Biosynthesis of hue-tunable indigo dyes
By biosynthesizing indigo dye and using genetically engineered microorganisms to catalyze the production of indigo and indirubin from L-tryptophan, the problems of color fastness and environmental pollution associated with traditional dyes have been solved, achieving efficient and environmentally friendly dye production and color adjustment.
Patent Information
- Application Number
- CN202510735492.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-05-26
- Filing Date
- 2025-06-04
- Publication Date
- 2026-06-05
AI Technical Summary
Traditional plant dyes suffer from uneven color, poor color fastness, and the use of toxic chemicals in the production process. Synthetic indigo dyes, on the other hand, cause environmental pollution and are difficult to treat by-products, making it difficult to achieve efficient and environmentally friendly dye production.
Indigo and indirubin were synthesized using a biosynthetic method by genetically engineered microorganisms based on L-tryptophan through tryptophanase and flavin monooxygenase. The ratio of indigo to indirubin was adjusted by controlling bioactive additives such as cysteine and 2-hydroxyindole, and staining was performed directly in the culture medium.
It has achieved efficient and environmentally friendly production of indigo dyes, with increased output, adjustable hue, excellent color fastness and wash resistance, reduced environmental pollution and dependence on chemicals, and lower energy consumption and carbon footprint.
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Figure CN122146811A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the biosynthesis of indigo dyes, and more specifically, to the biosynthesis of indigo dyes with tunable hues. Background Technology
[0002] Textile dyeing is the second leading cause of global water pollution. With the textile industry's increasing focus on sustainable development and stringent government regulations on water pollution, the demand for plant-based dyes as alternatives to synthetic dyes is constantly growing. Due to this increasing demand, the plant-based dye market is worth approximately $5 billion and is projected to grow annually.
[0003] Good wash resistance and colorfastness are the most attractive features driving the continued sales growth of fashion products in the market. However, traditional plant dyes often result in uneven color and poor colorfastness, making them difficult to apply to fashion. In addition, some products still require the use of toxic chemicals in the dyeing process, such as amines, sodium sulfide, and caustic soda.
[0004] Among plant dyes, the extraction rate of indigo dye from plants does not exceed 1%, and the indigo content does not exceed 20%. Indigo dye extracted from Polygonum leaves can produce beautiful colors on denim. However, Polygonum leaves require at least one year to harvest.
[0005] Synthetic indigo dyes can also be produced through the chemical synthesis of petrochemical precursors. The production of synthetic indigo dyes typically begins with aniline, a derivative of benzene derived from petroleum. Aniline is a highly reactive compound, making it an ideal precursor for the synthetic indigo pathway. Aniline undergoes several chemical reactions, most notably chlorination, to produce intermediates such as N-phenylglycine. This intermediate is crucial for the formation of the indigo molecule. N-phenylglycine then undergoes an alkaline melting process, which facilitates its conversion into indoxyl, a precursor of indigotin. Aniline is also a precursor of isatitin, whose oxidative coupling can form indirubin.
[0006] Indophenol molecules typically undergo a final oxidation reaction in the presence of oxygen to form indigo dye (the blue dye component of indigo). This oxidation reaction causes two indophenol molecules to couple and form the characteristic blue color.
[0007] However, the production of synthetic indigo involves several hazardous intermediates and byproducts, such as formaldehyde, cyanide, and aniline derivatives, which are toxic and must be carefully managed to minimize their environmental impact. Furthermore, wastewater from synthetic indigo production may contain heavy metals and chloride compounds, requiring extensive treatment to meet environmental safety standards. While recent advances in green chemistry aim to reduce the toxicity of indigo production by developing alternative pathways to avoid some of the more hazardous intermediates, these methods have not yet largely replaced traditional processes, primarily due to cost and the existing infrastructure for conventional synthetic indigo production.
[0008] Therefore, there is a need in the art for a dye with minimal environmental impact that exhibits consistent hue and good colorfastness when applied to fabrics. Furthermore, there is a need to select specific hues based on the dye production process. This invention fulfills this need.
[0009] This invention also provides a technique for increasing the yield of indirubin, a bisindole antitumor drug that has inhibitory effects on various transplanted animal tumors, including the destruction of leukemia cells, and is therefore an important substance with independent uses. Summary of the Invention
[0010] This invention provides a biosynthetic indigo dye that uses L-tryptophan as a starting material, with an indigo content of at least 30%, which is 1.5 times higher than that of plant-derived indigo. During the biosynthesis process, the total conversion rate of L-tryptophan can reach up to 18.9%. The indigo dye disclosed in this invention can be harvested in less than a week, while plant-derived indigo requires a year to harvest, thus shortening the production time and increasing yield. The indigo byproduct, indirubin, can impart a unique hue to the indigo dye. This invention also discloses a composition that, through careful control of the biosynthetic environment, provides a higher ratio of indirubin to indigo dye in the indigo dye.
[0011] A first aspect of the present invention provides a biosynthetic method for producing indigo dye comprising indigo and indirubin, comprising generating tryptophanase (TRP) and flavin monooxygenase (FMO) from a transgenic organism having a vector encoding tryptophanase and flavin monooxygenase; and catalyzing the conversion of L-tryptophan to indigo dye by the generated TRP and FMO in the presence of one or more bioactive additives selected from cysteine, 2-oxindole, or 2-indoxyl to form a dye having a weight ratio of indigo to indirubin between 1:0.1 and 1:4. The dye has a hue of X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, Z: 0.5543 to 0.5501 as defined in the CIE XYZ color space.
[0012] In another aspect, the present invention provides a composition for the biosynthesis of indigo dye, the dye comprising one or more of indigo and indirubin. The composition comprises a genetically engineered microorganism encoding tryptophanase (TRP) and flavin monooxygenase (FMO) and a bioactive additive at a concentration of 0 to 1.33 g / L (0 to 10 mM). The bioactive additive is one or more of cysteine, 2-hydroxyindole, or 2-indophenol. The composition is capable of regulating the biosynthesis from L-tryptophan to indigo in a yield of 2 to 18.9%, and the biosynthesis from L-tryptophan to indirubin in a yield of 3 to 7.65%, such that the weight ratio of indigo to indirubin is between 1:0.1 and 1:4, and the indigo dye has a hue of X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, Z: 0.5543 to 0.5501 as defined in the CIE XYZ color space. Attached Figure Description
[0013] Embodiments of the invention are described in more detail below with reference to the accompanying drawings, in which:
[0014] Figure 1 The pathways for the formation of indigo and indirubin from tryptophan precursors are illustrated schematically.
[0015] Figure 2 Another pathway for the formation of indigo in the presence of cysteine is illustrated.
[0016] Figure 3 This is a graph comparing the ratio of indigo to indigoferazone with the concentration of cysteine.
[0017] Figure 4 The graph shows the relationship between the ratio of indirubin to indigo and the concentration of 2-indophenol in the presence of 0.030 g / L (0.25 mM) cysteine.
[0018] Figure 5 A graph showing the ratio of indigo to indigo to cysteine concentration in the presence of 0.40 g / L (3 mM) 2-indophenol.
[0019] Figure 6 This is a schematic diagram illustrating the process by which bacteria form on textile fibers, followed by the formation of dyes on the textile fibers.
[0020] Figure 7 This is a graph showing the relationship between the ratio of indigo red to indigo pigment used for dyeing wool fibers and the concentration of cysteine.
[0021] Figure 8 It displays the dye colors defined in the CIE XYZ color space.
[0022] Figure 9 The images show wool yarns and fabrics dyed according to cysteine concentration. Detailed Implementation
[0023] This invention provides a biosynthesis of indigo dye, specifically allowing for freely adjustable ratios of indigo pigment (blue) and indirubin (red). Traditionally, the color of indigo dye primarily derives from the blue indigo pigment, but introducing a significant amount of indirubin will shift the hue towards a reddish-purple, which is highly useful for achieving unique color blends. By adjusting dye production conditions to select specific dye hues, customized dye production can be facilitated.
[0024] 1. Overview of Indigo Dye Biosynthesis
[0025] The biosynthesis of indigo dye relies on genetically engineered host materials that provide enzymes to convert selected raw materials into an indigo dye of selected color. The indigo dye used herein is a dye containing indigo pigment and indirubin in selected mass ratios. An exemplary starting material is L-tryptophan. This amino acid is cost-effective and can be produced by microbial fermentation of glucose or glycerol. L-tryptophan is a major precursor to indole, an intermediate required for the production of indigo and indirubin. Tryptophanase (TRP) catalyzes the conversion of L-tryptophan to indole. This reaction is a crucial first step because indole is a key substrate for the further conversion to the indolephenol intermediate.
[0026] Flavin monooxygenase (FMO) oxidizes indole to 2-indolephenol and 3-indolephenol (3-indoxyl). This enzyme catalyzes selective hydroxylation at different positions on the indole ring to generate precursor intermediates of indigo and indirubin.
[0027] In an oxygen-containing environment, 3-indophenol can undergo self-dimerization to form indigo (blue indigo). The reaction between 2-indophenol and 3-indophenol produces indirubin. An overview of this reaction process is as follows: Figure 1 As shown.
[0028] 2. Microbial hosts used for enzyme expression
[0029] Several microorganisms can be used as hosts for the production of indigo and indirubin biosynthetic enzymes, each with specific advantages in terms of enzyme compatibility, ease of genetic manipulation, and metabolic efficiency.
[0030] I. Escherichia coli
[0031] Escherichia coli is the most widely used microbial host for indigo biosynthesis due to its favorable genetic characteristics, ease of manipulation, and rapid growth. It can be engineered to express high levels of tryptophanase and flavin monooxygenase, enabling it to efficiently catalyze the conversion of L-tryptophan to indole and indophenol, subsequently to indigo or indirubin.
[0032] II. Pseudomonas putida
[0033] Pseudomonas has a robust metabolic network and is tolerant to aromatic compounds, making it a good candidate for the synthesis and accumulation of indole and indophenol intermediates.
[0034] Non-bacterial hosts, such as yeast, can also be used.
[0035] Plasmids (such as TRP-FMO pET29b) contain genes encoding TRP and FMO, enabling the microbial host to produce both enzymes and drive the production of indole and oxindole intermediates from L-tryptophan.
[0036] 3. Adjusting the ratio of indigo to indigo red through environmental control.
[0037] Because this invention provides a controlled ratio of indigo to indirubin, various factors and combinations of factors affecting indirubin production are identified to produce the desired ratio, as discussed in detail in the embodiments below. Figure 2 As shown, the formation of indirubin can be promoted by adding cysteine as a bioactive additive. Cysteine is converted from indophenol and hydroxyindole to cysteine indoleone, thereby driving the synthetic pathway towards indirubin instead of indigo. Cysteine also acts as a protecting group, helping to maintain the reactivity of indophenol and its availability for indirubin formation.
[0038] 2-Indophenol and 2-hydroxyindole can be added directly as precursors to indirubin to drive the reaction to form indirubin. This addition method can replenish the natural metabolic flux and produce a higher local concentration of indirubin precursors, thereby increasing the likelihood of indirubin forming beyond indigo.
[0039] Other bioactive molecules (such as 2-indophenol and 2-hydroxyindole) in varying amounts (0.067 to 1.33 g / L) can be used to drive the reaction environment to form indirubin. Indole intermediates are oxygen-sensitive, and the presence of oxygen strongly influences whether they combine to form indigo or indirubin.
[0040] 4. Color selection
[0041] This invention provides a biosynthesis of indigo and indigo red in a weight ratio of 1:0.1 to 1:4. The selected ratio creates a unique color, combining deep blue with a magenta hue, resulting in a distinctive tone that is both visually appealing and suitable for modern textiles. This mixture achieves a “soft blue” or “magenta” tone, offering subtle complexity compared to the traditional deep blue of pure indigo. The blend of the deep blue of indigo and the reddish-purple of indigo red produces a more subtle color than pure indigo. This mixture achieves a more complex tone. This tone differs from the more commonly used indigo. It can give denim a softer, vintage look, similar to naturally worn denim or distressed textiles. This tone is particularly appealing in garment styles seeking to capture a faded or “beloved” look. For silk and wool, indigo pigment and indigo red dye can also produce different subtle tones.
[0042] The selected colors can be represented using the CIE XYZ color space. The CIE XYZ color space was one of the first color spaces defined mathematically, established by the International Commission on Illumination (CIE) in 1931. CIE XYZ uses three coordinates, X, Y, and Z, corresponding to the human eye's perception of red, green, and blue, respectively. Using the CIE XYZ color space provides a standardized method for representing colors in three-dimensional space, making it ideal for comparing and modifying colors produced through biosynthetic processes.
[0043] 5. Direct dyeing of yarn or fabric
[0044] This invention allows for the direct dyeing of fabrics or yarns in a culture medium used for the biosynthesis of indigo and indirubin. Because indigo and indirubin are produced in situ in the culture medium, the dye molecules are in close contact with the textile substrate, which improves the absorption and adhesion of the dyes, especially for natural fibers such as cotton and wool. The continuous exposure of the fabric or yarn to the newly synthesized dyes in the culture medium promotes consistent coloring and allows the dyes to penetrate deeper into the fibers, potentially resulting in brighter and longer-lasting colors. By eliminating the need for dye extraction and purification before application, the total processing steps are reduced, saving time and labor. This method bypasses the typically energy-intensive extraction and dissolution steps traditionally required for indigo dyes. Since the biosynthesized indigo dye exists in the culture medium as an indophenol intermediate, it may adhere more readily without the need for additional chemicals to dissolve it, thus minimizing the environmental impact and complexity of the dyeing process.
[0045] If the dyeing process can be carried out directly in the culture medium, the need for large amounts of water rinsing and chemical waste disposal required for conventional dyes from production to application can be reduced. Since direct dyeing in the culture medium eliminates the heat treatment often used in indigo dyeing, energy can be saved, especially if the process is carried out at room temperature.
[0046] Example
[0047] Transgenic microorganisms were constructed to produce enzymes for converting L-tryptophan to indigo through various transformations described below.
[0048] Escherichia coli was selected as the host microorganism, and a plasmid encoding DNA containing tryptophanase and flavin monooxygenase was inserted into E. coli. The DNA construct includes, in the 5' to 3' direction of transcription, (i) a promoter that functions in the organism; (ii) a first transcribed nucleic acid sequence encoding the tryptophanase according to SEQ ID No:01, or a nucleotide sequence at least 90% identical thereto and capable of catalyzing the conversion of L-tryptophan to indole; a second transcribed nucleic acid sequence encoding the flavin monooxygenase according to SEQ ID No:02, or a polypeptide at least 90% identical thereto and encoding the catalytic formation of 3-indolephenol or 2-indolephenol from indole; and (iii) a transcription termination sequence. Table 1 below provides the sequence listings for SEQ ID No:01 and SEQ ID No:02.
[0049] In some implementations, the TRP-FMO pET29b plasmid is used. The TRP-FMO pET29b plasmid contains a T7 promoter, a lactose operon, tryptophanase and flavin monooxygenase genes, and a kanamycin resistance gene. The T7 promoter is a sequence that can be recognized by T7 RNA polymerase to transcribe the plasmid. The lactose operon allows gene transcription to regulate the formation of recombinant proteins in the presence of lactose. Tryptophanase catalyzes the conversion of L-tryptophan to the intermediate compound indole. Flavin monooxygenase then further catalyzes the conversion of indole to 2-hydroxyindole and 3-hydroxyindole (3-oxindole). The kanamycin resistance gene is used to select bacteria containing the plasmid.
[0050] In some embodiments, the growth medium includes a bioactive additive, including cysteine, 2-indophenol, and 2-hydroxyindole, for adjusting the ratio between indigo and indirubin. Cysteine at concentrations of 0.030 to 0.48 g / L (0.25 to 4 mM) can adjust the indigo to indirubin ratio from 1:0.1 to 1:4, where the CIE hue range under D65 light source is X: 0.1920–0.2481, Y: 0.2537–0.2019, and Z: 0.5543–0.5501.
[0051] General procedures for the production of indigo dye
[0052] The following steps describe the co-expression of tryptophanase and flavin monooxygenase in bacteria. The TRP-FMOpET29b plasmid was transformed into... In 10-beta competent *E. coli* cells, 1 to 5 μL of the TRP-FMO pET29b plasmid (50 pg to 100 ng) was transfected into 50 μL of competent cells. A mutant *E. coli* strain purchased from TWIST Bioscience was used to carry the TRP-FMO pET29b. Tryptophanase (SEQ ID No: 01) was used in the examples. Hexanin monooxygenase (SEQ ID No:02).
[0053] The bacterial culture was placed on ice for 30 minutes, then heat-shocked at 42°C for 30 seconds, and then placed on ice for 5 minutes. It was then shaken at 250 rpm at 37°C for 60 minutes. The diluted bacterial culture was spread onto selection plates containing lactose, kanamycin, and L-tryptophan and incubated overnight. Single colonies growing on Luria-Bertani (LB) agar plates containing 50 μg / mL kanamycin were inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin and incubated overnight at 30°C with shaking at 250 rpm. The culture was considered complete when the OD600 at 600 nm reached 0.8 (using Jenway). TM When measuring at 600 nm using a Genova Bio 72 series spectrophotometer, 1 mL of this cell culture was added to 100 mL of fermentation medium (20 g / L LB medium, 50 μg / mL kanamycin, 1.7 g / L lactose, and 2 g / L L-tryptophan). These components had been optimized for indigo dye production. The culture was further incubated at 30°C with shaking at 180 rpm for 72 hours. After 72 hours, a water-insoluble blue precipitate was observed in the medium.
[0054] The suspension was collected and centrifuged at 8000 rpm for 10 minutes. The precipitate was washed twice with deionized water and then freeze-dried. The solid collected after freeze-drying was defined as the crude solid. The weight of the crude solid was measured. The crude solid was dissolved in dimethyl sulfoxide (DMSO), filtered through a 0.45 μm PTFE filter, and then analyzed by high-performance liquid chromatography (HPLC). The signals of indirubin and indigo were observed at wavelengths of 500 nm and 620 nm, respectively, using a photodiode array detector.
[0055] Examples 1 to 7 describe the production of indigo dye using different concentrations of cysteine in a culture medium.
[0056] The formation of indirubin can be promoted by adding cysteine. Cysteine is converted from hydroxyindole and indophenol to 2-cysteine-indoleone, thereby driving the indirubin-but-indole-forming pathway. Cysteine also acts as a protecting group, helping to maintain the reactivity of indophenol and the availability of indirubin formation.
[0057] Table 2 lists the results for different cysteine concentrations in Examples 1 to 7, the separation yield of indigo dye, the content of indirubin, the content of indigo pigment, the conversion of L-tryptophan to indirubin and indigo pigment, and the ratio between indigo pigment and indirubin.
[0058] "Indigo dye separation yield" is defined as the mass of crude solids that can be produced in 1 L of culture medium.
[0059] The indigo and indigo dye contents are the percentages of the target compounds in the crude solids. This is obtained by dividing the concentration of each indigo dye by the concentration (200 ppm) of the crude solution dissolved in dimethyl sulfoxide.
[0060] The conversion rate (percentage) from L-tryptophan to indigo dye is calculated by multiplying the indigo content or indigo dye content by the yield of indigo dye separation, and then dividing by the theoretical yield of indigo dye separated from L-tryptophan.
[0061] The ratio between indigo and indirubin was calculated by analyzing the concentrations of indigo and indirubin using high-performance liquid chromatography.
[0062] Figure 3 The results showed that the yield of indirubin was higher than that of indigo dye as the cysteine content increased. The theoretical yield of indirubin increased from 3.37% to 7.65%, while the theoretical yield of indigo dye decreased from 18.9% to 2.14%.
[0063] 2-Indophenol and 2-hydroxyindole can be added directly as precursors to indirubin to drive the reaction to form indirubin. This addition method can replenish the natural metabolic flux and produce a higher local concentration of indirubin precursors, thereby increasing the likelihood of indirubin forming beyond indigo.
[0064] Indigo is also a precursor to indirubin, formed through oxidative coupling. Adding indigo can advance the reaction to form indirubin.
[0065] Examples 8, 9 and Comparative Example 10 describe the production of indigo dye in the presence of 10 mM 2-indophenol (1.33 g / L), 2-hydroxyindole (1.33 g / L) or indigo (1.47 g / L) in a culture medium.
[0066] Table 3 lists the indigo dye separation yield, indirubin content, indigo dye content, separation yield of indirubin and indigo dye, and the ratio of indigo dye to indirubin for Examples 8, 9 and Comparative Example 10.
[0067] Due to the high concentration of the bioactive additive, only indirubin was detected in Examples 8 and 9, but the separation yield was low. High concentrations of the bioactive additive may inhibit the growth of *E. coli*.
[0068] Figure 4 The results showed that, at a fixed cysteine concentration (0.030 g / L or 0.25 mM), increasing the concentration of 2-indophenol (0, 0.067, 0.13, 0.27 and 0.40 g / L, equivalent to 0, 0.5, 1, 2 and 3 mM) increased the yield of indirubin compared to indigo, but did not increase it by the same increment when cysteine was increased alone.
[0069] Figure 5 The results showed that, with a fixed 2-indophenol concentration (0.40 g / L or 3 mM), increasing the cysteine concentration (0, 0.030, 0.061, 0.091 and 0.12 g / L, equivalent to 0, 0.25, 0.5, 0.75 and 1 mM) reduced the yield of indirubin, while the yield of indigoferazone decreased.
[0070] 2-Indophenol did not significantly promote the formation of indirubin and may have an inhibitory effect. Cysteine alone is preferred.
[0071] Examples 11 to 13 describe the production and in-situ dyeing of indigo dye directly on wool yarn in a medium containing different concentrations of cysteine.
[0072] The staining process is as follows Figure 6 As shown. First, using 10 μL of cryopreserved transformed *E. coli*, a 5 mL inoculum solution was prepared using fermentation medium containing 20 g / L LLB medium, 2 g / L L-tryptophan, 1.71 g / L lactose, and 50 mg / L kanamycin stock solution. The solution was then activated at 30°C for 12 hours. 1 g of pre-autoclaved wool yarn was immersed in this inoculum solution and placed in an incubator at 30°C for 24 hours. This process allowed the transformed *E. coli* to adhere to the wool yarn.
[0073] In the second step, the wool yarns were transferred to freshly prepared fermentation medium (100 mL) with different cysteine concentrations (0, 0.061, and 0.36 g / L, equivalent to 0, 0.5, and 3 mM). They were then incubated at 30 °C for 72 hours with a stirring speed of 180 rpm. The resulting indigo dye adhered to the wool yarns. The yarns were sterilized at 60 °C for 30 minutes, washed several times with distilled water, and then air-dried. The medium and wool yarns were then extracted with dimethyl sulfoxide to quantify the indigo dye. The resulting solution was then filtered through a 0.45 μm PTFE filter and analyzed by high-performance liquid chromatography (HPLC).
[0074] Examples 11 to 13 summarize the indigo dye content in wool yarns. Table 4 lists the results for different concentrations of cysteine (0, 0.061, and 0.36 g / L, equivalent to 0, 0.5, and 3 mM) in Examples 11 to 13, as well as the ratio between indigo dye and indirubin.
[0075] Figure 7 This indicates that increased cysteine concentration increased the yield of indigo red in wool yarn, exceeding the yield of indigo pigment. The overall ratio of indigo red to indigo pigment in wool yarn was higher than that in the culture medium used for cultivation. The highest ratio between indigo pigment and indigo red was 1:15.8, which is much higher than the indigo dye produced in a culture medium without wool yarn. Wool fabrics dyed with indigo dye using the method disclosed in this invention achieved a color fastness to rubbing (ISO 105X-12) of 4 to 5.
[0076] The indigo dye was further quantified using the CIE XYZ color space.
[0077] Table 5 lists the CIE XYZ values of different concentrations of cysteine (0, 0.030, 0.061, 0.12, 0.24, 0.36, and 0.48 g / L, equivalent to 0, 0.25, 0.5, 1, 2, 3, and 4 mM) in Examples 1 through 7. Indigo dye was dissolved in dimethyl sulfoxide at a fixed concentration (200 ppm).
[0078] At a given cysteine concentration, the ratio of indirubin to indigo can be adjusted from 1:0.1 to 1:4, with CIE values of X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, and Z: 0.5543 to 0.5501 under a D65 light source.
[0079] Table 1:
[0080] Table 2: *Fermentation medium composition: [L-tryptophan] = 2 g / L; [Luria-Bertani (LB) medium] = 20 g / L; [lactose] = 1.7 g / L; [kanamycin]: 50 mg / L; Fermentation medium volume = 100 mL **High-performance liquid chromatography analysis based on the separation of indigo dye** Data are expressed as mean ± SD (n = 3)
[0081] Table 3: Example 8 Example 9 Comparative Example 10 *Bioactive additives 2-Indophenol 2-Hydroxyindole Indigo Indigo dye separation yield (g / L) 0.114 0.124 0.128 Indigo content (%)** 1.00 1.08 nd Indigo content (%)** nd nd nd Indigo pigment to indigo red ratio 0:1 0:1 nd [Bioactive additive] = 10mM *The fermentation medium contains: [L-tryptophan] = 2 g / L; [Luria-Bertani (LB) medium] = 20 g / L; [lactose] = 1.7 g / L; [kanamycin]: 50 mg / L; fermentation medium volume = 100 mL **High-performance liquid chromatography analysis based on the separation of indigo dye** nd: Not detected
[0082] Table 4: *Fermentation medium composition: [L-tryptophan] = 2 g / L; [Luria-Bertani (LB) medium] = 20 g / L; [lactose] = 1.7 g / L; [kanamycin]: 50 mg / L; Fermentation medium volume = 100 mL
[0083] Table 5:
[0084] 6. Industrial practicality and advantages
[0085] The biosynthetic indigo-based dyes of this invention have several unique advantages over conventional synthetic indigo, particularly in terms of sustainability, reproducibility, and environmental safety.
[0086] As mentioned above, biosynthetic methods allow for precise control of reaction pathways, resulting in more consistent color across batches. By adjusting factors such as enzyme expression, intermediate concentrations, and reaction conditions, manufacturers can achieve reproducible indigo to indirubin ratios, ensuring uniform color in each batch.
[0087] Precision in color adjustment: Biosynthesis allows for control over the content of indigo red and indigo, providing unprecedented capabilities for customized hues. By measuring the color in the CIE XYZ values, manufacturers can precisely standardize the color, thereby reducing the variability common in synthetic and plant-derived indigo dyes.
[0088] Improving dye quality: While the colorfastness of indigo largely depends on its chemical structure, controlling dye purity and reducing impurities during biosynthesis can contribute to more consistent colorfastness. This allows the dye to adhere more evenly to the fiber, thus providing better wash and lightfastness.
[0089] Better control over additives: Biosynthetic pathways allow for the addition of stabilizers and modifiers at specific points in the metabolic process, potentially enhancing color retention without affecting the natural properties of the dye.
[0090] Direct production of indophenol precursors: Traditional synthetic indigo requires a chemical reduction step to dissolve the dye in water for textile applications, which often involves toxic reducing agents such as sodium dithionite. However, biosynthetic indigo can be produced in the form of indophenol, which simplifies the dyeing process by reducing the need for additional chemicals.
[0091] The potential of cryogenic processing: Biosynthetic dyes can be used at lower temperatures, thereby reducing the energy requirements of the dyeing process. Since energy use is a major cost factor, the potential of cryogenic processing is particularly beneficial at the industrial application level of this technology.
[0092] Eliminating harmful reducing agents: Synthetic indigo production has traditionally relied on toxic substances, including aniline and heavy metals. In contrast, biosynthetic indigo production occurs in microbial cultures, eliminating the need for irritating chemicals and resulting in a cleaner production process with less harmful waste.
[0093] Reduced environmental impact: The production of biosynthetic indigo does not involve toxic precursors or reducing agents, meaning there are fewer pollutants in the wastewater. This is especially important for environmental protection, as dye industry wastewater can contain toxic compounds that can impact aquatic ecosystems.
[0094] Microbial production using renewable feedstocks: Biosynthetic indigo is produced using microorganisms (such as E. coli or Saccharomyces cerevisiae) that grow on renewable feedstocks like glucose. This offers far superior sustainability compared to synthetic methods that rely on petrochemical products and non-renewable resources.
[0095] Reducing the carbon footprint: Because biosynthesis can be carried out under milder conditions and at lower temperatures, less energy is required, thus reducing the overall carbon footprint of indigo dye production.
[0096] Industrial-scale fermentation: Microbial fermentation systems are well-suited for large-scale operations. Indigo and indirubin can be produced in large quantities on an industrial scale by culturing engineered bacteria in fermenters. Similar biosynthetic methods are already commercially used for compounds such as insulin and various amino acids, thus the scale of this pathway can be expanded using current biotechnology.
[0097] Fermentation efficiency: High-density fermentation methods, optimized nutrient feeding, and controlled oxygen levels are key to the efficient production of high-concentration indigo precursors. High-throughput screening of engineered strains can improve pathway efficiency and yield.
[0098] The above is only a brief description of several embodiments and their detailed features of the present invention. The embodiments described in this invention can easily serve as the basis for designing or modifying other processes and structures to achieve the same or similar objectives and / or obtain the same or similar advantages as described in the embodiments of this invention. Such equivalent constructions do not depart from the spirit and scope of the present invention, and various changes, substitutions, and modifications can be made without departing from the spirit and scope of the present invention.
[0099] The terms “approximately,” “substantially,” and “approximately” as used herein are used to describe and explain minute variations. When used in conjunction with an event or situation, the term can refer to an event or situation occurring precisely, or approximately. The term “approximately” as used herein with respect to a given value or range generally means within ±10%, ±5%, ±1%, or ±0.5% of the given value or range. A range can be expressed as from one endpoint to another or between two endpoints. Unless otherwise stated, all ranges disclosed in this disclosure include endpoints. The term “substantially coplanar” can refer to two surfaces located within a few micrometers (μm) of the same plane, such as within 10 μm, 5 μm, 1 μm, or 0.5 μm of the same plane. When referring to the same numerical value or characteristic, the term may refer to a value within ±10%, ±5%, ±1%, or ±0.5% of the average value.
Claims
1. A biosynthetic method for producing indigo dye comprising indigo dye and indirubin, characterized in that, include: Producing tryptophanase and flavin-containing amine oxidase from transgenic organisms with vectors encoding tryptophanase and flavin monooxygenase; as well as In the presence of one or more bioactive additives selected from cysteine, 2-hydroxyindole, and 2-indoleoxy, L-tryptophan is converted into indigo dye by the generated tryptophanase and flavin monooxygenase, wherein the weight ratio of indigo dye to indirubin in the indigo dye is between 1:0.1 and 1:4, and the dye has a hue of X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, Z: 0.5543 to 0.5501 as defined in the CIE XYZ color space.
2. The biosynthesis method according to claim 1, characterized in that, The bioactive additive includes 0.03 to 0.48 g / L of cysteine.
3. The biosynthesis method according to claim 1, characterized in that, The bioactive additive includes 1.33 g / L of 2-hydroxyindole.
4. The biosynthesis method according to claim 1, characterized in that, The bioactive additive includes 0.067 to 1.33 g / L of 2-indophenol.
5. The biosynthesis method according to claim 1, characterized in that, The carrier includes: According to the first transcribed nucleic acid sequence encoding tryptophanase according to SEQ ID No:01, or a nucleotide sequence that is at least 90% identical to it; and The second transcribed nucleic acid sequence encoding flavin monooxygenase according to SEQ ID No:02, or a nucleotide sequence that is at least 90% identical to it.
6. The biosynthesis method according to claim 1, characterized in that, The vector is the TRP-FMO pET29b plasmid.
7. An indigo dye, characterized in that, The indigo dye is manufactured by the biosynthesis method according to claim 1.
8. A composition comprising one or more of indigo dye and indirubin for the biosynthesis of indigo dye, characterized in that, include: A transgenic microorganism encoding tryptophanase and flavin monooxygenase; as well as Bioactive additives ranging from 0.03 to 1.33 g / L; The bioactive additive is selected from one or more of cysteine, 2-hydroxyindole, or 2-indophenol. The composition is capable of regulating the biosynthesis from L-tryptophan to indigo and from L-tryptophan to indirubin, wherein the yield of indigo is 2 to 18.9%, the yield of indirubin is 3 to 7.65%, and the weight ratio of indigo to indirubin is 1:0.1 to 1:4; and The indigo dye has hues ranging from X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, and Z: 0.5543 to 0.5501 in the CIE XYZ color space.
9. The composition according to claim 8, characterized in that, The bioactive additive includes 0.03 to 0.48 g / L of cysteine.
10. The composition according to claim 8, characterized in that, The bioactive additive includes 1.33 g / L of 2-hydroxyindole.
11. The composition according to claim 8, characterized in that, The bioactive additive includes 0.067 to 1.33 g / L of 2-indophenol.
12. The composition according to claim 8, characterized in that, The carrier includes: According to the first transcribed nucleic acid sequence encoding tryptophanase according to SEQ ID No:01, or a nucleotide sequence that is at least 90% identical to it; and The second transcribed nucleic acid sequence encoding flavin monooxygenase according to SEQ ID No:02, or a nucleotide sequence that is at least 90% identical to it.
13. The composition according to claim 8, characterized in that, The vector is the TRP-FMO pET29b plasmid.
14. A biosynthetic method for in-situ dyeing of fabrics with indigo dyes comprising indigo dyes including indigo pigment and indirubin, characterized in that, include: In the presence of the fabric or yarn to be dyed, tryptophanase and tryptophanase are produced from transgenic organisms that have encoding vectors for tryptophanase and flavin monooxygenase. In the presence of a fabric or yarn to be dyed, and one or more bioactive additives selected from cysteine, 2-hydroxyindole, and 2-indoleoxy, L-tryptophan is converted into indigo dye by the generated tryptophanase and flavin monooxygenase to form a dyed fabric or yarn with a weight ratio of indigo dye to indirubin between 1:1 and 1:16, wherein the indigo dye has a hue of X: 0.1920 to 0.2481, Y: 0.2537 to 0.2019, Z: 0.5543 to 0.5501 as defined in the CIE XYZ color space.
15. The biosynthetic method for in-situ dyeing of fabrics according to claim 14, characterized in that, In the presence of the fabric or yarn to be dyed, the fabric or yarn is immersed in a bacterial solution containing a transgenic organism encoding a vector of tryptophanase and flavin monooxygenase, allowing the transgenic organism to be absorbed by the fabric or yarn to be dyed. Subsequently, the L-tryptophan is converted into indigo dye by the produced tryptophanase and flavin monooxygenase.