Bio-indigo production method using recombinant bacteria having monooxygenase gene

Abstract

The present invention relates to a bio-indigo production method using an eco-friendly bio-process. A bio-indigo production process, which increases solubility of indole and indigo by using a surfactant and a reducing agent, and continuously bio-converts indole into indigo, has been developed. Adsorbents and reducing agents enabled efficient removal and solubilization of indigo, respectively, and thereby enabled high yields of the bio-indigo production process. An adsorbent removal process and the use of an eco-friendly reducing agent can be easily applied to mass production of bio-indigo, and are expected to be widely utilized industrially such as in adsorbent recovery, indigo separation, continuous reaction, and bio-dyeing processes.

Description

Method for producing bioindigo using recombinant bacteria having monooxygenase genes

[0001] One example of the present invention relates to a method for producing bio-indigo by co-culturing a recombinant bacterium having a monooxygenase gene with a surfactant; and bio-indigo obtained thereby.

[0002] Indigo is a well-known blue dye commonly used to dye jeans and various other fabrics. Traditionally, indigo was obtained from the Indigofera plant and used in the denim dyeing process.

[0003] When chemically synthesized, an indole ring is synthesized from aniline at high temperatures through a series of reactions including HCHO, HCN, and NaNH2 reactions and N-methylation, and indigo is formed through oxidative dimerization.

[0004] Recently, due to concerns about carbon neutrality and climate change, biological methods for synthesizing indigo using microorganisms with lower carbon emissions than chemically synthesized indigo have been attracting attention. One biological method synthesizes bioindigo using indole, formed during the metabolism of tryptophan, as a substrate. This process oxidizes indole at the C3 position to form 3-hydroxyindole (indoxyl). Once formed, indoxyl is converted to indigo through oxidative dimerization within the cell (Figure 1a). Several enzymes are known to catalyze indole-C3-hydroxylation, including flavin-containing monooxygenase (MaFMO), toluene 4-monooxygenase (PmT4MO), cytochrome P450 (CYP102G4), and various styrene monooxygenases and naphthalene dioxygenases.

[0005] However, bioindigo synthesis through bioconversion reactions has several limitations. First, the low solubility of the substrate (indole) and product (indigo) limits the bioconversion reaction. Indigo is poorly soluble in the aqueous solvents used for bioconversion. Specifically, when produced inside cells, it diffuses across the cell membrane to maintain homeostasis, but most of it remains bound to the cell membrane, resulting in production loss. Second, cytotoxicity due to high concentrations of the substrate or product is a major factor hindering high-concentration bioindigo production. Until now, bioconversion-based production of bioindigo has been difficult at the gram (g) level.

[0006] To overcome these problems, denim dyes were traditionally treated with powerful reducing agents, such as sodium dithionite, to convert them to a water-soluble leuco-form of indigo (a transparent yellow), which was then used for dyeing (Fig. 1b). However, the use of these reducing agents increases water treatment costs and is banned in some countries, leading to dyeing and reimportation through countries where these agents are permitted. Various attempts have been made to develop alternative methods that do not require reducing agents. However, none have fundamentally addressed the issue of overcoming the low solubility of indigo and the indole substrate.

[0007] One aspect provides a method for producing bio-indigo by co-cultivating a recombinant bacterium having a monooxygenase gene and a surfactant.

[0008] Another aspect is to provide bio-indigo obtained by the above method.

[0009] To overcome the above problems, the inventors of the present invention have completed a method for increasing the solubility and cell membrane permeability of indole and bio-indigo, and for optimizing the bioconversion process using indole or tryptophan as a substrate, thereby increasing the production of bio-indigo (Fig. 1c).

[0010] The present invention provides a method for producing bioindigo by co-cultivating a recombinant bacterium having a monooxygenase gene and a surfactant.

[0011] In one embodiment of the present invention, the culturing may be a method in which the culturing is performed at 15°C to 35°C for 15 to 50 hours.

[0012] Another embodiment of the present invention may be a method further comprising a step of adding indole, tryptophan or a combination thereof as a substrate during the co-culturing.

[0013] In another embodiment of the present invention, the substrate may be at a concentration of 0.1 to 30 mM.

[0014] In another embodiment of the present invention, the method may be one in which the monooxygenase gene is flavin-containing monooxygenase (MaFMO) or toluene monooxygenase (PmT4MO).

[0015] In another embodiment of the present invention, the method may be one or more strains selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, Salmonella Typhimurium, and Serratia marcescens.

[0016] In another embodiment of the present invention, the method may be such that the E. coli is E. coli BL21 (DE3).

[0017] In another embodiment of the present invention, the surfactant may be at least one selected from the group consisting of Tween 20, Span 20, Tween 40, Span 40, Tween 80, Span 60, Tween 85, Span 80, Pluronic 68, Brij 35, and Triton X-100.

[0018] In another embodiment of the present invention, the method may be one in which the surfactant is added at a concentration of 0.1% to 20%.

[0019] In another embodiment of the present invention, the method may further include a step of separating bio-indigo from the recombinant bacteria using an adsorbent.

[0020] In another embodiment of the present invention, the method may be such that the adsorbent is a hydrophobic polymer resin.

[0021] In another embodiment of the present invention, the method may further include a step of adding a reducing agent during the co-culturing.

[0022] In another embodiment of the present invention, the method may be one in which the reducing agent is thiourea dioxide.

[0023] In addition, the present invention aims to provide bioindigo obtained by the above method.

[0024] In one embodiment of the present invention, the bio-indigo may be a bio-indigo that is leuco indigo obtained by adding a reducing agent.

[0025] The present inventors developed a bio-indigo production process that increases the solubility of indole and indigo using a surfactant and a reducing agent, respectively, in the absence of NaOH, and continuously bioconverts indole into indigo. As a result, the concentration of bio-indigo produced was significantly increased compared to the initial production level. The use of an adsorbent and a reducing agent enabled the efficient removal and solubilization of indigo, respectively, resulting in a high yield in the bio-indigo production process. The adsorbent removal process and the use of an eco-friendly reducing agent are not only readily applicable to the mass production of bio-indigo, but are also expected to have wide-ranging industrial applications, including adsorbent recovery, indigo separation, continuous reaction, and bio-dyeing processes.

[0026] Figure 1a shows the pathway for converting tryptophan to indoxyl using the enzymes that produce indigo from tryptophan, flavin-containing monooxygenase of Methylophaga aminisulfidivorans (MaFMO) and toluene dioxygenase of Pseudomonas mendocina (PmT4MO), Figure 1b shows that when indigo is reduced to leucoindigo, indigo becomes soluble and can be oxidized back to insoluble indigo when exposed to air, Figure 1c schematically illustrates the present invention, and Figure 1d shows the ionic structure of indigo under pH conditions and the reduced structure due to electron donation.

[0027] Figure 2a shows the effect of indigo production according to the addition of Tween 80, and Figures 2b and 2c are data measuring the biotransformation of high concentration indole and tryptophan under semi-optimal induction and surfactant conditions. Figure 2b shows the degree of biotransformation of indole at a concentration ranging from 10 to 50 mM by adjusting the induction conditions such as pH, temperature, and addition of inducer. Figure 2c shows the degree of biotransformation of tryptophan at a concentration of 20 mM in the presence of Tween 80 surfactant.

[0028] Figure 3 shows that indigo production can be increased by removing indigo produced during the biotransformation process using an adsorbent.

[0029] Figure 4a shows the degree of indigo reduction over time using 0.02 mM reducing agent in the presence of 7% Tween 80, Figures 4b and 4c show the indigo production and pH change profiles during indole biotransformation of 5 mM indole (b) and tryptophan (c), Figure 4d shows that indigo was reduced to leucoindigo and formed a transparent layer in the supernatant, and Figure 4e shows the results of checking the optimal reducing agent supply conditions (NaOH concentration and reducing agent supply time) to achieve the highest indigo production concentration by offsetting the insolubility of indigo.

[0030] Hereinafter, the present invention will be described in detail.

[0031] The present invention provides a method for producing bioindigo by co-cultivating a recombinant bacterium having a monooxygenase gene and a surfactant.

[0032] In one embodiment of the present invention, the culturing may be a method in which the culturing is performed at 15°C to 35°C for 15 to 50 hours.

[0033] Another embodiment of the present invention may be a method further comprising a step of adding indole, tryptophan or a combination thereof as a substrate during the co-culturing.

[0034] In another embodiment of the present invention, the substrate may be at a concentration of 0.1 to 30 mM.

[0035] In another embodiment of the present invention, the method may be one in which the monooxygenase gene is flavin-containing monooxygenase (MaFMO) or toluene monooxygenase (PmT4MO).

[0036] In another embodiment of the present invention, the method may be one or more strains selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, Salmonella Typhimurium, and Serratia marcescens.

[0037] In another embodiment of the present invention, the method may be such that the E. coli is E. coli BL21 (DE3).

[0038] In another embodiment of the present invention, the surfactant may be at least one selected from the group consisting of Tween 20, Span 20, Tween 40, Span 40, Tween 80, Span 60, Tween 85, Span 80, Pluronic 68, Brij 35, and Triton X-100.

[0039] In another embodiment of the present invention, the method may be one in which the surfactant is added at a concentration of 0.1% to 20%.

[0040] The term "indigo" in the present invention refers to a deep blue crystalline powder. Due to the intramolecular hydrogen bonding between the oxygen of the ketone and the -NH and the structural symmetry resulting in a low dipole moment, it has a low solubility of less than 0.1 g / 100 mL in water at 25°C. It is slightly soluble in ethanol and glycerol, but insoluble in most oils. Due to its low solubility, it is converted to leucoindigo during the dyeing process using a reducing agent, as shown in Figure 1b.

[0041] The term "leucoindigo" in the present invention refers to a compound in which the ketone group at the C3 position of the indole ring is replaced with a hydrophilic hydroxyl group. This reduction reaction typically utilizes a strong reducing agent, such as sodium dithionite, hydroxyacetone, or a direct electron donor. The reduction reaction of indigo is significantly affected by pH, as it is protonated below pH 5.5 and deprotonated above pH 5.5, increasing its solubility in the form of an ion. Furthermore, the reduction reaction at low pH reduces the NH bond of indole and, through continuous electron donor, converts it to a dehydrated indigo form (Figure 1d).

[0042] In the present invention, the term "bio-indigo" may refer to indigo produced by microorganisms.

[0043] The term "surfactant" in the present invention refers to a nonionic, non-toxic, non-irritating polyoxyethylene sorbitan fatty acid ester series emulsifier, which is used for forming oil and water emulsions and stabilizing protein preparations, etc. The surfactant may correspond to one or more selected from the group consisting of Tween 20, Span 20, Tween 40, Span 40, Tween 80, Span 60, Tween 85, Span 80, Pluronic 68, Brij 35, and Triton X-100.

[0044] When adding the above surfactant to the medium, the concentration of the surfactant is preferably 1 to 20%, but is not limited thereto. Specifically, when 10% surfactant is added, the yield increases compared to the control group to which no surfactant was added, so adding 10% surfactant is more preferable.

[0045] The term "cultivation" in the present invention refers to a method of growing microorganisms under appropriately artificially controlled environmental conditions. In one embodiment, the culturing may be performed using a method widely known in the art, and may be continuously cultured in a batch process, fed batch process, or repeated fed batch process. Specifically, the culturing may be batch culture, fed-batch culture, or a combination thereof.

[0046] In one aspect, the medium used for the above cultivation can satisfy the requirements of a specific strain in an appropriate manner by controlling temperature, pH, etc. under aerobic conditions in a general medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc. As a carbon source that can be used, a mixed sugar of glucose and xylose is used as a main carbon source, and in addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or as a mixture. As a nitrogen source that can be used, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, and peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products can be used. These nitrogen sources can be used alone or in combination. The medium can contain potassium phosphate monobasic, potassium phosphate dibasic, and the corresponding sodium-containing salts as phosphorus. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus. In addition, inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate can be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

[0047] Additionally, suitable precursors may be used in the culture medium. The above-mentioned raw materials may be added to the culture in a batch, fed-batch, or continuous manner during the culture process, but are not particularly limited thereto. The pH of the culture may be adjusted using basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acid compounds such as phosphoric acid or sulfuric acid, in an appropriate manner.

[0048] Specifically, the medium of the present invention may be, but is not limited to, M9 medium. The M9 medium is a minimum nutrient medium composed of 33.7 mM Na2HPO4, 22 mM KH2PO4, 8.55 mM NaCl, 9.35 mM NH4Cl, 0.4% glucose, 1 mM MgSO4, 0.3 mM CaCl2, and trace element solution (134 μM EDTA, 31 μM FeCl3-6H2O, 6.2 μM ZnCl2, 0.76 μM CuCl2-2H2O, 0.42 μM CoCl2-2H2O, 1.62 μM H3BO3, 0.08 μM MnCl2-4H2O), and such a medium is a medium containing a mixture of inorganic nutrients that provide essential elements such as nitrogen, magnesium, calcium, etc. as well as glucose to supply carbon and energy. The above M9 medium is used when bacterial cultures must grow under precisely controlled conditions, as all components are known. The above M9 medium may be used interchangeably with the term "M9 minimal medium." Additionally, the M9 medium of the present invention may additionally contain necessary components, depending on the intended purpose.

[0049] In the present invention, the term "indole" means an aromatic heterocyclic organic compound having a molecular formula of C8H7N, which is widely distributed in the natural environment and can be used as a culture substrate for the production of bio-indigo.

[0050] When adding the above indole to the medium, it is preferable that the concentration of indole be included in the medium at a concentration of 1 to 30 mM, but is not limited thereto, and as the concentration of indole increases, the production of indole may decrease. Specifically, when indole is added at 30 mM or more, the production of bio-indigo decreases rapidly, so it is preferable to add indole at 30 mM or less.

[0051] In the present invention, the term "tryptophan" means one of the 20 or so amino acids encoded by the genetic code, contains an indole group, and can be used as a culture substrate for the production of bio-indigo.

[0052] When adding the above tryptophan to the medium, it is preferable that the concentration of tryptophan be included in the medium at a concentration of 1 to 30 mM, but is not limited thereto, and as the concentration of tryptophan increases, the production of tryptophan may increase.

[0053] In another embodiment of the present invention, the method may further include a step of separating bio-indigo from the recombinant bacteria using an adsorbent.

[0054] In another embodiment of the present invention, the method may be such that the adsorbent is a hydrophobic polymer resin.

[0055] The term "adsorbent" in the present invention refers to a solid material on which adsorption occurs, a phenomenon in which atoms, molecules, or ions in a gaseous, liquid, or dissolved state adhere to the surface. It is a porous adsorbent composed of a nonionic cross-linked aromatic polymer, specifically a hydrophobic polymer resin.

[0056] When adding the above adsorbent, the concentration of the adsorbent is preferably included at a concentration of 10 g / L, but is not limited thereto.

[0057] In another embodiment of the present invention, the method may further include a step of adding a reducing agent during the co-culturing.

[0058] In another embodiment of the present invention, the method may be one in which the reducing agent is thiourea dioxide.

[0059] In the present invention, the term "reducing agent" refers to a compound that reduces a molecule in its environment, i.e., a compound that changes a molecule to a more reduced state or a more reduced state in its environment. A reducing agent acts by donating electrons, thereby causing the reducing agent itself to become oxidized after the substrate is reduced. Therefore, a reducing agent is an active substance that donates electrons. The reducing agent may be at least one selected from the group consisting of thiourea dioxide, dithiothreitol (DTT), mercaptoethanol, cysteine, thioglycolic acid, cysteamine (2-mercaptoethylamine: 2-MEA), glutathione (GSH), TCEP (tris(2-carboxyethyl)phosphine), and sodium borohydride.

[0060]

[0061] In addition, the present invention aims to provide bioindigo obtained by the above method.

[0062] In one embodiment of the present invention, the bio-indigo may be a bio-indigo that is leuco indigo obtained by adding a reducing agent.

[0063] The terms used in this bio-indigo are consistent with the terms described in the above method.

[0064]

[0065] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0066]

[0067] Experimental method

[0068] 1. Chemical substances

[0069] Tryptophan, indole, isopropyl-β-d-thiogalactoside (IPTG), DMSO, adsorbent (Amberlite XAD4; Cas No. 37380-42-0), formamidinesulfinic acid (thiourea dioxide; Cas No. 1758-73-2), and NaOH were purchased from Sigma-Aldrich Korea (Suwon, South Korea). M9 minimal medium and LB medium were purchased from Difco (Seoul, South Korea). All chemical reagents used in this study were of analytical grade or higher.

[0070]

[0071] 2. Cultivation of bacterial strains for indigo production

[0072] The gene sequences of MaFMO and PmT4MO were obtained from Methylophaga aminisulfidovorans and Pseudomonas mendocinaKR1, respectively. Both genes were chemically synthesized and optimized for codon bias in Escherichia coli by L&C Bio (Seoul, South Korea). Each synthetic gene was cloned into the commercial pET-28a(+) vector under the control of the T7 promoter. For indigo production, E. coli strain BL21(DE3) was used as a host. The prepared cells were inoculated into LB medium and cultured overnight for use as a seed culture. The seed culture was further inoculated into M9 minimal medium supplemented with 2% glucose, 0.5% yeast extract, and kanamycin and cultured at 37°C until induced with IPTG. When OD600 reached approximately 0.8, various concentrations of IPTG (0.02–0.04 mM) and indole or tryptophan (5–30 mM) as substrates were added to the medium for induction, and then optimization was performed by further culturing at 20–30°C for up to 44 hours.

[0073]

[0074] 3. Indigo extraction from cell culture

[0075] Extraction and purification of indigo produced from whole-cell biotransformation were performed as follows. After induction, indigo-containing cells were collected by centrifugation at 8,000 rpm for 15 minutes. The collected cells were washed three times with distilled water. DMSO (2% v / v) was then added to the dried cells, the mixture was thoroughly mixed, and the mixture was incubated at 60°C for 16 hours. The bio-indigo in the supernatant was carefully pipetted to separate it.

[0076]

[0077] 4. Solubilization using surfactants for optimizing indigo production

[0078] The addition of Tween 80 to solubilize the indole substrate and indigo product was optimized by adding 1, 5, and 10% during indole biotransformation. The optimal combination of induction conditions and surfactants for solubilization was evaluated by quantifying the indigo concentration produced in terms of concentration and yield (%).

[0079]

[0080] 5. Indigo adsorption removal during indole biotransformation

[0081] The commercial indigo adsorbent used in the present invention is a hydrophobic polymer resin, Amberlite XAD4 with a 20-60 mesh size. To determine the optimal adsorbent concentration, the adsorption capacity of the adsorbent was calculated, and an adsorbent concentration of 10 g / L was used for indigo removal. To initiate biotransformation, 15 mM indole substrate was supplied to the medium together with 7% Tween 80 surfactant. The adsorbent was added to the culture medium when indigo production peaked during indole biotransformation (18 or 42 h after IPTG induction, respectively). After biotransformation, the adsorbent beads were separated using a 0.25 mm filter, and the filtered indigo was extracted using the DMSO extraction protocol described in Experimental Method 3. The indigo adsorbed on the adsorbent surface was separated by vigorous mixing with DMSO and then combined with the filtered indigo. The amount of indigo produced was evaluated after balancing the extraction amount and concentration.

[0082]

[0083] 6. Indigo reduction reaction during indole biotransformation

[0084] To initiate biotransformation, 5–150 mM indole substrate was supplied to the medium with 7% Tween 80 surfactant. Thiourea dioxide (15 mM) was added to the culture medium when indigo production peaked during indole biotransformation (16 or 44 h after IPTG induction, respectively). For optimization, 75, 100, and 125 mM NaOH were also added. To initiate the reduction reaction, the culture medium was stationary incubated for 5 min, and then incubated at 140 rpm for 24–72 h after confirming the formation of leuco-indigo. After biotransformation, the DMSO extraction protocol described in Experimental Method 3 was used for quantification.

[0085]

[0086] Experimental results

[0087]

[0088] 1. Co-cultivation of surfactants during bio-indigo production

[0089] To enhance the nonspecific permeability of cells, surfactants were used to improve the transport of substrates and products. Among the various surfactants tested, Tween 80 was selected due to its common use and ease of use. First, the effect of adding Tween 80 at various concentrations (1, 5, and 10%) to 5 mM indole was evaluated to enhance transport efficiency. Surprisingly, the addition of Tween 80 increased indigo production to 0.45 g / L, resulting in a conversion rate of 66.2% (Fig. 2a). In particular, the highest yield was achieved when 10% Tween 80 was added, representing a 27.3% increase compared to the control group without Tween 80.

[0090] The effect of Tween 80 was further confirmed at high substrate concentrations. Under previously optimized conditions, bioconversion was investigated by varying the indole concentration from 10 to 50 mM, and 10% Tween 80 was added along with the substrate. As a result, the highest production concentration (0.83 g / L) was achieved at 15 mM indole, and the highest yield (53.8%) was achieved at 10 mM indole (Fig. 2b). As the substrate concentration increased above 20 mM, bio-indigo production slightly decreased, but at concentrations above 30 mM, the production concentration decreased rapidly.

[0091] To evaluate the effect of surfactants on the potential of tryptophan as a substrate to replace indole, the same biotransformation reaction was performed on tryptophan. Unlike indole, tryptophan has a relatively high solubility of 11.4 g / L at 25°C (https: / / pubchem.ncbi.nlm.nih.gov) and is less toxic to the production host. Biotransformation of 20 mM tryptophan over a Tween 80 concentration range of 1–20% yields resulted in a maximum concentration of 0.83 g / L with a yield of 31.5% at 10% Tween 80 (Fig. 2c). These results suggest that using tryptophan as a substrate could be an alternative to indole due to its higher solubility without the same cytotoxicity. Furthermore, replacing indole with tryptophan is highly attractive considering the availability and availability of tryptophan.

[0092]

[0093] These experimental results imply that the addition of surfactants during culture can be useful in an eco-friendly, high-concentration bio-indigo production process that increases the solubility of indole and indigo and enhances cell permeability, thereby continuously bioconverting indole into indigo.

[0094]

[0095] 2. Separation of bioindigo using an adsorbent

[0096] To address the low solubility of indigo, we attempted to remove bio-indigo from the culture medium by adsorption. Among various commercially available adsorbents, we selected the Amberlite XAD4 adsorbent, which has a high adsorption capacity for aromatic substances. To optimize adsorption and bio-indigo production, 10 g / L of Amberlite adsorbent was applied. As a result of using the adsorbent, bio-indigo production increased over time, reaching a maximum of 1.11 g / L in 42 h (Fig. 4a). The bioconversion yield of 15 mM indole substrate was estimated to be 56.3%. Indigo adsorbed on the Amberlite XAD4 beads is shown in Fig. 3, which is combined with the indigo extracted with DMSO by Experimental Method 3. By using the adsorbent in the process, we demonstrated highly efficient indigo production with a yield increase of more than 78% compared to the existing optimized conditions, and the concentration could also be achieved at more than 1.0 g / L.

[0097]

[0098] These experimental results imply that the method of adding an adsorbent can be usefully utilized in an environmentally friendly, high-concentration bioindigo production process that physically separates bioindigo from cells and continuously bioconverts it.

[0099]

[0100] 3. Bioindigo reduction reaction

[0101] The inventors sought to develop a method for more efficiently dissolving indigo and preventing its bioconversion inhibition. They speculated that using a reducing agent to convert indigo into a water-soluble form during indigo dyeing could be applied to the bioconversion process.

[0102] Instead of sodium dithionite, which is toxic and has environmental concerns, thiourea dioxide was used. Thiourea dioxide is reported to be environmentally friendly and environmentally benign, and does not increase wastewater load compared to sodium dithionite. Various concentrations of thiourea dioxide were applied to cells to compare the degree of indigo reduction. After optimizing indigo reduction conditions, the reduction strategy was applied to indole bioconversion following bioindigo production. The results showed a concentration of 1.04 g / L and a yield of 53.0% (Fig. 4a). The use of thiourea dioxide to solubilize indigo had a significantly positive effect on indigo production.

[0103] The time-dependent profile of indigo production showed a similar trend to the bioconversion of indigo products using adsorbents. Here, the bioconversion of 15 mM indole and 15 mM tryptophan was investigated separately. As a result, when indole was used as a substrate, bioindigo production was highest at 1.35 g / L after 24 hours. Simultaneously, to maintain a high pH, ​​the pH of the medium was observed when 400 mM NaOH was treated as a reducing agent. The pH increased from 7.4 to 8.5 and then decreased to 8.2, at which point the highest production was achieved (Fig. 4b). However, when tryptophan was used as a substrate, indigo production decreased significantly to 0.61 g / L at high NaOH concentrations (Fig. 4c). The pH showed a steady increase from 7.6 to 8.5. This suggests that the use of high concentrations of NaOH may affect the stability or cellular transport of tryptophan.

[0104] Investigating the indole bioconversion reaction upon treatment with thiourea dioxide, we observed that bioindigo was produced in the culture medium and reduced to leucoindigo, which formed a clear layer in the supernatant (Fig. 4d). Experiments on bioindigo production using a reducing agent revealed that the reduction reaction and bioindigo production were significantly affected by pH, with leucoindigo remaining stable for longer periods when the pH was maintained above 8.0. However, high pH levels lead to cytotoxicity and substrate deatomization, necessitating further optimization of the production process.

[0105] The bio-indigo production concentration was further optimized by controlling the NaOH concentration and reducing agent supply time. The NaOH concentration was varied from 0 to 125 mM, and the reducing agent supply time was adjusted to 16 or 44 hours during the late bioconversion stage, and the bio-indigo production was observed. Here, 15 mM thiourea dioxide and 15 mM indole substrate were used for bioconversion. When the reducing agent was added after 44 hours without NaOH, after indigo production reached 1 g / L, the highest production concentration of 1.54 g / L was achieved (Fig. 4e). The yield level was measured to be 78.1%. Adding the reducing agent after 44 hours of cultivation resulted in a higher indigo production concentration than adding it after 16 hours.

[0106]

[0107] These experimental results imply that the method using the reducing agent of the present invention can be usefully utilized in an environmentally friendly, high-concentration bioindigo production process that continuously bioconverts indole into indigo by increasing the solubility of separated indigo and preventing reverse conversion.

[0108]

[0109] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A method for producing bio-indigo by co-cultivating a recombinant bacterium having a monooxygenase gene and a surfactant.

2. A method according to claim 1, wherein the culturing is performed at 15°C to 35°C for 15 to 50 hours.

3. A method according to claim 1, further comprising a step of adding indole, tryptophan or a combination thereof as a substrate during the co-cultivation.

4. A method according to claim 3, wherein the substrate has a concentration of 0.1 to 30 mM.

5. A method according to claim 1, wherein the monooxygenase is a flavin-containing monooxygenase (MaFMO) or toluene monooxygenase (PmT4MO).

6. A method according to claim 1, wherein the bacteria is at least one strain selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, Salmonella Typhimurium, and Serratia marcescens.

7. A method according to claim 6, wherein the E. coli is E. coli BL21 (DE3).

8. A method according to claim 1, wherein the surfactant is at least one selected from the group consisting of Tween 20, Span 20, Tween 40, Span 40, Tween 80, Span 60, Tween 85, Span 80, Pluronic 68, Brij 35, and Triton X-100d.

9. A method according to claim 1, wherein the surfactant is added at a concentration of 0.1% to 20%.

10. A method according to claim 1, further comprising a step of separating bio-indigo from the recombinant bacteria using an adsorbent.

11. A method according to claim 10, wherein the adsorbent is a hydrophobic polymer resin.

12. A method according to claim 1, further comprising a step of adding a reducing agent during the co-culturing.

13. A method according to claim 12, wherein the reducing agent is thiourea dioxide.

14. Bio-indigo obtained by the method of paragraph 1.

15. In the 14th paragraph, the bio-indigo is leuco indigo obtained by adding a reducing agent.

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