A method for synthesizing indigo and its derivatives by a two-enzyme cascade catalysis

Abstract

The application discloses a method for synthesizing indigo and derivatives thereof through a double-enzyme cascade catalysis, and the method comprises the following steps: taking an aminophenethyl alcohol compound I as a substrate, and then performing a catalytic reaction through an alcohol dehydrogenase and a flavin-containing monooxygenase to generate the indigo and derivatives III. The method is convenient to operate, reduces the use of auxiliary factors, and has a higher yield, and therefore, has a good industrial application prospect in the biological catalysis preparation of the indigo and derivatives thereof.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for the synthesis of indigo and its derivatives via a dual-enzyme cascade catalysis, and more particularly to a method for the synthesis of indigo and its derivatives via a cascade catalysis of alcohol dehydrogenase and flavin-containing monooxygenase. Background Technology

[0002] Indigo and its derivatives are non-azo colorants. Indigo is one of the oldest known pigments and is widely used in the food, pharmaceutical, and printing and dyeing industries. Indigo derivatives have broad application research value in dyes, semiconductor materials, and other fields.

[0003] Currently, the chemical methods for synthesizing indigo require aniline, a toxic compound extracted from petroleum product benzene, as a raw material. The synthesis process involves hazardous chemicals such as formaldehyde, hydrogen cyanide, sodium amide, and strong alkalis, with harsh reaction conditions, high pollution, and low safety. Biological methods offer milder reaction conditions, but the cost of substrates is relatively high. Furthermore, there are very few reported methods for synthesizing indigo derivatives. Therefore, finding new green methods for synthesizing indigo and its derivatives is of significant scientific and social value for promoting the green upgrading of existing chemical processes and strengthening environmental protection.

[0004] Aminophenylethanol and substituted 2-aminophenylethanol are important chemical raw materials, characterized by their easy availability and low price. They are used as raw materials for the synthesis of indigo and its derivatives, and have good economic benefits. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for the synthesis of indigo and its derivatives by a two-enzyme cascade catalysis, which addresses the shortcomings of the prior art.

[0006] To address the aforementioned technical problems, this invention discloses a method for synthesizing indigo and its derivatives via a dual-enzyme cascade catalysis, using aminophenylethanol compound I as a substrate, which is catalyzed by alcohol dehydrogenase and flavin-containing monooxygenase to generate indigo and its derivative III.

[0007]

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

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

[0010] When the catalytic reaction is added in steps, the method includes the following steps:

[0011] S1: Using aminophenylethanol compound I as a substrate, indole compound II is generated by alcohol dehydrogenase catalysis;

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

[0013]

[0014] Among them, R1-R4 are independently selected from H, F, Cl, Br, I, Me, OMe, NO2, COOH, CN, NH2 or OH.

[0015] In the above method, the alcohol dehydrogenase is any one or a combination of several of the following a1-a9;

[0016] a1, derived from the alcohol dehydrogenase HLADH from horse liver, has the amino acid sequence UniProtKB P00327.

[0017] a2 is derived from the alcohol dehydrogenase ADH-A of Rhodococcus ruber DSM 44541, with the amino acid sequence UniProtKB being Q8KLT9.

[0018] a3, derived from the alcohol dehydrogenase YsADH of Yokenella sp. WZY002, has the amino acid sequence UniProtKB as W6CX26;

[0019] a4 is derived from the alcohol dehydrogenase TeSADH of Thermoanaerobacter ethanolicus, with the amino acid sequence UniProtKB being P77990.

[0020] a5, derived from the alcohol dehydrogenase SyADH of Sphingobium yanoikuyae DSM 6900, has the amino acid sequence UniProtKB A0A084E9B3.

[0021] a6 is derived from the alcohol dehydrogenase AaADH of Aromatoleum aromaticum bacterium strain EbN1, and its amino acid sequence UniProtKB is Q5P1J5.

[0022] a7, derived from the alcohol dehydrogenase PpADH of Paracoccus pantotrophus, has the amino acid sequence UniProtKB as A0A1I5GPJ0.

[0023] a8 is derived from the alcohol dehydrogenase LkADH of Lentilactobacillus kefiri (Lactobacillus kefiri), and the amino acid sequence UniProtKB is Q6WVP7.

[0024] a9 is derived from the alcohol dehydrogenase APDH of the RMM Microcompartment of Mycobacterium smegmatis, with the amino acid sequence UniProtKB being A0QP46.

[0025] In the above method, the flavin-containing monooxygenase is any one or a combination of several of the following b1-b18;

[0026] b1 is derived from the flavin-containing monooxygenase mFMO of Methylophaga sp.strain SK1, with the amino acid sequence UniProtKB being Q83XK4;

[0027] b2, derived from the flavin-containing monooxygenase NiFMO of Nitrincola lacisaponensis, has the amino acid sequence UniProtKB as A0A063Y6V3;

[0028] b3 is derived from the flavin-containing monooxygenase cFMO of Corynebacterium glutamicum (Brevibacterium saccharolyticum), with the amino acid sequence UniProtKB being A0A6L2RF42.

[0029] b4, derived from the flavin-containing monooxygenase SMFMO of Stenotrophomonas maltophilia, has the amino acid sequence UniProtKB as B2FLR2.

[0030] b5 is derived from the flavin-containing monooxygenase mutant mFMO (C78I) of Methylophaga sp.strain SK1, which is to mutate the 78th cysteine ​​of wild-type mFMO to isoleucine.

[0031] b6 is derived from the flavin-containing monooxygenase mutant mFMO (C78V) of Methylophaga sp.strain SK1, which is to mutate the 78th cysteine ​​of wild-type mFMO to valine.

[0032] b7 is derived from the flavin-containing monooxygenase mutant mFMO (C78L) of Methylophaga sp.strain SK1, which is to mutate the 78th cysteine ​​of wild-type mFMO to leucine.

[0033] b8 is derived from the flavin-containing monooxygenase mutant mFMO (C78A) of Methylophaga sp.strain SK1, which is a mutation of cysteine ​​at position 78 of wild-type mFMO to alanine.

[0034] b9 is derived from the flavin-containing monooxygenase mutant mFMO (W319A) of Methylophaga sp.strain SK1, which is to mutate tryptophan at position 319 of wild-type mFMO to alanine;

[0035] b10 is derived from the flavin-containing monooxygenase mutant mFMO (W319F) of Methylophaga sp.strain SK1, which is to mutate tryptophan at position 319 of wild-type mFMO to phenylalanine;

[0036] b11 is derived from the flavin-containing monooxygenase mutant mFMO (Y207W) of Methylophaga sp.strain SK1, which is to mutate the tyrosine at position 207 of wild-type mFMO to tryptophan;

[0037] b12 is derived from the flavin-containing monooxygenase mutant mFMO (Y207W / W319A) of Methylophaga sp.strain SK1, which mutates tyrosine at position 207 of wild-type mFMO to tryptophan and tryptophan at position 319 to alanine.

[0038] b13 is derived from the flavin-containing monooxygenase mutant mFMO (C78I / Y207W / W319A) of Methylophaga sp.strain SK1, which mutates cysteine ​​at position 78 of wild-type mFMO to isoleucine, tyrosine at position 207 to tryptophan, and tryptophan at position 319 to alanine.

[0039] b14 is derived from the flavin-containing monooxygenase mutant mFMO (N291T) of Methylophaga sp.strain SK1, which is to mutate the asparagine at position 291 of wild-type mFMO to threonine;

[0040] b15 is derived from the flavin-containing monooxygenase mutant mFMO (K223R) of Methylophaga sp.strain SK1, which is to mutate the lysine at position 223 of wild-type mFMO to arginine;

[0041] b16 is derived from the flavin-containing monooxygenase mutant mFMO (K223R / D317S) of Methylophaga sp.strain SK1, which mutates lysine at position 223 of wild-type mFMO to arginine and aspartic acid at position 317 to serine.

[0042] b17 is derived from the flavin-containing monooxygenase mutant mFMO (K223R / D317M) of Methylophaga sp.strain SK1, which mutates lysine at position 223 of wild-type mFMO to arginine and aspartic acid at position 317 to methionine.

[0043] b18 is derived from the flavin-containing monooxygenase mutant mFMO (K223R / D317A) of Methylophaga sp.strain SK1, which mutates lysine at position 223 of wild-type mFMO to arginine and aspartic acid at position 317 to alanine.

[0044] The alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of recombinant cells, crude enzyme solution, 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 solution, crude enzyme powder, or pure enzyme is as follows: expressing alcohol dehydrogenase or flavin-containing monooxygenase in host cells to obtain recombinant cells; preferably, introducing nucleic acid molecules capable of expressing alcohol dehydrogenase or flavin-containing monooxygenase into host cells, and obtaining recombinant cells after induction of expression; preferably, lysing the recombinant cells to obtain crude enzyme solution; preferably, drying the crude enzyme solution to obtain crude enzyme powder; preferably, purifying the crude enzyme powder with resin to obtain pure enzyme.

[0045] The induction of expression involves adding IPTG to the culture system to a final concentration of 0.1-0.6 mM (e.g., 0.3 mM) and inducing culture at 20-35℃ (e.g., 30℃) for 12-24 h (e.g., 20 h).

[0046] The nucleic acid molecule capable of expressing alcohol dehydrogenase or flavin monooxygenase is introduced into the host cell in the form of a recombinant vector; the recombinant vector may be a bacterial plasmid, bacteriophage, yeast plasmid or retroviral packaging plasmid carrying the coding gene of the alcohol dehydrogenase or flavin monooxygenase; the recombinant extraction specifically refers to the recombinant plasmid obtained by replacing the small fragment between the restriction sites NdeI and XhoI of the pET28a vector with the coding gene of the alcohol dehydrogenase or flavin monooxygenase.

[0047] 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).

[0048] The alcohol dehydrogenase and flavin monooxygenase are used in a system of 1-5 U / mL, preferably 3 U / mL. When catalyzing in the form of recombinant cells, the dosage is 10-100 mg wet cells / mL. Preferably, when the alcohol dehydrogenase and flavin monooxygenase are used in the form of crude enzyme solution or crude enzyme powder, the final concentration of alcohol dehydrogenase and flavin monooxygenase in the reaction system is 10-30 g / L (e.g., 20 g / L). Preferably, when the alcohol dehydrogenase and flavin monooxygenase are used in the form of pure enzymes, the final concentration of alcohol dehydrogenase and flavin monooxygenase in the reaction system is 0.1-2 g / L (e.g., 0.4 g / L).

[0049] The final concentration of the aminophenylethanol compound I in the reaction system is 1-10 mM (e.g., 1 mM).

[0050] The catalytic reaction is carried out in a buffer solution; preferably, the buffer solution is a CHES buffer solution with a concentration of 50-100 mM and a pH value of 8-10, and / or a phosphate buffer solution with a concentration of 50-100 mM and a pH value of 6-8; in some embodiments, the buffer solution is a CHES buffer solution with a concentration of 50 mM and a pH value of 9.0 and / or a phosphate buffer solution with a concentration of 50 mM and a pH value of 7.0.

[0051] In the alcohol dehydrogenase catalyzed reaction, the reaction also includes a coenzyme; specifically, the coenzyme of the alcohol dehydrogenase may be oxidized coenzyme I (i.e., NAD). + ) or oxidized coenzyme II (i.e., NADP) + ), preferably NAD + The coenzyme containing flavin monooxygenase is specifically reduced coenzyme I (i.e., NADH) or reduced coenzyme II (i.e., NADPH).

[0052] In the reaction system, NAD + and / or NADP + The final concentration is 0.1-2.0 mM (e.g., 0.5 mM).

[0053] The temperature of the catalytic reaction is 25–35°C (specifically, 30°C).

[0054] The catalytic reaction takes 2 to 48 hours (specifically 24 hours).

[0055] This invention establishes a method for the synthesis of indigo and its derivatives via a dual-enzyme cascade catalysis of alcohol dehydrogenase and flavin-containing monooxygenase. The provided method for synthesizing indigo includes a cofactor self-circulation system, such as... Figure 1 As shown, the cofactor regeneration system is a process in which alcohol dehydrogenase catalyzes the oxidation of aminophenylethanol compound I to indole compound II, NAD... + or NADP + It is reduced to NADH or NADPH; flavin-containing monooxygenases catalyze the formation of indole compounds II into indigo and its derivatives III, and NADH or NADPH is oxidized to NAD. + or NADP + The generated NAD + or NADP + It re-engages in the process of enzyme A.

[0056] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0057] This invention provides a novel green biosynthetic route for the synthesis of indigo from aminophenylethanol compound I via a two-enzyme cascade catalysis. This invention is easy to operate, reduces the use of cofactors, and achieves high yields, showing promising industrial application prospects in the biocatalytic preparation of indigo and its derivatives. Attached Figure Description

[0058] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0059] Figure 1 A schematic diagram of the reaction for preparing indigo and its derivatives by coupling alcohol dehydrogenase with flavin-containing monooxygenase. Detailed Implementation

[0060] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0062] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0063] Example 1: Preparation of engineered bacteria expressing alcohol dehydrogenase HLADH and engineered bacteria expressing flavin monooxygenase mFMO

[0064] The coding genes for alcohol dehydrogenase HLADH and flavin-containing monooxygenase mFMO were synthesized using *E. coli* as the host, with codon optimization performed as needed. The synthesized genes were then ligated into various expression vectors to construct the recombinant expression vector. The expression vectors used were various conventional vectors in the art. Specifically, the vector in this invention was pET28a. A small DNA fragment between the NdeⅠ and XhoⅠ restriction sites of pET28a was replaced with the coding gene for the relevant enzyme after full gene synthesis, resulting in the recombinant expression vector.

[0065] The recombinant expression vector was transformed into a suitable microbial host using a heat shock transformation method. The specific procedure for heat shock transformation was as follows: 5 μL of the recombinant plasmid was added to 100 μL of thawed competent microbial host cells, shaken well, placed on ice for 30 minutes, then heat-shocked in a 42°C water bath for 90 seconds, followed by 5 minutes on ice. Then, 900 μL of LB medium was added, mixed well, and incubated at 37°C for 45 minutes. After centrifugation at 8000 rpm for 5 minutes, 800 μL of supernatant was removed, and the remaining 200 μL was mixed and spread onto a kanamycin-resistant plate. The plate was incubated at 37°C for 12 hours to obtain transformants. Transformants were picked, cultured, and sent to a gene company for sequencing to confirm successful transformation, resulting in a genetically engineered strain. The microorganisms used were various conventional host microorganisms in the field, as long as they could stably replicate the recombinant expression vector and effectively express the alcohol dehydrogenase or flavin-containing monooxygenase gene. The host microorganism was *Escherichia coli* BL21(DE3).

[0066] The coding gene for flavin monooxygenase mFMO was mutated at a site using a site-directed mutagenesis kit to obtain a mutant of the flavin monooxygenase mFMO gene. Then, following the steps described above, the corresponding genetically engineered strains expressing flavin monooxygenase b5-b18 were obtained.

[0067] Example 2: Expression of alcohol dehydrogenase HLADH and flavin-containing monooxygenase mFMO engineered bacteria, and preparation of recombinant cells, crude enzyme solution, crude enzyme powder and pure enzyme.

[0068] 1. Take 10 μL of the genetically engineered strain obtained in Example 1 and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 200 rpm for 12 h with shaking. Then, inoculate it into LB medium containing 50 μg / mL kanamycin at a volume percentage of 1% and incubate at 37°C until the OD600 reaches 0.6-1.0. Add IPTG to a final concentration of 0.3 mmol / L and induce expression at 30°C and 200 rpm for 20 h. Centrifuge at 4°C and 8000 rpm for 5 min and collect the precipitated bacterial cells (i.e., recombinant cells). Resuspend the collected bacterial cells in phosphate buffer (50 mmol / L, pH 7.0) to obtain a bacterial cell suspension. Then, sonicate the bacterial cells under ice bath conditions to obtain the broken sample (i.e., crude enzyme solution).

[0069] 2. Centrifuge the crude enzyme solution at 4℃ and 4000rpm for 40min, collect the supernatant, freeze part of the supernatant at -80℃ and then freeze-dry it using a vacuum dryer to obtain crude enzyme powder.

[0070] 3. Add the remaining supernatant to a gravity-type protein purification column containing 2 mL of Ni-NTA agarose purification resin, shake for 2 min to ensure complete binding, and elute sequentially with different concentrations of imidazole in 50 mmol / L, pH 7.0 phosphate buffer (20 mmol / L, 50 mmol / L, 100 mmol / L, 200 mmol / L, 500 mmol / L imidazole).

[0071] 4. Concentrate and wash the collected eluent (200 mmol / L imidazole) to obtain pure enzymes. The activity of alcohol dehydrogenase HLADH is 0.118 U / ul, and the activity of flavin monooxygenase mFMO is 0.882 U / ul. Enzyme activity is defined as the amount of enzyme required to generate / consume NADH cofactor per minute.

[0072] Example 3: The alcohol dehydrogenase HLADH catalyzes the formation of indole from 2-aminophenylethanol.

[0073] The purified enzyme of HLADH, the alcohol dehydrogenase prepared in Example 2, catalyzed the formation of indole from 2-aminophenylethanol.

[0074] 1. Add the following to the reaction system sequentially: 50 mM CHES buffer (pH 9.0), 1 mM 2-aminophenylethanol, and 1 mM NAD+. + or NADP + The reaction mixture contained 3 U / mL of purified HLADH alcohol dehydrogenase. The reaction system was incubated at 30°C for 24 hours to obtain the reaction solution.

[0075] 2. Thoroughly mix 200 μL of ethyl acetate and 200 μL of the reaction solution, then centrifuge at 10,000 rpm for 1 min. Collect the supernatant and filter it through a filter membrane. Analyze the filtrate using high-performance liquid chromatography (HPLC). The conversion rates were 99% (NAD...). + ) and 20% (NADP) + The yields were 99% (NAD). + ) and 20% (NADP) + The results showed that the alcohol dehydrogenase HLADH could catalyze the formation of indole from 2-aminophenylethanol.

[0076] Example 4: The flavin-containing monooxygenase mFMO catalyzes the formation of indole from indole to indigo.

[0077] The purified enzyme containing flavin monooxygenase mFMO prepared in Example 2 was used to catalyze the formation of indole from indole.

[0078] 1. Add the following to the reaction system sequentially: 50 mM phosphate buffer (pH 7.0), 1 mM indole, 1 mM NADH or NADPH, and 3 U / mL of purified enzyme containing flavin monooxygenase mFMO. Incubate the reaction system at 30°C for 24 hours to obtain the reaction solution.

[0079] 2. Mix sufficient DMF and 200 μL of reaction solution thoroughly to dissolve the blue precipitate completely. Centrifuge at 10,000 rpm for 1 min, collect the supernatant, and filter through a filter membrane. Analyze the filtrate using high-performance liquid chromatography (HPLC). The conversion rates were 99% (NADH) and 99% (NADPH), with yields of 75% (NADH) and 80% (NADPH), respectively. The results indicate that the flavin monooxygenase mFMO can catalyze the formation of indole from indole.

[0080] Example 5: The dual-enzyme cascade of alcohol dehydrogenase HLADH and flavin-containing monooxygenase mFMO catalyzes the production of indigo from 2-aminophenylethanol.

[0081] The pure enzymes prepared in Example 2, HLADH (an alcohol dehydrogenase) and mFMO (a flavin-containing monooxygenase), catalyze the production of indigo from 2-aminophenylethanol.

[0082] 1. Add the following to the reaction system sequentially: 50 mM phosphate buffer (pH 7.0), 1 mM 2-aminophenylethanol, and 0.5 mM NAD+. + or NADP + The reaction mixture contained 3 U / mL of alcohol dehydrogenase HLADH and 3 U / mL of flavin-containing monooxygenase mFMO. The reaction system was incubated at 30°C for 24 hours to obtain the reaction solution.

[0083] 2. Mix sufficient DMF and 200 μL of reaction solution thoroughly to dissolve the blue precipitate completely. Centrifuge at 10,000 rpm for 1 min, collect the supernatant, and filter through a filter membrane. Analyze the filtrate using high-performance liquid chromatography (HPLC). The conversion rates were 99% (NAD... + ) and 25% (NADP) + The yields were 90% (NAD). + ) and 17% (NADP + The results showed that pure enzymes of alcohol dehydrogenase HLADH and flavin-containing monooxygenase mFMO could catalyze the production of indigo from 2-aminophenylethanol.

[0084] Example 6

[0085] Similar to Example 5, different aminophenylethanol compounds I, alcohol dehydrogenases, and flavin-containing monooxygenases were used in 50 mM phosphate buffer (pH 7.0) and 0.5 mM NAD+. + The reaction was carried out at 30℃ for 24 hours, and the results are shown in Table 1.

[0086] The synthesis methods of different aminophenylethanol compounds I are as follows: 2g of substituted 2-nitrotoluene, 114mg of paraformaldehyde, and 182.4ul of Triton-B were dissolved in 2mL of DMSO and reacted at 90℃ for 2h. After dilution with saturated ammonium chloride, extraction with ethyl acetate, washing with saturated NaCl, drying with anhydrous sodium sulfate, concentration under reduced pressure, and separation by ethyl acetate / n-hexane 1:5 column chromatography, substituted 2-nitrophenylethanol was obtained. 0.5mmol of substituted 2-nitrophenylethanol, 5mmol of ammonium formate, and 5mmol of zinc powder were dissolved in 3mL of MeOH and reacted at room temperature for 1h. After filtration with diatomaceous earth, washing with methanol, evaporation to dryness, extraction with ethyl acetate / water, drying with anhydrous sodium sulfate, and separation by ethyl acetate / n-hexane 1:10 column chromatography, substituted 2-aminophenylethanol was obtained. The NMR results of substituted 2-aminophenylethanol are shown in Table 2.

[0087] Table 1

[0088]

[0089]

[0090] Note: The amounts of aminophenylethanol compound I, alcohol dehydrogenase, and flavin monooxygenase are the same as in Example 5; the alcohol dehydrogenase and flavin monooxygenase are pure enzymes, and the preparation methods are the same as in Examples 1 and 2.

[0091] Table 2. NMR data of aminophenylethanol compound I

[0092]

[0093]

[0094] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for the synthesis of indigo and its derivatives via a two-enzyme cascade catalysis, characterized in that, Using aminophenylethanol compound I as a substrate, indigo and its derivative III are generated through a reaction catalyzed by alcohol dehydrogenase and flavin-containing monooxygenase; wherein the alcohol dehydrogenase and flavin-containing monooxygenase are added simultaneously to catalyze the reaction; the reaction also includes a coenzyme, wherein the coenzyme is NAD. + ; Among them, R1-R4 are independently selected from H, F, Cl, Br, I, Me, OMe, NO2, COOH, CN, NH2 or OH; The flavin-containing monooxygenase is any one or a combination of several of the following: b1, b3, b10-b13, b17, and b18. b1, derived from the flavin-containing monooxygenase mFMO of Methylophaga sp. strain SK1, has an amino acid sequence of UniProtKB of Q83XK4; b3 is a flavin-containing monooxygenase cFMO derived from Corynebacterium glutamicum (Brevibacterium saccharolyticum), with the amino acid sequence UniProtKB being A0A6L2RF42. b10 is derived from the flavin-containing monooxygenase mutant mFMO (W319F) of Methylophaga sp. strain SK1, which is to mutate tryptophan at position 319 of wild-type mFMO to phenylalanine; b11 is derived from the flavin-containing monooxygenase mutant mFMO (Y207W) of Methylophaga sp. strain SK1, which is to mutate the tyrosine at position 207 of wild-type mFMO to tryptophan; b12 is derived from the flavin-containing monooxygenase mutant mFMO (Y207W / W319A) of Methylophaga sp. strain SK1, which is to mutate tyrosine at position 207 of wild-type mFMO to tryptophan and tryptophan at position 319 to alanine. b13 is derived from the flavin-containing monooxygenase mutant mFMO (C78I / Y207W / W319A) of Methylophaga sp. strain SK1, which mutates cysteine ​​at position 78 of wild-type mFMO to isoleucine, tyrosine at position 207 to tryptophan, and tryptophan at position 319 to alanine. b17 is derived from the flavin-containing monooxygenase mutant mFMO (K223R / D317M) of Methylophaga sp. strain SK1, which is to mutate lysine at position 223 of wild-type mFMO to arginine and aspartic acid at position 317 to methionine. b18 is derived from the flavin-containing monooxygenase mutant mFMO (K223R / D317A) of Methylophaga sp. strain SK1, which mutates lysine at position 223 of wild-type mFMO to arginine and aspartic acid at position 317 to alanine. The alcohol dehydrogenase is any one or a combination of several of the following: a1, a5, a6, and a8. a1, derived from the alcohol dehydrogenase HLADH from horse liver, has the amino acid sequence UniProtKB P00327. a5, derived from the alcohol dehydrogenase SyADH of Sphingobium yanoikuyae DSM 6900, has the amino acid sequence UniProtKB A0A084E9B3. a6 is derived from the alcohol dehydrogenase AaADH of Aromatoleum aromaticum bacterium strain EbN1, and its amino acid sequence UniProtKB is Q5P1J5. a8 is derived from the alcohol dehydrogenase LkADH of Lentilactobacillus kefiri (Lactobacillus kefiri), and its amino acid sequence UniProtKB is Q6WVP7.

2. The method according to claim 1, characterized in that, The aminophenylethanol compound I is 4-bromo-2-aminophenylethanol, the alcohol dehydrogenase is a1, derived from the horse liver alcohol dehydrogenase HLADH, and the amino acid sequence UniProtKB is P00327; the flavin-containing monooxygenase is b18, derived from the flavin-containing monooxygenase mutant mFMO (K223R / D317A) of Methylophaga sp. strain SK1, that is, the lysine at position 223 of wild-type mFMO is mutated to arginine, and the aspartic acid at position 317 is mutated to alanine.

3. The method according to claim 1, characterized in that, The alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of recombinant cells, crude enzyme solution, crude enzyme powder or pure enzyme for catalysis.

4. The method according to claim 3, characterized in that, The preparation methods for alcohol dehydrogenase and flavin-containing monooxygenase in the form of recombinant cells, crude enzyme solution, crude enzyme powder or pure enzyme are as follows: alcohol dehydrogenase or flavin-containing monooxygenase is expressed in host cells to obtain recombinant cells.

5. The method according to claim 4, characterized in that, The preparation method specifically involves: introducing a nucleic acid molecule capable of expressing alcohol dehydrogenase or flavin monooxygenase into a host cell, and obtaining recombinant cells after induction of expression; lysing the recombinant cells to obtain a crude enzyme solution; drying the crude enzyme solution to obtain crude enzyme powder; and purifying the crude enzyme powder with resin to obtain pure enzyme.

6. The method according to claim 5, characterized in that, The host cell was Escherichia coli BL21 (DE3).

7. The method according to claim 3, 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; when the alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of crude enzyme solution or crude enzyme powder for catalysis, the final concentration of alcohol dehydrogenase and flavin-containing monooxygenase in the reaction system is 10-30 g / L; when the alcohol dehydrogenase and flavin-containing monooxygenase are present in the form of pure enzymes for catalysis, the final concentration of alcohol dehydrogenase and flavin-containing monooxygenase in the reaction system is 0.1-2 g / L.

8. The method according to claim 1, characterized in that, The final concentration of the aminophenylethanol compound I in the reaction system is 1-10 mM.

9. The method according to claim 1, characterized in that, In the reaction system, NAD + The final concentration is 0.1-2.0 mM.

10. The method according to claim 1, characterized in that, The temperature of the catalytic reaction is 25~35℃.

11. The method according to claim 1, characterized in that, The catalytic reaction takes 2 to 48 hours.

12. A method for the synthesis of indigo and its derivatives via a two-enzyme cascade catalysis, characterized in that, Indigo is produced by reacting aminophenylethanol compounds as substrates with alcohol dehydrogenase and flavin-containing monooxygenase; the alcohol dehydrogenase and flavin-containing monooxygenase are added simultaneously to catalyze the reaction; the reaction also includes a coenzyme, namely NAD+. + ; Among them, the aminophenylethanol compound is 2-aminophenylethanol, the alcohol dehydrogenase is a7, which is derived from the alcohol dehydrogenase PpADH of Paracoccus pantotrophus, and the amino acid sequence UniProtKB is A0A1I5GPJ0; the flavin monooxygenase is b1, which is derived from the flavin monooxygenase mFMO of Methylophaga sp. strain SK1, and the amino acid sequence UniProtKB is Q83XK4.

Images