Method for synthesizing indigo and derivatives thereof by means of dual-enzyme cascade catalysis

Abstract

Disclosed in the present invention is a method for synthesizing indigo and derivatives thereof by means of dual-enzyme cascade catalysis, comprising: using an aminophenethyl alcohol compound (I) as a substrate to generate indigo and derivatives (III) thereof by means of a reaction catalyzed by alcohol dehydrogenase and flavin-containing monooxygenase. The present invention is convenient to operate, reduces the use of cofactors, and has high yield and good industrial application prospects in biocatalytic preparation of indigo and derivatives thereof.

Description

A method for synthesizing indigo and its derivatives by double enzyme cascade catalysis Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for synthesizing indigo and its derivatives by dual-enzyme cascade catalysis, and in particular to a method for synthesizing indigo and its derivatives by utilizing alcohol dehydrogenase and flavin-containing monooxygenase in a cascade catalysis. Background Art

[0002] Indigo and its derivatives are non-azo colorants. One of the oldest pigments known to humanity, indigo is widely used in the food, pharmaceutical, and dyeing industries. Indigo derivatives have broad research applications in dyes, semiconductor materials, and other fields.

[0003] Currently, chemical methods for synthesizing indigo require the use of aniline, a toxic compound extracted from the petroleum product benzene, as a raw material. The synthesis process involves hazardous chemicals such as formaldehyde, hydrogen cyanide, sodium amide, and strong bases, resulting in harsh reaction conditions, high pollution, and a low safety margin. Biological methods offer milder reaction conditions, but the substrate costs are relatively high. Furthermore, there are few reports on the synthesis of indigo derivatives. Therefore, the search for new green methods to synthesize indigo and its derivatives has important scientific and social significance for promoting the green upgrading of existing chemical processes and strengthening environmental protection.

[0004] Aminophenylethanol and substituted 2-aminophenylethanol are important chemical raw materials with the characteristics of easy availability and low price. They are used as raw materials for the synthesis of indigo and its derivatives with good economic benefits.

[0005] Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for synthesizing indigo and its derivatives by dual-enzyme cascade catalysis in view of the shortcomings of the existing technology.

[0007] In order to solve the above technical problems, the present invention discloses a method for synthesizing indigo and its derivatives by dual-enzyme cascade catalysis, wherein an aminophenylethanol compound I is used as a substrate, and indigo and its derivative III are produced by the catalytic reaction of alcohol dehydrogenase and flavin-containing monooxygenase;

[0008] wherein R1-R4 are independently selected from H, F, Cl, Br, I, Me, OMe, NO2, COOH, CN, NH2 or OH.

[0009] Wherein, the alcohol dehydrogenase and flavin-containing monooxygenase are added to the catalytic reaction simultaneously or in steps.

[0010] Wherein, when adopting stepwise addition catalytic reaction, the method comprises the following steps:

[0011] S1: Using aminophenylethanol compound I as substrate, alcohol dehydrogenase catalyzes the reaction to generate indole compound II;

[0012] S2: Using indole compound II as substrate, indigo and its derivative III are generated through flavin monooxygenase-catalyzed reaction;

[0013] wherein R1-R4 are independently selected from H, F, Cl, Br, I, Me, OMe, NO2, COOH, CN, NH2 or OH.

[0014] In the above method, the alcohol dehydrogenase is any one or a combination of the following a1-a9;

[0015] a1, alcohol dehydrogenase HLADH from Horse liver, the amino acid sequence of which is P00327 in UniProtKB;

[0016] a2, alcohol dehydrogenase ADH-A from Rhodococcus ruber DSM 44541, the amino acid sequence of UniProtKB is Q8KLT9;

[0017] a3, alcohol dehydrogenase YsADH from Yokenella sp. WZY002, with the amino acid sequence of UniProtKB as W6CX26;

[0018] a4, alcohol dehydrogenase TeSADH from Thermoanaerobacter ethanolicus, with the amino acid sequence of UniProtKB as P77990;

[0019] a5, alcohol dehydrogenase SyADH from Sphingobium yanoikuyae DSM 6900, with the amino acid sequence of UniProtKB being A0A084E9B3;

[0020] a6, alcohol dehydrogenase AaADH from Aromatoleum aromaticum bacterium strain EbN1, with the amino acid sequence of UniProtKB being Q5P1J5;

[0021] a7, alcohol dehydrogenase PpADH from Paracoccus pantotrophus, with the amino acid sequence of UniProtKB being A0A1I5GPJ0;

[0022] a8, alcohol dehydrogenase LkADH from Lentilactobacillus kefiri ( Lactobacillus kefiri ), the amino acid sequence of UniProtKB is Q6WVP7;

[0023] a9, alcohol dehydrogenase APDH from RMM Microcompartment of Mycobacterium smegmatis, with the amino acid sequence of UniProtKB as A0QP46.

[0024] In the above method, the flavin-containing monooxygenase is any one or a combination of the following b1-b18;

[0025] b1, flavin-containing monooxygenase mFMO from Methylophaga sp. strain SK1, with the amino acid sequence of UniProtKB being Q83XK4;

[0026] b2, flavin-containing monooxygenase NiFMO from Nitrincola lacisaponensis, with the amino acid sequence of UniProtKB being A0A063Y6V3;

[0027] b3, flavin-containing monooxygenase cFMO from Corynebacterium glutamicum (Brevibacterium saccharolyticum), with the amino acid sequence of UniProtKB being A0A6L2RF42;

[0028] b4, flavin-containing monooxygenase SMFMO from Stenotrophomonas maltophilia, with the amino acid sequence of UniProtKB being B2FLR2;

[0029] b5, flavin monooxygenase mutant mFMO(C78I) from Methylophaga sp. strain SK1, in which the cysteine ​​at position 78 of the wild-type mFMO was mutated to isoleucine;

[0030] b6, flavin-containing monooxygenase mutant mFMO (C78V) from Methylophaga sp. strain SK1, in which the cysteine ​​at position 78 of the wild-type mFMO was mutated to valine;

[0031] b7, flavin-containing monooxygenase mutant mFMO (C78L) from Methylophaga sp. strain SK1, in which the cysteine ​​at position 78 of the wild-type mFMO was mutated to leucine;

[0032] b8, flavin-containing monooxygenase mutant mFMO (C78A) from Methylophaga sp. strain SK1, in which the cysteine ​​at position 78 of the wild-type mFMO was mutated to alanine;

[0033] b9, flavin monooxygenase mutant mFMO (W319A) from Methylophaga sp. strain SK1, in which the tryptophan at position 319 of the wild-type mFMO was mutated to alanine;

[0034] b10, flavin-containing monooxygenase mutant mFMO (W319F) from Methylophaga sp. strain SK1, in which the tryptophan at position 319 of the wild-type mFMO was mutated to phenylalanine;

[0035] b11, flavin monooxygenase mutant mFMO (Y207W) from Methylophaga sp. strain SK1, in which the tyrosine 207 of the wild-type mFMO was mutated to tryptophan;

[0036] b12, flavin-containing monooxygenase mutant mFMO (Y207W / W319A) from Methylophaga sp. strain SK1, in which tyrosine at position 207 of wild-type mFMO was mutated to tryptophan, and tryptophan at position 319 was mutated to alanine;

[0037] b13, a flavin-containing monooxygenase mutant mFMO (C78I / Y207W / W319A) from Methylophaga sp. strain SK1, in which the cysteine ​​at position 78 of the wild-type mFMO was mutated to isoleucine, the tyrosine at position 207 was mutated to tryptophan, and the tryptophan at position 319 was mutated to alanine;

[0038] b14, flavin-containing monooxygenase mutant mFMO (N291T) from Methylophaga sp. strain SK1, in which the asparagine at position 291 of the wild-type mFMO was mutated to threonine;

[0039] b15, flavin-containing monooxygenase mutant mFMO (K223R) from Methylophaga sp. strain SK1, in which the lysine 223 of the wild-type mFMO was mutated to arginine;

[0040] b16, flavin-containing monooxygenase mutant mFMO (K223R / D317S) from Methylophaga sp. strain SK1, in which the lysine 223 at position A was mutated to arginine, and the aspartic acid 317 at position B was mutated to serine;

[0041] b17, a flavin-containing monooxygenase mutant mFMO (K223R / D317M) from Methylophaga sp. strain SK1, in which the lysine 223 at position A was mutated to arginine, and the aspartic acid 317 at position B was mutated to methionine;

[0042] b18, a flavin-containing monooxygenase mutant mFMO (K223R / D317A) from Methylophaga sp. strain SK1, wherein the lysine at position 223 of the wild-type mFMO is mutated to arginine, and the aspartic acid at position 317 is mutated to alanine.

[0043] Wherein, the alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of recombinant cells, crude enzyme liquid, crude enzyme powder or pure enzyme for catalysis; preferably, the preparation method of the alcohol dehydrogenase and flavin-containing monooxygenase in the form of recombinant cells, crude enzyme liquid, crude enzyme powder or pure enzyme is as follows: expressing the alcohol dehydrogenase or flavin-containing monooxygenase in a host cell to obtain a recombinant cell; preferably, introducing a nucleic acid molecule capable of expressing the alcohol dehydrogenase or flavin-containing monooxygenase into the host cell, and inducing expression to obtain a recombinant cell; preferably, lysing the recombinant cell to obtain a crude enzyme liquid; preferably, the crude enzyme liquid is dried to obtain a crude enzyme powder; preferably, the crude enzyme powder is purified by a resin to obtain a pure enzyme.

[0044] The induced expression is to add IPTG to the culture system to a final concentration of 0.1-0.6 mM (specifically 0.3 mM), and to induce culture at 20-35° C. (specifically 30° C.) for 12-24 h (specifically 20 h).

[0045] The nucleic acid molecule capable of expressing the alcohol dehydrogenase or the flavin-containing monooxygenase is introduced into the host cell in the form of a recombinant vector; the recombinant vector may be a bacterial plasmid, a bacteriophage, a yeast plasmid or a retroviral packaging plasmid carrying the gene encoding the alcohol dehydrogenase or the flavin-containing monooxygenase; the recombinant vector is specifically a recombinant plasmid obtained by replacing the gene encoding the alcohol dehydrogenase or the flavin-containing monooxygenase with a small fragment between the restriction sites NdeI and XhoI of the pET28a vector.

[0046] The host cell may be a prokaryotic cell or a eukaryotic cell; the prokaryotic cell may be a bacterium, and the eukaryotic cell may be a yeast cell; the bacterium may be Escherichia coli; the host cell is Escherichia coli, preferably E. coli BL21 (DE3).

[0047] The alcohol dehydrogenase and flavin-containing monooxygenase are used in an amount of 1-5 U / mL, preferably 3 U / mL. For example, when the alcohol dehydrogenase and flavin-containing monooxygenase are used in the form of recombinant cells for catalysis, the amount is 10-100 mg wet cells / mL. Preferably, when the alcohol dehydrogenase and flavin-containing monooxygenase are used in the form of crude enzyme liquid or crude enzyme powder for catalysis, the final concentration of the alcohol dehydrogenase and flavin-containing monooxygenase in the reaction system is 10-30 g / L (such as 20 g / L). Preferably, when the alcohol dehydrogenase and flavin-containing monooxygenase are used in the form of pure enzymes for catalysis, the final concentration of the alcohol dehydrogenase and flavin-containing monooxygenase in the reaction system is 0.1-2 g / L (such as 0.4 g / L).

[0048] The final concentration of the aminophenylethanol compound I in the reaction system is 1-10 mM (eg, 1 mM).

[0049] Wherein, the catalytic reaction is carried out in a buffer; preferably, the buffer is a CHES buffer with a concentration of 50-100 mM and a pH of 8-10, and / or a phosphate buffer with a concentration of 50-100 mM and a pH of 6-8; in some embodiments, the buffer is a CHES buffer with a pH of 9.0 and / or a phosphate buffer with a pH of 7.0.

[0050] Wherein, in the alcohol dehydrogenase catalytic reaction process, the reaction also includes a coenzyme; the coenzyme of the alcohol dehydrogenase can be specifically oxidized coenzyme I (ie NAD + ) or oxidized coenzyme II (ie NADP + ), preferably NAD + ; The coenzyme of the flavin-containing monooxygenase is specifically reduced coenzyme I (i.e. NADH) or reduced coenzyme II (i.e. NADPH).

[0051] Wherein, in the reaction system, NAD + and / or NADP + The final concentration is 0.1-2.0 mM (such as 0.5 mM).

[0052] Wherein, the temperature of the catalytic reaction is 25-35°C (specifically 30°C).

[0053] The catalytic reaction time is 2 to 48 hours (specifically 24 hours).

[0054] The present invention establishes a method for synthesizing indigo and its derivatives by a dual enzyme cascade catalysis of alcohol dehydrogenase and flavin-containing monooxygenase. In the provided method for synthesizing indigo, there is a cofactor self-circulating system. As shown in FIG1 , the cofactor regeneration system is a system in which alcohol dehydrogenase catalyzes the oxidation of aminophenylethanol compound I to indole compound II, and NAD + or NADP + Reduced to NADH or NADPH; flavin monooxygenase catalyzes indole compounds II to produce indigo and its derivatives III, and NADH or NADPH is oxidized to NAD + or NADP + , the generated NAD + or NADP + Re-engage in the process of enzyme A.

[0055] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0056] The present invention provides a novel green biosynthetic route for synthesizing indigo using aminophenylethanol compound I as a starting material via a dual-enzyme cascade catalysis. The method is easy to operate, reduces the use of cofactors, and offers high yields. It has promising industrial application prospects in the biocatalytic preparation of indigo and its derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0058] FIG1 is a schematic diagram of the reaction of alcohol dehydrogenase coupled with flavin-containing monooxygenase to prepare indigo and its derivatives. DETAILED DESCRIPTION

[0059] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0060] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0061] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0062] Example 1. Preparation of engineered bacteria expressing alcohol dehydrogenase HLADH and flavin monooxygenase mFMO

[0063] The genes encoding alcohol dehydrogenase HLADH and flavin-containing monooxygenase mFMO are codon-optimized and synthesized using Escherichia coli as the host, respectively, and then ligated into respective expression vectors to construct the recombinant expression vectors. These expression vectors are commonly used in the art. Specifically, the vector described in the present invention is pET28a. A small DNA fragment between the enzyme recognition sites NdeI and XhoI of pET28a is replaced with the fully synthesized genes encoding the relevant enzymes to produce the recombinant expression vectors.

[0064] The above-mentioned recombinant expression vector is transferred to a suitable microbial host by heat shock transformation. The specific operation of heat shock transformation is to add 5ul of recombinant plasmid to 100ul of thawed competent microbial host cells, shake well, place on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and then place on ice for 5 minutes. Then, add 900ul of LB culture medium, mix well, and culture at 37°C for 45 minutes. After centrifugation at 8000rpm for 5 minutes, remove 800ul of supernatant, and mix the remaining 200ul and spread it on a kanamycin-resistant plate. Culture at 37°C for 12 hours to obtain transformants. The transformants are picked and cultured and sent to a genetic company for sequencing to confirm successful transformation, thereby obtaining a genetically engineered strain. The microorganisms are various conventional host microorganisms in the field, as long as they can meet the requirements of stable self-replication of the above-mentioned recombinant expression vector and effective expression of the alcohol dehydrogenase or flavin monooxygenase gene. The host microorganism is Escherichia coli BL21 (DE3).

[0065] The site-directed mutagenesis kit was used to perform site-directed mutagenesis on the coding gene of flavin monooxygenase mFMO to obtain a mutant of the flavin monooxygenase mFMO gene, and then the above steps were followed to obtain the corresponding genetically engineered strain expressing flavin monooxygenase b5-b18.

[0066] Example 2. Expression of alcohol dehydrogenase HLADH and flavin monooxygenase mFMO engineered bacteria and preparation of recombinant cells, crude enzyme solution, crude enzyme powder and pure enzyme

[0067] 1. 10 μl of the genetically engineered strain obtained in Example 1 was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, shaken overnight at 37°C and 200 rpm for 12 h, and then inoculated into LB medium containing 50 μg / mL kanamycin at a volume percentage of 1%. The culture was cultured at 37°C until the OD600 was 0.6-1.0, IPTG was added at a final concentration of 0.3 mmol / L, and expression was induced at 30°C and 200 rpm for 20 h. The culture was then centrifuged at 4°C and 8000 rpm for 5 min to collect the precipitated cells (i.e., recombinant cells). The collected cells were resuspended in phosphate buffer (50 mmol / L, pH 7.0) to obtain a cell suspension; the cells were then ultrasonically disrupted in an ice bath to obtain a disrupted sample (i.e., a crude enzyme solution).

[0068] 2. Centrifuge the crude enzyme solution at 4°C and 4000 rpm for 40 min, collect the supernatant, freeze part of the supernatant at -80°C, and then freeze-dry it in a vacuum dryer to obtain crude enzyme powder.

[0069] 3. Add the remaining supernatant to a gravity protein purification column containing 2 mL of Ni-NTA agarose purification resin, shake for 2 minutes to allow it to fully bind, and elute with 50 mmol / L, pH 7.0 phosphate buffer with different imidazole concentrations (20 mmol / L, 50 mmol / L, 100 mmol / L, 200 mmol / L, 500 mmol / L imidazole).

[0070] 4. The collected eluate (200 mmol / L imidazole) was concentrated and washed to obtain pure enzyme. The enzyme activity of alcohol dehydrogenase HLADH was 0.118 U / ul, and the enzyme activity of flavin monooxygenase mFMO was 0.882 U / ul. Enzyme activity is defined as the amount of enzyme required to generate / consume NADH cofactor per minute.

[0071] Example 3: Alcohol dehydrogenase HLADH catalyzes 2-aminophenylethanol to produce indole

[0072] The pure alcohol dehydrogenase HLADH prepared in Example 2 was used to catalyze the production of indole from 2-aminophenylethanol.

[0073] 1. Add 50mM CHES buffer (pH 9.0), 1mM 2-aminophenylethanol, and 1mM NAD in the reaction system. + or NADP + and 3 U / mL of pure alcohol dehydrogenase HLADH enzyme. The reaction system was reacted at 30° C. for 24 hours to obtain a reaction solution.

[0074] 2. Mix 200ul of ethyl acetate and 200ul of reaction solution thoroughly, then centrifuge at 10000rpm for 1min, collect the supernatant and filter with a filter membrane. The filtrate was tested by liquid chromatography (HPLC). The conversion rates were 99% (NAD + ) and 20% (NADP + ), the yields were 99% (NAD + ) and 20% (NADP + The results showed that alcohol dehydrogenase HLADH could catalyze the conversion of 2-aminophenylethanol to indole.

[0075] Example 4: Flavin-containing monooxygenase mFMO catalyzes the conversion of indole to indigo

[0076] The pure enzyme containing flavin monooxygenase mFMO prepared in Example 2 catalyzed the conversion of indole to indigo.

[0077] 1. Add 50 mM phosphate buffer (pH 7.0), indole to a final concentration of 1 mM, NADH or NADPH to a final concentration of 1 mM, and 3 U / mL of pure flavin monooxygenase mFMO to the reaction system. Incubate the reaction system at 30°C for 24 hours to obtain a reaction solution.

[0078] 2. Thoroughly mix sufficient DMF and 200 μl of the reaction solution to dissolve the blue precipitate. Centrifuge at 10,000 rpm for 1 minute, collect the supernatant, and filter through a membrane. The filtrate is analyzed by liquid chromatography (HPLC). Conversion rates are 99% (NADH) and 99% (NADPH), respectively, and yields are 75% (NADH) and 80% (NADPH), respectively. These results demonstrate that mFMO, a flavin-containing monooxygenase, can catalyze the conversion of indole to indigo.

[0079] Example 5: Alcohol dehydrogenase HLADH and flavin-containing monooxygenase mFMO dual enzyme cascade catalyzes the production of indigo from 2-aminophenylethanol

[0080] The alcohol dehydrogenase HLADH prepared in Example 2 and the pure enzyme containing flavin monooxygenase mFMO were used to catalyze the conversion of 2-aminophenylethanol to indigo.

[0081] 1. Add 50mM phosphate buffer (pH 7.0), 1mM 2-aminophenylethanol, and 0.5mM NAD to the reaction system. + or NADP + , 3 U / mL of alcohol dehydrogenase HLADH and 3 U / mL of pure flavin monooxygenase mFMO. The reaction system was reacted at 30°C for 24 hours to obtain a reaction solution.

[0082] 2. Mix enough DMF and 200ul of the reaction solution thoroughly to dissolve the blue precipitate, then centrifuge at 10000rpm for 1min, collect the supernatant and filter with a filter membrane. The filtrate was tested by liquid chromatography (HPLC). The conversion rates were 99% (NAD + ) and 25% (NADP + ), with yields of 90% (NAD + ) and 17% (NADP + The results showed that pure enzymes of alcohol dehydrogenase HLADH and flavin monooxygenase mFMO could catalyze the conversion of 2-aminophenylethanol to indigo.

[0083] Example 6

[0084] The same as Example 5, using different aminophenylethanol compounds I, alcohol dehydrogenase and flavin monooxygenase in 50mM phosphate buffer (pH 7.0), 0.5mM NAD + , reacted at 30℃ for 24h, and the results are shown in Table 1.

[0085] The synthesis method for different aminophenylethanol compounds I is as follows: 2 g of substituted 2-nitrotoluene, 114 mg of paraformaldehyde, and 182.4 μl of Triton-B were dissolved in 2 mL of DMSO and reacted at 90°C for 2 h. The mixture was diluted with saturated ammonium chloride, extracted with ethyl acetate, washed with saturated NaCl, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography using ethyl acetate / n-hexane (1:5) to obtain substituted 2-nitrophenylethanol. 0.5 mmol of substituted 2-nitrophenylethanol, 5 mmol of ammonium formate, and 5 mmol of zinc powder were dissolved in 3 mL of MeOH and reacted at room temperature for 1 h. The mixture was filtered through celite, washed with methanol, and dried by spin drying. The mixture was then extracted with ethyl acetate / water, dried over anhydrous sodium sulfate, and separated by column chromatography using ethyl acetate / n-hexane (1:10) to obtain substituted 2-aminophenylethanol. The NMR results of the substituted 2-aminophenylethanol are shown in Table 2.

[0086] Table 1

[0087] Note: The dosage of the aminophenylethanol compound I, alcohol dehydrogenase and flavin-containing monooxygenase is the same as that in Example 5; the alcohol dehydrogenase and flavin-containing monooxygenase are both pure enzymes, and the preparation method is the same as that in Examples 1 and 2.

[0088] Table 2 NMR data of aminophenylethanol compound I

[0089] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for synthesizing indigo and its derivatives by double enzyme cascade catalysis, characterized in that: Using aminophenylethanol compound I as substrate, indigo and its derivative III are produced through the catalytic reaction of alcohol dehydrogenase and flavin-containing monooxygenase; Wherein, R1-R4 are independently selected from H, F, Cl, Br, I, Me, OMe, NO2, COOH, CN, NH2 or OH.

2. The method according to claim 1, characterized in that: The alcohol dehydrogenase and the flavin-containing monooxygenase are added to the catalytic reaction simultaneously or in steps.

3. The method according to claim 2, characterized in that: When the catalytic reaction is added step by step, the method comprises the following steps: S1: Using aminophenylethanol compound I as substrate, an indole compound II is generated through alcohol dehydrogenase-catalyzed reaction; S2: Using indole compound II as substrate, indigo and its derivative III are generated through flavin monooxygenase-catalyzed reaction; Wherein, R1-R4 are independently selected from H, F, Cl, Br, I, Me, OMe, NO2, COOH, CN, NH2 or OH.

4. The method according to any one of claims 1 to 3, characterized in that: The alcohol dehydrogenase is any one or a combination of the following a1-a9; a1, alcohol dehydrogenase HLADH from Horse liver, the amino acid sequence of which is P00327 in UniProtKB; a2, alcohol dehydrogenase ADH-A from Rhodococcus ruber DSM 44541, the amino acid sequence of UniProtKB is Q8KLT9; a3, alcohol dehydrogenase YsADH from Yokenella sp. WZY002, the amino acid sequence of which is W6CX26 in UniProtKB; a4, alcohol dehydrogenase TeSADH from Thermoanaerobacter ethanolicus, with the amino acid sequence of UniProtKB as P77990; a5, alcohol dehydrogenase SyADH from Sphingobium yanoikuyae DSM 6900, the amino acid sequence of which is A0A084E9B3 in UniProtKB; a6, alcohol dehydrogenase AaADH from Aromatoleum aromaticum bacterium strain EbN1, with the amino acid sequence of UniProtKB as Q5P1J5; a7, alcohol dehydrogenase PpADH from Paracoccus pantotrophus, with the amino acid sequence of UniProtKB A0A1I5GPJ0; a8, alcohol dehydrogenase LkADH from Lentilactobacillus kefiri (Lactobacillus kefiri), the amino acid sequence of UniProtKB is Q6WVP7; a9, alcohol dehydrogenase APDH from RMM Microcompartment of Mycobacterium smegmatis, the UniProtKB amino acid sequence is A0QP46.

5. The method according to any one of claims 1 to 3, characterized in that: The flavin-containing monooxygenase is as follows Any one or combination of b1-b18; b1, flavin-containing monooxygenase mFMO from Methylophaga sp. strain SK1, with the amino acid sequence of UniProtKB as Q83XK4; b2, flavin-containing monooxygenase NiFMO from Nitrincola lacisaponensis, with the amino acid sequence of UniProtKB A0A063Y6V3; b3, flavin-containing monooxygenase cFMO from Corynebacterium glutamicum (Brevibacterium saccharolyticum), the amino acid sequence of UniProtKB is A0A6L2RF42; b4, flavin-containing monooxygenase SMFMO from Stenotrophomonas maltophilia, with the amino acid sequence of UniProtKB as B2FLR2; b5, flavin monooxygenase mutant mFMO (C78I) from Methylophaga sp. strain SK1, i.e., the cysteine ​​at position 78 of wild-type mFMO was mutated to isoleucine; b6, flavin monooxygenase mutant mFMO (C78V) from Methylophaga sp. strain SK1, i.e., the cysteine ​​at position 78 of wild-type mFMO was mutated to valine; b7, flavin monooxygenase mutant mFMO (C78L) from Methylophaga sp. strain SK1, i.e., the cysteine ​​at position 78 of wild-type mFMO was mutated to leucine; b8, flavin-containing monooxygenase mutant mFMO (C78A) from Methylophaga sp. strain SK1, i.e., the cysteine ​​at position 78 of wild-type mFMO was mutated to alanine; b9, flavin-containing monooxygenase mutant mFMO (W319A) from Methylophaga sp. strain SK1, i.e., the tryptophan at position 319 of the wild-type mFMO was mutated to alanine; b10, flavin-containing monooxygenase mutant mFMO (W319F) from Methylophaga sp. strain SK1, i.e., the tryptophan at position 319 of the wild-type mFMO was mutated to phenylalanine; b11, flavin monooxygenase mutant mFMO (Y207W) from Methylophaga sp. strain SK1, i.e., the tyrosine at position 207 of the wild-type mFMO was mutated to tryptophan; b12, flavin monooxygenase mutant mFMO (Y207W / W319A) from Methylophaga sp. strain SK1, i.e., the tyrosine at position 207 of the wild-type mFMO was mutated to tryptophan, and the tryptophan at position 319 was mutated to alanine; b13, flavin monooxygenase mutant mFMO (C78I / Y207W / W319A) from Methylophaga sp. strain SK1, i.e., the cysteine ​​at position 78 of the wild-type mFMO was mutated to isoleucine, the tyrosine at position 207 was mutated to tryptophan, and the tryptophan at position 319 was mutated to alanine; b14, flavin monooxygenase mutant mFMO (N291T) from Methylophaga sp. strain SK1, i.e., the asparagine at position 291 of the wild-type mFMO was mutated to threonine; b15, flavin-containing monooxygenase mutant mFMO (K223R) from Methylophaga sp. strain SK1, i.e., the lysine 223 of wild-type mFMO was mutated to arginine; b16, flavin-containing monooxygenase mutant mFMO (K223R / D317S) from Methylophaga sp. strain SK1, i.e., the lysine 223 of wild-type mFMO was mutated to arginine, and the aspartic acid 317 was mutated to serine; b17, flavin-containing monooxygenase mutant mFMO (K223R / D317M) from Methylophaga sp. strain SK1, i.e., the lysine 223 of wild-type mFMO was mutated to arginine, and the aspartic acid 317 was mutated to methionine; b18, a flavin monooxygenase mutant mFMO (K223R / D317A) derived from Methylophaga sp. strain SK1, wherein the lysine at position 223 of the wild-type mFMO was mutated to arginine, and the aspartic acid at position 317 was mutated to alanine.

6. The method according to any one of claims 1 to 3, characterized in that: The alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of recombinant cells, crude enzyme liquid, crude enzyme powder or pure enzyme for catalysis; preferably, the preparation method of the alcohol dehydrogenase and flavin-containing monooxygenase in the form of recombinant cells, crude enzyme liquid, crude enzyme powder or pure enzyme is as follows: expressing alcohol dehydrogenase or flavin-containing monooxygenase in a host cell to obtain a recombinant cell; preferably, introducing a nucleic acid molecule capable of expressing alcohol dehydrogenase or flavin-containing monooxygenase into the host cell, and obtaining a recombinant cell after inducing expression; preferably, lysing the recombinant cell to obtain a crude enzyme liquid; preferably, the crude enzyme liquid is dried to obtain a crude enzyme powder; preferably, the crude enzyme powder is purified by resin to obtain a pure enzyme; preferably, the host cell is Escherichia coli, preferably E.coli BL21 (DE3).

7. The method according to claim 6, characterized in that: When the alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of recombinant cells for catalysis, the dosage is 10-100 mg wet cells / mL; preferably, when the alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of crude enzyme liquid or crude enzyme powder for catalysis, the final concentration of the alcohol dehydrogenase and flavin-containing monooxygenase in the reaction system is 10-30 g / L; preferably, when the alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of pure enzymes for catalysis, the final concentration of the alcohol dehydrogenase and flavin-containing monooxygenase in the reaction system is 0.1-2 g / L.

8. The method according to claim 1 or 2, characterized in that: The final concentration of the aminophenylethanol compound I in the reaction system is 1-10 mM; preferably, the catalytic reaction is carried out in a buffer; preferably, the buffer is a CHES buffer with a concentration of 50-100 mM and a pH of 8-10, and / or a phosphate buffer with a concentration of 50-100 mM and a pH of 6-8.

9. The method according to claim 1 or 2, characterized in that: In the alcohol dehydrogenase catalyzed reaction process, the reaction also includes NAD + and / or NADP + Preferably, in the reaction system, NAD + and / or NADP + The final concentration is 0.1-2.0 mM.

10. The method according to claim 1 or 2, characterized in that: The temperature of the catalytic reaction is 25 to 35° C.; preferably, the time of the catalytic reaction is 2 to 48 hours.

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