Biosynthesis of indigo dyes that allow for hue adjustment

Abstract

This invention provides a biosynthesis method and composition for producing indigo dyes containing indigotin and indirubin. [Solution] Tryptophanase (TRP) and flavin-containing monooxygenase (FMO) are produced from a genetically modified organism having vectors encoding tryptophanase and flavin-containing monooxygenase. The conversion of L-tryptophan to indigo dye is carried out by the produced TRP and FMO in the presence of one or more bioactive additives selected from cysteine, 2-oxindole, or 2-indoxyl, forming a dye in which the weight ratio of indigotine to indirubin is 1:0.1 to 1:4. The dye has hues defined in the CIE XYZ color space, ranging from X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, and Z: 0.5543 to 0.5501. A selectable and reproducible hue is provided.

Description

【Technical Field】 【0001】 The present invention relates to the biosynthesis of indigo dyes, and more specifically, to the biosynthesis of indigo dyes with adjustable hue. 【0002】 Reference regarding sequence disclosure: 【0003】 The sequence listing file named "P3315US01_Sequence Listing.xml" (ST.26 XML file format, file size 5.51 KB, creation date May 12, 2025, filing date May 26, 2025) is incorporated herein by reference. 【Background Art】 【0004】 The dyeing of fibers is the second leading cause of water pollution worldwide. As the fiber industry increasingly focuses on sustainable development and along with strict government environmental regulations regarding water pollution, the demand for plant-derived dyes as an alternative to synthetic dyes is also increasing. The plant-derived dye market is approximately $5 billion in size and is expected to increase annually due to the growing demand for these dyes. 【0005】 However, conventional plant-derived dyes often exhibit color unevenness and a decrease in color fastness, making it difficult to apply them to fashion clothing. Excellent colorfastness to fading is the most attractive feature for promoting the continuous market growth of fashion clothing products. Furthermore, in some products, the use of toxic chemicals such as amines, sodium sulfide, and caustic substances is still necessary in the dyeing process. 【0006】 Among plant-derived dyes, the blue dye extracted from plants has an extraction rate of 1% or less and an indigotin content of 20% or less. The blue dye extracted from the leaves of Polygonum tinctorium can dye denim in a beautiful color. However, it takes at least one year to harvest the leaves. 【0007】 Synthetic indigo dyes can also be produced through chemical synthesis from petrochemical raw materials. The production of synthetic indigo dyes typically begins with aniline, a benzene derivative derived from petroleum. Because aniline is a highly reactive compound, it serves as an ideal precursor in the synthetic indigo pathway. Aniline undergoes several chemical reactions, particularly chlorination, to produce intermediates such as N-phenylglycine. These intermediates play a crucial role in the formation of the indigo molecule. Next, N-phenylglycine is subjected to an alkali fusion process, facilitating its conversion to indoxyl, a precursor of indigotine. Aniline is also a precursor of isatin, and its oxidative coupling leads to the formation of indirubin. 【0008】 Indigotin is formed by oxidative coupling: Indoxyl molecules typically undergo a final oxidation reaction in the presence of oxygen to form indigotin (the blue dye component of indigo). This oxidation causes two indoxyl molecules to bond, creating the characteristic blue color. 【0009】 However, the production of synthetic indigo involves several toxic and hazardous intermediates and by-products, such as formaldehyde, cyanide compounds, and aniline derivatives, which must be carefully managed to minimize environmental impact. Furthermore, wastewater from synthetic indigo production may contain heavy metals and chlorine compounds, requiring thorough treatment to meet environmental safety standards. Recent advances in green chemistry aim to mitigate the toxicity of indigo production by developing alternative routes that avoid some of the more hazardous intermediates, but these methods have not yet come close to replacing conventional processes on a large scale, mainly due to cost and the established infrastructure of classical synthetic indigo production. 【0010】 Therefore, in this technical field, there is a need for dyes that have a consistent color tone and good colorfastness when applied to fabrics, and that have little environmental impact. Furthermore, it is necessary to select specific hues based on the dye production process. This invention addresses this need. 【0011】 The present invention further provides a technique for enhancing the production of indirubin. Indirubin is a bisindole-based antitumor drug that has inhibitory effects on various tumors in transplant animals, including the destruction of leukemia cells, and is therefore an important substance with distinct applications. [Overview of the Initiative] 【0012】 This invention provides a biosynthetic indigo dye using L-tryptophan as a starting material, having an indigotin content of at least 30%, which is 1.5 times higher than that of plant-extracted indigo. During biosynthesis, the total conversion rate from L-tryptophan can reach up to 18.9%. The indigo dye disclosed in this invention can be recovered in less than a week, compared to one year for plant-derived indigo extraction, resulting in reduced production time and improved yield. Indirubin, a byproduct of indigo, can impart a unique hue to the indigo dye. This invention also discloses a composition in which the indirubin content relative to indigotin in the indigo dye is relatively increased by carefully controlling the biosynthetic environment. 【0013】 In a first aspect, the present invention relates to a biosynthetic method for producing an indigo dye comprising indigotine and indirubin, comprising: generating tryptophanase (TRP) and flavin-containing monooxygenase (FMO) from a genetically modified organism having vectors encoding tryptophanase and flavin-containing monooxygenase; and using the generated TRP and FMO, catalyzing the conversion of L-tryptophan to an indigo dye in the presence of one or more bioactive additives selected from the group consisting of cysteine, 2-oxindole, and 2-indoxyl, to form a dye having a weight ratio of indigotine to indirubin of 1:0.1 to 1:4. The dye has hues defined in the CIE XYZ color space, ranging from X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, and Z: 0.5543 to 0.5501. 【0014】 In a further embodiment, the present invention provides a composition for the biosynthesis of indigo dyes comprising one or more indigotin and indirubin. The composition comprises a genetically modified microorganism for expressing tryptophanase (TRP) and flavin-containing monooxygenase (FMO), and a bioactive additive in a concentration of 0 to 1.33 g / L (0 to 10 mM). The bioactive additive is one or more cysteine, 2-oxindole, or 2-indoxyl. This composition is adjustable to biosynthesize indigotin from L-tryptophan in a yield of 2-18.9% and indirubin in a yield of 3-7.65%, with a weight ratio of indigotin to indirubin in the range of 1:0.1-1:4, and the indigo dye has a hue defined in the CIE XYZ color space (X: 0.1920-0.2481, Y: 0.2537-0.2019, Z: 0.5543-0.5501). [Brief explanation of the drawing] 【0015】 This patent or application document includes at least one color drawing. A copy of this patent or published patent application with a color drawing will be provided by the Office upon request and payment of the prescribed fee. [Figure 1] Figure 1 schematically shows the pathway by which indigotine and indirubin are produced from tryptophan precursors. [Figure 2] Figure 2 schematically illustrates a further pathway for indirubin production in the presence of cysteine. [Figure 3] Figure 3 is a graph showing the ratio of indirubin to indigotine relative to cysteine ​​concentration. [Figure 4] Figure 4 is a graph showing the ratio of indirubin to indigotine relative to the 2-indoxyl concentration in the presence of 0.030 g / L (0.25 mM) cysteine. [Figure 5]Figure 5 is a graph showing the ratio of indirubin to indigotine to cysteine ​​concentration in the presence of 0.40 g / L (3 mM) 2-indoxyl. [Figure 6] Figure 6 schematically illustrates the process of dye generation on textile fibers, following seed formation. [Figure 7] Figure 7 is a graph showing the ratio of indirubin to indigotine to the cysteine ​​concentration used in dyeing wool fibers. [Figure 8] Figure 8 shows the colors of the dyes based on the CIE XYZ color space. [Figure 9] Figure 9 shows dyed wool yarns and wool fabrics according to cysteine ​​concentration. [Modes for carrying out the invention] 【0016】 This invention provides a biosynthesis of indigo dyes with controlled ratios of indigotine (blue) and indirubin (red). Conventionally, the color of indigo dyes is primarily derived from the blue indigotine, but by significantly including indirubin, the hue shifts to a reddish-purple range, which can be useful in obtaining unique color profiles. By adjusting the dye production conditions, specific hues can be selected, facilitating the manufacture of custom dyes. 【0017】 1. Overview of Indigo Dye Biosynthesis 【0018】 The biosynthesis of indigo dyes relies on a genetically modified host material that provides enzymes for converting selected raw materials into indigo dyes with desired hues. As used herein, "indigo dyes" refers to dyes containing indigotin and indirubin in a selected mass ratio. An exemplary starting material is L-tryptophan. This amino acid is cost-effective and can be produced by microbial fermentation from glucose or glycerol. L-tryptophan functions as a major precursor for indole, an intermediate necessary for the production of indigo and indirubin. The enzyme tryptophanase (TRP) catalyzes the reaction that converts L-tryptophan to indole. This reaction is an important initial step because indole functions as the major substrate for the further conversion to indoxyl intermediates. 【0019】 Flavin-containing monooxygenase (FMO) oxidizes indole to form 2-indoxyl and 3-indoxyl. This enzyme catalyzes selective hydroxylation at various positions of the indole ring, generating intermediates that are precursors to indigotin and indirubin. 【0020】 In the presence of oxygen, 3-indoxyl undergoes self-dimerization to produce indigotin (blue indigo). The reaction between 2-indoxyl and 3-indoxyl generates indirubin. The outline of this reaction process is shown in Figure 1. 【0021】 2. Microbial host for enzyme expression 【0022】 For the production of enzymes in the biosynthesis of indigotin and indirubin, multiple microorganisms can be used as hosts, each having unique advantages in terms of compatibility with enzymes, ease of genetic manipulation, and metabolic efficiency. 【0023】 a. Escherichia coli (E. coli) 【0024】 E. coli is the most widely used microbial host in indigo biosynthesis because its genetic characteristics are well understood, it is easy to manipulate, and it grows rapidly. E. coli can be genetically modified to highly express TRP and FMO, and as a result, it can efficiently catalyze the reaction that converts L-tryptophan to indole, indoxyl, and then to indigotine or indirubin. 【0025】 b. Pseudomonas putida 【0026】 Pseudomonas putida possesses a robust metabolic network resistant to aromatic compounds, making it a suitable candidate for the synthesis and accumulation of indole and indoxyl intermediates. 【0027】 Non-bacterial hosts such as yeast can also be used. 【0028】 Plasmids such as TRP-FMO pET29b, which contain genes encoding TRP and FMO, enable the production of both enzymes in a microbial host, allowing for the generation of indole and oxyindole intermediates from L-tryptophan. 【0029】 3. Environmental control to adjust the ratio of indigotin to indirubin 【0030】 The present invention makes it possible to control the ratio of indigotine to indirubin, and in order to obtain the desired ratio, various factors and their combinations that affect indirubin production have been identified. These will be described in detail in the following examples. As shown in Figure 2, indirubin production can be promoted by adding cysteine ​​as a bioactive additive. Cysteine ​​is converted from indoxyl and oxindole to cysteinylindorenionone, promoting a pathway toward indirubin rather than indigotine. Cysteine ​​also functions as a protecting group, contributing to the retention of the reactivity of indoxyl and its availability for indirubin production. 【0031】 2-Indoxyl and 2-oxindole can be added directly as indirubin precursors, promoting the reaction toward indirubin production. This additive approach complements the natural metabolic flow and increases the likelihood of indirubin production over indigotine by increasing the local concentration of indirubin precursors. 【0032】 Other bioactive molecules, such as 2-indoxyl and 2-oxindole, can also be added in concentrations ranging from 0.067 to 1.33 g / L to promote the reaction environment toward indirubin formation. Indoxyl intermediates are highly sensitive to oxygen, and the presence of oxygen strongly influences whether they form indigotine or indirubin. 【0033】 4. Color selection 【0034】 This invention provides a biosynthesis in which the weight ratio of indigotine to indirubin is 1:0.1 to 1:4. The ratio selected in this invention produces a unique color that combines deep blue and reddish-purple tones, providing a visually appealing and versatile hue applicable to modern textile products. This mixing ratio yields shades such as "soft blue" or "violet blue," expressing a more delicate and complex hue compared to the conventional deep blue of pure indigo. The mixing of the deep blue of indigotine and the reddish-purple of indirubin produces a more nuanced hue than pure indigotine. This mixing results in a more complex hue. This hue is distinct from more commonly used indigo. In denim, it can mimic the softer, vintage look of naturally worn denim or aged textiles. This hue is particularly appealing in clothing styles that aim to express a worn-in texture and a "familiar" feel. For silk and wool, indigotine and indirubin dyes can produce different, more subtle shades. 【0035】 The selected color can be represented in the CIE XYZ color space. The CIE XYZ color space is one of the first mathematically defined color spaces, established in 1931 by the International Commission on Illumination (CIE). CIE XYZ uses three coordinates, X, Y, and Z, which correspond to the human eye's perception of red, green, and blue. By using the CIE XYZ color space, it becomes possible to standardize and represent colors in three-dimensional space, providing an ideal means for comparing and adjusting colors produced by biosynthesis processes. 【0036】 5. Direct dyeing of yarn or fabric 【0037】 In this invention, a method can be used to directly dye fabrics or yarns in a culture medium used for biosynthesis of indigotin and indirubin. Because indigo and indirubin are produced in situ in the culture medium, the dye molecules are in close contact with the fiber substrate, which may improve the adsorption and fixation of the dye, especially to natural fibers such as cotton and wool. Continuous exposure of the fabric or yarn to the newly synthesized dye in the culture medium may promote uniform coloring of the fibers and deeper penetration of the dye, potentially resulting in a more vivid and long-lasting color. Since there is no need to extract and purify the dye before application, the overall number of processing steps can be reduced, potentially saving time and labor. This method avoids the extraction and dissolution steps that are often required with conventional indigo dyes, which require a lot of energy. Because the biosynthesized indigo dye exists as an indoxyl intermediate in the culture medium, it may adhere to fibers more easily without the use of additional chemicals for dissolution, which can reduce the environmental impact and complexity of the dyeing process. 【0038】 When the staining process is carried out directly in the culture medium, it may be possible to reduce the need for the large-scale water rinsing and chemical waste treatment typically required when transitioning from dye production to application. Direct staining in the culture medium eliminates the need for the heat treatment often used in indigo application, which can lead to energy savings, especially when the process is carried out at room temperature. [Examples] 【0039】 Genetically modified microorganisms are constructed with the aim of producing enzymes that convert L-tryptophan to indigo through the various transformations described below. 【0040】 E. coli is selected as the host microorganism. Plasmids containing DNA encoding tryptophanase (TRP) and flavin-containing monooxygenase (FMO) are introduced into E. coli. The DNA construct in the 5' to 3' direction of transcription includes: (i) a promoter that functions within the organism; (ii) a first transcribable nucleic acid sequence encoding tryptophanase (TRP) as shown in SEQ ID NO: 01, or a nucleotide sequence having at least 90% homology thereto and catalytically converting L-tryptophan to indole; and a second transcribable nucleic acid sequence encoding flavin-containing oxygenase (FMO) as shown in SEQ ID NO: 02, or a nucleotide sequence having at least 90% homology thereto and encoding a polypeptide that catalyzes the production of 3-indoxyl or 2-indoxyl from indole; and (iii) a transcription termination sequence. Table 1 shows the sequence lists for SEQ ID NO: 01 and SEQ ID NO: 02. 【0041】 In certain embodiments, the TRP-FMO pET29b plasmid is used. The TRP-FMO pET29b plasmid contains a T7 promoter, a lactose operon, TRP and FMO genes, and a kanamycin resistance gene. The T7 promoter is a sequence that can be identified by T7 RNA polymerase for plasmid transcription. The lactose operon enables the transcription of genes that form recombinant proteins controlled by the presence of lactose. TRP catalyzes the conversion of L-tryptophan to the intermediate compound indole. Subsequently, FMO continues to catalyze the conversion of indole to 2-oxindole and 3-oxindole. The kanamycin resistance gene is used for selecting bacteria containing the plasmid. 【0042】 In some embodiments, the culture medium contains bioactive additives including cysteine, 2-indoxyl, and 2-oxindole to adjust the ratio of indigotin to indirubin. A cysteine ​​concentration of 0.030–0.48 g / L (0.25–4 mM) allows for adjustment of the indirubin to indigotin ratio within the range of 1:0.1–1:4, with a CIE color space range of X:0.1920–0.2481, Y:0.2537–0.2019, and Z:0.5543–0.5501 under a D65 light source. 【0043】 In certain embodiments, wool yarn is immersed in a microbial growth solution and stained in situ. In growth media containing cysteine ​​at different concentrations (corresponding to 0-0.36 g / L, or 0-3 mM), the ratio of indigotin to indirubin on the wool yarn becomes 1:1 to 1:16. 【0044】 General manufacturing procedure for indigo dye 【0045】 The following procedure describes the co-expression of TRP and FMO in bacteria. Competent E. coli cells for NEB® 10-beta transformation were introduced with the TRP-FMO pET29b plasmid. 1–5 μL of TRP-FMO pET29b plasmid (50 pg–100 ng) was introduced into 50 μL of competent cells. A mutant E. coli strain purchased from TWIST Bioscience was used as the host for TRP-FMO pET29b. TRP (SEQ ID NO: 01) and FMO (SEQ ID NO: 02) were used in the example. 【0046】 The bacterial mixture was placed on ice for 30 minutes, then subjected to a heat shock at 42°C for 30 seconds, and then placed on ice for another 5 minutes. It was then cultured with shaking at 250 rpm at 37°C for 60 minutes. The resulting mixture was diluted and spread onto screening agar plates containing lactose, kanamycin, and L-tryptophan, and cultured overnight. Single colonies grown on Luria-Bertani(LB) agar containing 50 μg / mL kanamycin were inoculated into 10 mL of LB liquid medium also containing 50 μg / mL kanamycin, and cultured overnight with shaking at 30°C and 250 rpm. When the optical density (OD600) of the resulting cell culture medium reached 0.8 (Jenway TM (Measured at 600 nm using a 72 Series Genova Bio spectrophotometer), 1 mL of this cell culture was taken and added to 100 mL of fermentation medium (20 g / L LB liquid medium, 50 μg / mL kanamycin, 1.7 g / L lactose, and 2 g / L L-tryptophan). These components are optimized for indigo dye production. The culture was further incubated with shaking at 30°C and 180 rpm for 72 hours. After 72 hours, a water-insoluble blue precipitate was observed in the culture. 【0047】 The suspension was collected and centrifuged at 8000 rpm for 10 minutes. The precipitate was washed twice with deionized water and lyophilized. Crude solid was defined as the solid obtained after lyophilization. The weight of the crude solid was measured. The crude solid was dissolved in DMSO, filtered through a 0.45 μm PTFE filter, and analyzed by HPLC. The signals for indirubin and indigotine were observed using a photodiode array detector at wavelengths of 500 nm and 620 nm, respectively. 【0048】 Examples 1-7 describe the production of indigo dye when the cysteine ​​concentration in the culture medium is varied. 【0049】 The addition of cysteine ​​can promote the formation of indirubin. Cysteine ​​is converted from indoxyl and oxindole to 2-cysteinyl indorenionone, facilitating a pathway toward indirubin rather than indigotine. Cysteine ​​also functions as a protecting group, contributing to the preservation of indoxyl's reactivity and availability for indirubin production. 【0050】 Table 2 lists the isolation yield of indigo dye, indirubin content, indigotin content, conversion rate from L-tryptophan to indirubin and indigotin, and the ratio of indigotin to indirubin, corresponding to the differences in cysteine ​​concentration in Examples 1 to 7. 【0051】 The isolation yield of the indigo dye was defined as the mass of crude solid obtained from 1 liter of culture medium. 【0052】 The indirubin and indigotin content were defined as the proportion of the target compound contained in the crude solid. This was obtained by dividing the concentration of each indigo dye by the concentration of the crude solution dissolved in DMSO (200 ppm). 【0053】 The conversion rate (%) from L-tryptophan to indirubin and indigotin was calculated by multiplying the indirubin content or indigotin content by the isolation yield of the indigo dye and dividing the result by the theoretical yield obtained from L-tryptophan. 【0054】 The ratio of indigotin to indirubin was calculated based on the concentrations of indigotin and indirubin obtained by HPLC analysis. 【0055】 Figure 3 shows that increased cysteine ​​levels promote the production of indirubin in relation to indigotine. The theoretical isolation yield of indirubin increases from 3.37% to 7.65%. The theoretical isolation yield of indigotine decreases from 18.9% to 2.14%. 【0056】 2-Indoxyl and 2-oxindole can be added directly as indirubin precursors, promoting the reaction toward indirubin production. This additive approach complements the natural metabolic flow and increases the likelihood of indirubin production over indigotine by increasing the local concentration of indirubin precursors. 【0057】 Isatin is also a precursor in the production of indirubin via oxidative coupling. Adding isatin can accelerate the reaction toward indirubin production. 【0058】 Examples 8 and 9 and Comparative Example 10 describe the production of indigo dye when 2-indoxyl (1.33 g / L), 2-oxindole (1.33 g / L), or isatin (1.47 g / L) is present in the culture medium at a concentration of 10 mM. 【0059】 Table 3 lists the isolation yield of the indigo dye, the indirubin content, the indigotin content, the isolation yield of indirubin and indigotin, and the ratio of indigotin to indirubin for Examples 8, 9, and Comparative Example 10. 【0060】 In Examples 8 and 9, only indirubin was detected due to the high concentration of the bioactive additive, but the isolation yield was low. High concentrations of bioactive additives may act as growth inhibitors of E. coli. 【0061】 Figure 4 shows that increasing the 2-indoxyl concentration (0, 0.067, 0.13, 0.27, and 0.40 g / L, i.e., 0, 0.5, 1, 2, and 3 mM) under a constant cysteine ​​concentration (0.030 g / L or 0.25 mM) promotes the production of indirubin relative to indigotin, but it does not show a consistent increasing trend like that observed when cysteine ​​alone is increased. 【0062】 Figure 5 shows that, under a constant 2-indoxyl concentration (0.40 g / L or 3 mM), increasing the cysteine ​​concentration (0, 0.030, 0.061, 0.091, and 0.12 g / L, i.e., 0, 0.25, 0.5, 0.75, and 1 mM) resulted in a relative increase in indigotine production relative to indirubin and a decrease in indirubin production. 【0063】 The effect of 2-indoxyl on promoting indirubin production is not significant, and it may even show an inhibitory effect. The use of cysteine ​​alone is preferable. 【0064】 Examples 11-13 describe methods for generating indigo dye in culture media with different cysteine ​​concentrations and for directly dyeing wool yarn in situ. The staining process is shown in Figure 6. In the first step, 10 μL of cryopreserved transformed E. coli was used to prepare 5 mL of seed solution in a fermentation medium containing 20 g / L LB liquid medium, 2 g / L L-tryptophan, 1.71 g / L lactose, and 50 mg / L kanamycin stock solution, and then activated at 30°C for 12 hours. Wool yarn (1 g) that had been pre-autoclaved at 121°C was immersed in this seed solution and left to stand in a 30°C incubator for 24 hours. Through this process, the transformed E. coli was adsorbed onto the wool yarn. 【0065】 In the second stage, these wool yarns were transferred to newly prepared fermentation media (100 mL) with different cysteine ​​concentrations (0, 0.061, and 0.36 g / L, i.e., 0, 0.5, and 3 mM). They were then incubated at 30°C for 72 hours with stirring at 180 rpm. The resulting indigo dye was fixed to the wool yarns. They were sterilized at 60°C for 30 minutes, washed several times with distilled water, and then air-dried. The media and wool yarns were then extracted with DMSO to quantify the indigo dye. The resulting solution was filtered through a 0.45 μm PTFE filter before HPLC analysis. 【0066】 Examples 11-13 summarize the indigo dye content in wool yarn. Table 4 lists the results for Examples 11-13 with different cysteine ​​concentrations (0, 0.061, and 0.36 g / L, i.e., 0, 0.5, and 3 mM), along with the ratios of indigotin to indirubin. 【0067】 Figure 7 shows that increasing the cysteine ​​concentration in wool yarn promotes the production of indirubin relative to indigotine. Compared to the culture medium used for incubation, the ratio of indirubin content to indigotine in wool yarn is generally higher. The maximum ratio of indigotine to indirubin was 1:15.8. This ratio is significantly higher than that of indigo dye produced in culture medium without wool yarn. Wool fabrics dyed with indigo dye by the method disclosed herein achieved abrasion fastness (ISO 105 X-12) of grade 4 to 5. 【0068】 The indigo dye was further quantified using the CIE XYZ color space. 【0069】 Table 5 lists the CIE XYZ values ​​for Examples 1-7 with different cysteine ​​concentrations (0, 0.030, 0.061, 0.12, 0.24, 0.36, and 0.48 g / L, i.e., 0, 0.25, 0.5, 1, 2, 3, and 4 mM). Indigo dye was dissolved in DMSO at a constant concentration (200 ppm). 【0070】 At a specific cysteine ​​concentration, the ratio of indirubin to indigotine can be adjusted within the range of 1:0.1 to 1:4, and the CIE values ​​under a D65 light source are X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, and Z: 0.5543 to 0.5501. 【0071】 [Table 1-1] 【0072】 [Table 1-2] 【0073】 [Table 2] 【0074】 [Table 3] 【0075】 [Table 4] 【0076】 [Table 5] 【0077】 6. Industrial Applicability and Benefits 【0078】 The biosynthesized indigo dyes of the present invention have several clear advantages over conventional synthetic indigo, particularly in terms of sustainability, reproducibility, and environmental safety. 【0079】 As mentioned above, biosynthesis allows for precise control of the reaction pathway, leading to consistent color across batches. By controlling factors such as enzyme expression levels, intermediate concentrations, and reaction conditions, manufacturers can reproducibly obtain the ratio of indigotine to indirubin, ensuring uniform color across all lots. 【0080】 Color Adjustment Accuracy: By controlling the content of indirubin and indigotin, biosynthesis offers unprecedented color customization. By measuring color using CIE XYZ values, manufacturers can standardize color with high precision, reducing the color variability commonly seen in synthetic and plant-derived indigo dyes. 【0081】 Improving dye quality: While the lightfastness and colorfastness of indigo are primarily determined by its chemical structure, controlling the purity of the dye and reducing impurities during the biosynthesis process can contribute to more consistent colorfastness. This can lead to more uniform dye adhesion to the fibers, potentially improving wash fastness and lightfastness. 【0082】 Better management of additives: In the biosynthetic pathway, stabilizers and modifiers can be added at specific stages of the metabolic process, potentially improving color retention without compromising the inherent properties of the dye. 【0083】 Direct generation of indoxyl precursors: Conventional synthetic indigo requires a reduction process to make it water-soluble for application to fibers, and this process typically involves the use of harmful reducing agents such as sodium dithionite. However, since biosynthesized indigo can be produced in the indoxyl form, the need for additional chemicals can be reduced and the dyeing process can be simplified. 【0084】 Potential for low-temperature processing: Biosynthetic dyes can be applied at low temperatures, reducing energy consumption in the dyeing process. This is particularly beneficial in industrial applications where energy use is a major cost factor. 【0085】 Elimination of harmful reducing agents: Conventional synthetic indigo production relies on harmful substances such as aniline and heavy metals. In contrast, biosynthesis of indigo is carried out in microbial cultures, does not require harsh chemicals, and is a clean manufacturing process with less harmful waste. 【0086】 Lower environmental impact: The production of biosynthetic indigo does not use harmful precursors or reducing agents, resulting in less pollutants in the wastewater. This is particularly important because wastewater from the dye industry may contain harmful compounds that affect aquatic ecosystems. 【0087】 Microbial production using renewable raw materials: Biosynthetic indigo is produced by culturing microorganisms such as E. coli and Saccharomyces cerevisiae with renewable raw materials such as glucose. This is in contrast to synthesis methods that rely on petrochemicals or non-renewable resources. 【0088】 Reduction of carbon dioxide emissions: Biosynthesis can be carried out under milder conditions and at lower temperatures, requiring less energy and thus reducing the overall carbon footprint of indigo dye production. 【0089】 Industrial-scale fermentation: Microbial fermentation systems are suitable for large-scale operations. By culturing artificial bacteria in fermentation tanks, indigotin and indirubin can be produced in industrial quantities. Similar biosynthetic approaches are already commercially available for compounds such as insulin and various amino acids, and therefore, scaling up this biosynthetic pathway is feasible with current biotechnology. 【0090】 Fermentation efficiency: High-density fermentation, optimized nutrient supply, and controlled oxygen levels are key factors for the efficient production of indigo precursors at high concentrations. High-throughput screening of genetically modified strains can improve pathway efficiency and yield. 【0091】 Some embodiments and detailed features of the present disclosure are briefly described above. The embodiments described herein can be readily used as a basis for designing or modifying other processes and structures to achieve the same or similar objectives and / or to obtain the same or similar advantages introduced in the embodiments of the present disclosure. Such equivalent configurations can be modified, substituted, and altered in various ways without departing from the spirit and scope of the present disclosure. 【0092】 As used herein, the terms “approximately,” “basically,” “substantially,” and “about” are used to describe and explain small variations. When used in conjunction with an event or situation, the terms may refer to the event or situation occurring precisely or approximately. As used herein with respect to a given value or range, the term “about” generally means a range of ±10%, ±5%, ±1%, or ±0.5% of the given value or range. Ranges may be expressed herein as from one endpoint to another, or between two endpoints. Unless otherwise specified, all ranges disclosed herein include endpoints. The term “substantially coplanar” may refer to two surfaces within a few micrometers (μm) of each other that are aligned along the same plane, for example, within 10 μm, 5 μm, 1 μm, or 0.5 μm of each other that are aligned along the same plane. When referring to substantially the same numerical value or characteristic, the term may mean a value within ±10%, ±5%, ±1%, or ±0.5% of the mean value.

Claims

[Claim 1] A biosynthesis method for producing indigo dyes containing indigotin and indirubin, To produce tryptophanase (TRP) and flavin-containing monooxygenase (FMO) from genetically modified organisms having vectors encoding tryptophanase and flavin-containing monooxygenase; The generated TRP and FMO catalyze the conversion of L-tryptophan to an indigo dye in the presence of one or more physiologically active additives selected from the group consisting of cysteine, 2-oxindole, and 2-indoxyl, forming a dye in which the weight ratio of indigotin to indirubin is 1:0.1 to 1:4, and the dye has a hue defined in the CIE XYZ color space, in the range of X: 0.1920 to 0.2481, Y: 0.2537 to 0.019, and Z: 0.5543 to 0.5501. A method that includes [the following features]. [Claim 2] The biosynthesis method according to claim 1, wherein the physiologically active additive contains cysteine ​​in an amount of 0.03 to 0.48 g / L. [Claim 3] The biosynthesis method according to claim 1, wherein the physiologically active additive contains 2-oxindole in an amount of 1.33 g / L. [Claim 4] The biosynthesis method according to claim 1, wherein the physiologically active additive contains 2-indoxyl in an amount of 0.067 to 1.33 g / L. [Claim 5] The biosynthesis method according to claim 1, wherein the vector comprises a first transcribable nucleic acid sequence encoding a tryptophanase shown in SEQ ID NO: 01 or a nucleotide sequence having at least 90% homology thereto, and a second transcribable nucleic acid sequence encoding a flavin-containing oxygenase (FMO) shown in SEQ ID NO: 02 or a nucleotide sequence having at least 90% homology thereto. [Claim 6] The biosynthesis method according to claim 1, wherein the vector is a TRP-FMO pET29b plasmid. [Claim 7] An indigo dye produced by the method described in claim 1. [Claim 8] A composition for the biosynthesis of indigo dyes comprising one or more indigotin and indirubin, Genetically modified microorganisms encoding the expression of tryptophanase (TRP) and flavin-containing monooxygenase (FMO); With a physiologically active additive at a concentration of 0.03 to 1.33 g / L; Equipped with, The aforementioned physiologically active additive contains one or more of cysteine, 2-oxindole, or 2-indoxyl. The composition can be adjusted to biosynthesize indigotin from L-tryptophan in a yield of 2 to 18.9%, and indirubin from L-tryptophan in a yield of 3 to 7.65%. The weight ratio of indigotine to indirubin is 1:0.1 to 1:

4. The indigo dye has hues defined in the CIE XYZ color space, with X: 0.1920–0.2481, Y: 0.2537–0.019, and Z: 0.5543–0.5501. composition. [Claim 9] The composition according to claim 8, wherein the physiologically active additive contains cysteine ​​in an amount of 0.03 to 0.48 g / L. [Claim 10] The composition according to claim 8, wherein the physiologically active additive contains 2-oxindole in an amount of 1.33 g / L. [Claim 11] The composition according to claim 8, wherein the physiologically active additive contains 2-indoxyl in an amount of 0.067 to 1.33 g / L. [Claim 12] The composition according to claim 8, wherein the vector comprises a first transcribable nucleic acid sequence encoding a tryptophanase shown in SEQ ID NO: 01 or a nucleotide sequence having at least 90% homology thereto, and a second transcribable nucleic acid sequence encoding a flavin-containing oxygenase (FMO) shown in SEQ ID NO: 02 or a nucleotide sequence having at least 90% homology thereto. [Claim 13] The composition according to claim 8, wherein the vector is a TRP-FMO pET29b plasmid. [Claim 14] A biosynthesis method for in situ dyeing of fabrics with indigo dyes containing indigotine and indirubin, To produce tryptophanase (TRP) and flavin-containing monooxygenase (FMO) from a genetically modified organism having vectors encoding tryptophanase and flavin-containing monooxygenase in the presence of fabric or yarn to be dyed; In the presence of a fabric or yarn to be dyed, and in the presence of one or more bioactive additives selected from the group consisting of cysteine, 2-oxindole, and 2-indoxyl, L-tryptophan is converted into an indigo dye by the generated TRP and FMO to form a dyed fiber or yarn having a weight ratio of indigotin to indirubin of 1:1 to 1:16, wherein the dye has a hue defined in the CIE XYZ color space, in the range of X: 0.1920 to 0.2481, Y: 0.2537 to 0.019, and Z: 0.5543 to 0.5501; A method that includes [the following features]. [Claim 15] A biosynthesis method for dyeing a fabric according to claim 14, wherein the generation of TRP in the presence of the fabric or yarn to be dyed is derived from a seed solution of a genetically modified organism having a vector encoding tryptophanase and flavin-containing monooxygenase, and the method comprises the step of adsorbing the genetically modified organism onto a wool fabric or wool yarn, and then converting L-tryptophan into an indigo dye using the generated TRP and FMO.

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