitate polypeptide purification and detection (although there may be cases where such specific uses are not intended for the peptide tag) and do not impair the function of the polypeptide. Embodiments in which an enzyme is expressed as a fusion protein with a larger polypeptide are also contemplated, as long as enzymatic activity is not impaired.

[0031] Two or more genes encoding exogenous polypeptides may be contained together in the same plasmid molecule. This means that multiple genes are inserted together into a single plasmid vector and then introduced into E. coli cells. For example, any TAL gene, 4CL gene, CHS gene, and any EFP gene may be inserted into a single plasmid vector. Alternatively, any CHR gene, I2'MT gene, and UGT gene may be inserted into a single plasmid vector. It should be understood that these are merely examples and that the forms of practicable plasmids are not limited to these.

[0032] In addition to the viable E. coli cells, the composition of this embodiment preferably contains at least water, and more preferably further contains medium components to ensure the survival of E. coli over a sustained period of time. Numerous medium components that enable E. coli to survive or grow are known to those skilled in the art and can be included in the composition of this embodiment. Such medium components typically include at least inorganic salts and a carbon source. As an example, the composition may at least contain the composition of M9 medium. M9 medium contains 48 mM NaHPO, 2 mM KHPO, 19 mM NHCl, 8.6 mM NaCl, 0.4% (w / v) glucose, 1 mM MgSO, and 100 μM CaCl. The phrase "may at least contain" also encompasses cases where any of these components are present at a higher concentration. From the perspective of increasing product purity, using glucose as the sole carbon source may be advantageous. The composition may also contain antibiotics to promote the selective growth of cells that have incorporated a plasmid vector. Examples of antibiotics include, but are not limited to, ampicillin, kanamycin, streptomycin, and chloramphenicol. Selection of an appropriate antibiotic depending on the type of plasmid vector, etc., is within the ordinary skill of those skilled in the art.

[0033] In summary, the E. coli cells contained in the composition express 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), isoliquiritigenin 2'-O-methyltransferase (I2'MT), and UDP glycosyltransferase (UGT), and optionally, an EFP protein and / or tyrosine ammonia lyase (TAL). Suitable polypeptides for use in these compositions have at least 90% sequence identity to the amino acid sequences of the polypeptides specifically provided in the present disclosure, but are not limited to these. This sequence identity may be at least 95%, at least 98%, at least 99%, or even 100%. In another aspect, the present disclosure provides E. coli cells of the various embodiments described above.

[0034] In another aspect, the present disclosure provides a method for producing a chalcone dye, comprising adding p-coumaric acid to the above-described composition or E. coli cells. The 4CL enzyme activity converts p-coumaric acid to p-coumaroyl-CoA, and the CHS and CHR enzyme activities cooperate to produce isoliquiritigenin using one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA. The I2'MT enzyme activity then 2'-O-methylates isoliquiritigenin, and the UGT activity carries out glycosidation. The resulting product is 2'-O-methylated isoliquiritigenin glycoside.

[0035] Adding p-coumaric acid to the composition or E. coli cells of this embodiment essentially means incorporating p-coumaric acid into the culture medium. The concentration of p-coumaric acid after addition to the composition can be, for example, 0.1 to 10 mM, more preferably 0.2 to 5 mM, even more preferably 0.3 to 2 mM, and particularly preferably 0.5 to 1.5 mM. It is understood that a specific embodiment of the composition for producing a chalcone dye described above also includes a composition for producing a chalcone dye containing the E. coli cells of the above embodiment and p-coumaric acid. This composition can have the substrate concentration described above. After adding p-coumaric acid, shaking, aeration, temperature control, and the like can be performed during the period required for bioconversion, as in conventional E. coli culture. The temperature can be, for example, 15 to 42°C, preferably 18 to 38°C, more preferably 20 to 37°C, and even more preferably 25 to 32°C. The temperature can also be varied over time within these ranges. Those skilled in the art can appropriately select temperature conditions that allow E. coli to survive continuously.

[0036] After the addition of p-coumaric acid, a stable chalcone dye accumulates in the composition, for example, by allowing 3 hours or more, 10 hours or more, preferably 24 hours or more, and more preferably 30 hours or more to elapse. During this bioconversion period, medium components and / or substrates may be replenished as needed. Bioconversion can be continued virtually indefinitely by appropriately replenishing fresh medium components and substrates and subculturing, so there is no particular upper limit to the bioconversion time. Bioconversion may be continued for up to 2 days, 5 days, 10 days, or even 1 month. Typically, most of the glycosidated chalcone dye is recovered extracellularly, i.e., in the supernatant after centrifugation; however, glycosidated chalcone dyes may also be present intracellularly, i.e., in the solid fraction.

[0037] When using E. coli expressing TAL, chalcone pigments can be produced by adding L-tyrosine to the composition or E. coli cells instead of or in addition to p-coumaric acid. Adding L-tyrosine to the composition or E. coli cells of this embodiment essentially means including L-tyrosine in the culture medium. The concentration of L-tyrosine after addition to the composition can be, for example, 0.1 to 5 mM, preferably 0.2 to 2 mM, and more preferably 0.3 to 1 mM. It is understood that a specific embodiment of the above-described composition for producing chalcone pigments includes a composition for producing chalcone pigments containing E. coli cells of the above-described embodiment and L-tyrosine. This composition can have the substrate concentration described above. In particular, chalcone pigments can also be produced using a culture medium of a normal composition without supplementing L-tyrosine. Suitable culture conditions, such as culture temperature and culture time, are as described above.

[0038] The method for producing a chalcone pigment may further include a step of separating, from the culture supernatant of the above-described Escherichia coli, a chalcone pigment compound, which is a glycoside of isoliquiritigenin in which the hydroxyl group at the 2'-position is methylated. Techniques for separating and purifying hydrophilic flavonoid-related compounds from aqueous solutions are known to those skilled in the art or can be appropriately designed by those skilled in the art, and these techniques can also be applied to the 2'-O-methylated isoliquiritigenin glycoside of this embodiment. [Example]

[0039] The following examples further illustrate the embodiments of the present disclosure. However, these examples are for illustrative purposes only, and the present invention is not limited to these specific examples. In particular, it should be understood that the exogenous enzymes expressed in E. coli may be substituted with enzymes derived from organisms (plant species) other than those described in these examples, or with enzymes of different isoforms. As a clear example of this, the following CHR1 could be substituted with CHR5, which shares only 66% amino acid sequence identity with CHR1.

[0040] [Preparation of E. coli strains] Using conventional genetic engineering techniques, the plasmid vectors shown in the table below for expressing heterologous proteins were constructed, and then transformed into the Rosetta2 (DE3) strain to prepare E. coli strains 27, 28, 29, 38, and 39. Strain 27 was used as a control, transformed only with an empty plasmid vector that did not encode any of the heterologous proteins. [Table 1]

[0041] The gene abbreviations in the table indicate heterologous genes encoding the enzymes or proteins (i.e., polypeptides) shown below. Each heterologous gene is accompanied by a T7 promoter and a T7 terminator. HaTAL: tyrosine ammonia lyase from Herpetosiphon aurantiacus (SEQ ID NO: 19) Gm4CL3: 4-coumarate-CoA ligase 3 from soybean (Glycine max) (SEQ ID NO: 1) GmCHS1: chalcone synthase 1 from soybean (SEQ ID NO: 5) AmEFP: EFP from Antirrhinum majus (SEQ ID NO: 8) GmCHR1: chalcone reductase 1 from soybean (SEQ ID NO: 14) MsI2'MT: isoliquiritigenin 2'-O-methyltransferase from alfalfa (Medicago sativa) (SEQ ID NO: 16) AmC4'GT: chalcone 4' glucosyltransferase from Antirrhinum majus (SEQ ID NO: 18) GmUGT1: UDP glycosyltransferase 1 from soybean (SEQ ID NO: 17)

[0042] [Production of chalcone pigments using transformed E. coli] At least three replica experiments were performed for each of the E. coli strains listed in Table 1. First, these strains were inoculated into 3 mL of LB medium containing the appropriate antibiotic and pre-cultured overnight at 37°C and 120 rpm. Then, 200 μL of the pre-culture solution was added to 20 mL of LB medium containing the appropriate antibiotic and main culture was performed at 37°C and 120 rpm. OD 600 When the β-associated ATP concentration reached 0.4 to 0.5, IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, and the mixture was cultured at 18°C ​​and 180 rpm for 30 hours. At this stage, expression of the polypeptides shown in Table 1 above was induced.

[0043] The mixture was then centrifuged at 15,000 × g at 4 °C for 10 minutes to separate the supernatant and the bacterial cells. 600 μL of methanol:chloroform (2:1 ratio, containing 1 μM apigenin-7-O-glucoside (A7G) as an internal standard for LC-MS analysis) was added to the supernatant fraction to extract the product. 200 μL each of methanol and water were then added and mixed, followed by centrifugation at 15,000 × g at 4 °C for 10 minutes. The product was detected by LC-MS analysis of the supernatant after centrifugation.

[0044] The LC-MS (liquid chromatography / mass spectrometry) analysis conditions are as follows. Control software: Lab Solutions LCMS (SHIMADZU) Column: CAPCELL CORE C18 (UG120 2.1 mm ID x 100 mm, Shiseido) Column temperature: 40℃ Sample application volume: 5 μL Flow rate: 0.2 mL / min Detection (Experiments in Figures 1 and 2): MS (SIM: negative, 229, 255, 269, 271, 285, 417, 431, 433, 447) Detection (Experiment in Figure 3): PDA (190-800 nm) MS (scan: negative 100-600, positive 100-600) Developing solvent: Solution A (water + 0.05% formic acid), Solution B (acetonitrile + 0.05% formic acid) Deployment conditions: [Table 2]

[0045] Figure 1 shows LC-MS data demonstrating the production and accumulation of 2'-O-methylated isoliquiritigenin (m / z 269) by the transformed E. coli strains (*). Other by-products, including naringenin chalcone and methylated naringenin chalcone, were also detected in strains other than the control strain 27 (see Figure 3, for example). As shown in Table 2, the peaks appearing after 31 min in Figure 1 are not considered because they represent the column washing and equilibration steps after the analysis. Figure 2(a) shows LC-MS data demonstrating the production and accumulation of the target compound, i.e., 2'-O-methylated isoliquiritigenin (m / z 431) glycosylated (O-glucosylated) at the 4' position (**). It can be seen that the increased hydrophilicity of the compound leads to a loss of affinity for the hydrophobic column (C18), resulting in a shorter retention time for elution. As illustrated in Figure 2(b), when chalcone 4' glucosyltransferase (C4'GT) was used instead of UGT, no clear peak corresponding to the target chalcone pigment was obtained.

[0046] Figure 3 shows the data obtained by analyzing the solid-phase extract of the culture medium of strain No. 39 by LC-MS combined with a photodiode array detector. Solid-phase extraction was performed using a Sep-Pak cartridge (Waters WAT020515) as follows: (1) Column conditioning: 5 mL of 100% acetonitrile was applied; (2) Column equilibration: 5 mL of Milli-Q water was applied; (3) Culture supernatant application: 10 mL of the supernatant obtained after centrifugation of the culture medium was applied; (4) Column washing: 5 mL of Milli-Q water was applied; (5) Elution: 2 mL of 100% acetonitrile was applied.

[0047] The glycosidized methylated isoliquiritigenin (MIL-G: peak P1, MW 432) absorbs light at 350 nm, similar to the unglycosidized methylated isoliquiritigenin (MIL: peak P2, MW 270). However, naringenin (Nar: peak P3, MW 272) and an unidentified compound (peak P4, MW 405) essentially lack this absorption (Figure 3). Examination of the absorption spectra of the four compounds corresponding to these peaks revealed that glycosidized methylated isoliquiritigenin (P1) had an absorption peak near 350 nm and extended to the 400 nm range, whereas naringenin (P3) absorbed light only in the ultraviolet region (not shown). This observation is consistent with the fact that the former is yellowish, whereas the latter is colorless.

Claims

1. A composition for use in producing a chalcone dye compound from p-coumaric acid or tyrosine, in which the hydroxyl group at the 4'-position of isoliquiritigenin is methylated at the 2'-position, and the hydroxyl group at the 4'-position is glycosylated, comprising: The method includes the step of producing an Escherichia coli cell expressing 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), isoliquiritigenin 2'-O-methyltransferase (I2'MT), and UDP glycosyltransferase (UGT), For the production of the chalcone dye compound from tyrosine, the E. coli cells further express tyrosine ammonia lyase (TAL). composition.

2. The composition of claim 1 , wherein the E. coli cells further express an EFP protein.

3. 3. The composition of claim 1, wherein the UGT has (iso)flavone 7-O-glucosyltransferase activity.

4. A method for producing a chalcone dye, comprising adding p-coumaric acid to the composition according to any one of claims 1 to 3.

5. A method for producing a chalcone dye, comprising adding tyrosine to a composition described in any one of claims 1 to 3, in which the Escherichia coli cells express the TAL.

6. A chalcone pigment compound in which the hydroxyl group at the 4'-position of isoliquiritigenin is methylated at the 2'-position.

7. Escherichia coli cells expressing 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone reductase (CHR), isoliquiritigenin 2'-O-methyltransferase (I2'MT), and UDP glycosyltransferase (UGT).

8. 8. The E. coli cell of claim 7, further expressing a tyrosine ammonia lyase (TAL) and / or an EFP protein.

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