Construction of recombinant escherichia coli for synthesis of indole acetic acid ester and its application in dyeing

Abstract

This invention relates to the construction and staining application of recombinant Escherichia coli for synthesizing indoleacetic acid ester. The recombinant Escherichia coli for synthesizing indoleacetic acid ester is obtained by using recombinant Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1 as the initial strain. Then, the endogenous acetylesterase is knocked out, and the endogenous tryptophan hydrolase gene tnaA and the flavin monooxygenase gene maFMO are overexpressed, while simultaneously overexpressing the acetyltransferase gene. The indoleacetic acid ester yield of this invention is as high as 195.3 mg / L. Using indoleacetic acid ester for staining eliminates the need for reducing agents and enzyme solutions, significantly reducing staining costs.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the construction and staining application of recombinant Escherichia coli that synthesizes indoleacetic acid ester. Background Technology

[0002] Indigo is one of the most widely used dyes in the world, and it is widely used in the dyeing of denim fabrics. Currently, indigo dyeing usually involves synthesizing indigo using chemical or biological methods, followed by a reduction dyeing process. This process requires a large amount of reducing agent, which increases the cost of treating dyeing wastewater. To solve the problem of using reducing agents in indigo dyeing, some researchers have constructed recombinant strains that produce indole glycosides and proposed that cotton fabric be soaked with indole glycosides and then sprayed with glycosidase for dyeing. However, this method has a long enzymatic hydrolysis time, and the cost of using enzyme solution on a large industrial scale is still too high.

[0003] Therefore, a recombinant Escherichia coli strain that synthesizes indoleacetic acid was constructed, and staining with indoleacetic acid was performed. This process does not use reducing agents or enzyme solutions, which greatly reduces the staining cost. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects in the prior art and provide a method for constructing and staining recombinant Escherichia coli with synthetic indole acetate, which does not use reducing agents or enzyme solutions, thus greatly reducing staining costs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A recombinant Escherichia coli that synthesizes indole acetate was obtained by using an indigo-producing recombinant Escherichia coli as the initial strain, followed by sequential knockout of endogenous acetylesterase, overexpression of the endogenous tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO, and overexpression of the acetyltransferase gene.

[0007] As a further technical solution, the flavin-containing monooxygenase gene maFMO is derived from Methylophagaa minisulfidivorans;

[0008] As a further technical solution, the endogenous acetylesterase includes one or more of aes (Gene ID: 947514), yjfP (Gene ID: 948707), tesA (Gene ID: 945127), and nanS (Gene ID: 948835);

[0009] As a further technical solution, the acetyltransferase gene includes one or more of CAT, ATF1, and ATF2.

[0010] As a further technical solution, CAT is derived from Clostridium scindens;

[0011] As further technical solutions, ATF1 and ATF2 are derived from Saccharomyces cerevisiae.

[0012] As a further technical solution, the nucleotide sequence of CAT is shown in SEQ ID No. 1;

[0013] As a further technical solution, the nucleotide sequence of ATF1 is as shown in SEQ ID No. 2;

[0014] As a further technical solution, the nucleotide sequence of ATF2 is shown in SEQ ID No. 3;

[0015] As a further technical solution, the pCOLADuet-1 vector was used to simultaneously overexpress the tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO.

[0016] The acetyltransferase genes CAT, ATF1, and ATF2 were overexpressed using the pACYCDuet-1 vector.

[0017] As a further technical solution, the nucleotide sequence of the promoter tac is shown in SEQ ID NO.4.

[0018] As a further technical solution, the nucleotide sequence of the pCOLADuet-1 vector is shown in SEQ ID NO.5;

[0019] As a further technical solution, the nucleotide sequence of the pACYCDuet-1 vector is shown in SEQ ID NO.6;

[0020] As a further technical solution, the expression of the endogenous tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO is controlled by the promoter tac.

[0021] As a further technical solution, the nucleotide sequence of the promoter tac is shown in SEQ ID NO.4.

[0022] As a further technical solution, the construction of indigo-producing recombinant Escherichia coli involves using Escherichia coli as the starting strain, sequentially knocking out the bifunctional enzyme protein gene pheA (Gene ID: 947081), the tryptophan repressor protein gene trpR (Gene ID: 948917), the prebenzoic acid dehydratase gene tyrA (Gene ID: VWQ04783.1), and the aromatic amino acid transporter gene yddG (Gene ID: 945942) of Escherichia coli. Then, the flavin monooxygenase gene maFMO is integrated at the arsB and ldhA sites, the tryptophan hydrolase gene tnaA is integrated at the yghWX site, and the caveolin gene cav1 (CAA79476.1) is integrated at the poxB site, respectively, to obtain indigo-producing recombinant Escherichia coli.

[0023] As a further technical solution, the Escherichia coli includes any one of Escherichia coli MG1655, BL21(DE3), JM109, DH5α, and W3110;

[0024] As a further technical solution, the nucleotide sequence of the flavin monooxygenase gene maFMO is shown in SEQ ID NO.7;

[0025] As a further technical solution, the tryptophan hydrolase gene tnaA is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.8;

[0026] The recombinant Escherichia coli used to synthesize indoleacetic acid is used in the production of indoleacetic acid or in staining.

[0027] A method for synthesizing indoleacetic acid ester, using tryptophan as a substrate, involves fermenting recombinant *Escherichia coli* according to any one of claims 1-6 to OD0.05 in a fermentation medium at 35–38 °C. 600 After adding 0.6 to 0.8, add IPTG to a final concentration of 0.05 to 1.0 mM and induce culture at 28 to 32 °C for at least 48 h to generate indoleacetic acid ester, thus obtaining a fermentation broth containing indoleacetic acid ester.

[0028] As a further technical solution, the fermentation medium is M9 medium containing 10 g / L glucose and 5 g / L yeast extract;

[0029] As a further technical solution, the concentration of tryptophan is 2 g / L.

[0030] A method for staining with indoleacetic acid involves centrifuging the fermentation broth containing indoleacetic acid, soaking cotton cloth in the supernatant, air-drying it naturally, then applying an alkaline solution and allowing it to oxidize and air-dry at room temperature.

[0031] As a further technical solution, the alkaline solution is a 0.5-1 mol / L NaOH solution.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. This invention utilizes genetic engineering technology, using *Escherichia coli* MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1 as the initial strain. By knocking out endogenous acetylesterases aes, yjfP, tesA, and nanS, overexpressing the endogenous tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO, and overexpressing the acetyltransferase gene, a recombinant *E. coli* strain capable of synthesizing indoleacetate is obtained. Using tryptophan as a substrate, indoleacetate at a concentration of 195.3 mg / L can be obtained by fermenting in a shake flask for 48 hours.

[0034] This invention utilizes the supernatant of fermentation broth containing indole acetate to wet cotton cloth, which is then air-dried. By spraying alkaline solution and supplementing with natural air oxidation, indole acetate is converted into indole blue, thereby achieving indole blue dyeing. Compared with traditional processes, this method is simple and convenient, does not require the use of reducing agents, and solves a series of defects caused by the use of reducing agents in existing technologies. Furthermore, it does not use enzyme solutions, which greatly reduces dyeing costs. Attached Figure Description

[0035] Figure 1 Synthetic pathway of indoleacetic acid ester and synthesis diagram of indigo;

[0036] Figure 2 A schematic diagram illustrating the construction of plasmid ACYC-CAT;

[0037] Figure 3 A schematic diagram of the construction of plasmid pACYC-ATF1;

[0038] Figure 4 A schematic diagram of the construction of plasmid pACYC-ATF2;

[0039] Figure 5 Figure showing the effect of different acetyltransferases on the yield of indoleacetic acid ester;

[0040] Figure 6 The effect of knocking out acetylesterase on the production of indoleacetate is shown in the figure.

[0041] Figure 7 Image showing the effect of indoleacetic acid staining;

[0042] exist Figure 7In the table, A: 1 g / L indole acetate solution; B: IA1-2 indole acetate solution. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0045] In this invention,

[0046] 1. All commercially available products used, including PCR amplification enzymes, restriction enzymes, plasmids, DNA gel recovery kits, and column-based plasmid extraction kits, were operated according to the kit instructions. Routine molecular biology experimental procedures such as E. coli competent cell preparation, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation transformation, competent cell preparation, colony PCR, and bacterial genome extraction were performed according to Molecular Cloning Therapy.

[0047] The sequencing was performed using a Laboratory Manual (Fourth Edition). Sequencing of plasmids and PCR amplification products was completed by Suzhou Genewiz.

[0048] 2. Culture medium

[0049] (1) LB medium: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L peptone, and 15 g / L agar powder added to LB solid medium.

[0050] (2) M9 medium: 6.8 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 0.4929 g / L MgSO47H2O, 0.02 g / L CaCl2, 10 g / L glucose, 5 g / L yeast extract.

[0051] The fermentation mediums described above contain 50 mg / L kanamycin antibiotics and 100 mg / L ampicillin antibiotics, depending on the different plasmid vectors used, and 2 g / L tryptophan is added as a substrate in all of them.

[0052] Inducer concentration: During shake-flask fermentation, the final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) added was 0.2 mM.

[0053] 3. The production method of fermented indoleacetic acid ester is as follows:

[0054] During fermentation, single colonies with normal morphology were picked from the corresponding LB agar plates and transferred to 4 mL LB culture medium in test tubes containing the appropriate antibiotic. After culturing for 10-12 hours, the seed culture in the test tubes was transferred at an inoculation rate of 2% (v / v) to a shake flask containing 25 mL of fermentation medium and incubated at 37°C with shaking at 200 rpm. When the bacterial OD... 600 When the concentration reached 0.6–0.8, IPTG was added to a final concentration of 0.2 mM, and the mixture was fermented at 30 °C for 48 h. After fermentation, the fermentation broth was collected, samples were prepared, and indoleacetic acid ester was detected.

[0055] 4. The sample preparation and detection methods for indoleacetic acid esters are as follows:

[0056] Sample preparation: The fermentation broth obtained from fermentation was centrifuged at 12000 rpm for 8 min, the supernatant was collected, the supernatant was diluted with DMSO solution by a certain factor, filtered through a 0.22 μm filter membrane, and the sample was detected by Agilent high performance liquid chromatography.

[0057] Detection method: High-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II); Column: Agilent ZORBAX EclipsePlus C18 column, 4.6 × 250 mm, 5 μm; Mobile phase: Pump A was 0.1% formic acid in H2O, Pump B was acetonitrile containing 0.1% formic acid; Gradient elution program: 0 min 95% A / 5% B, 2 min 95% A / 5% B, 10 min 2% A / 98% B, 13 min 2% A / 98% B, 14 min...

[0058] 95% A / 5% B for 1 min, 95% A / 5% B for 16 min; Flow rate: 0.5 mL / min; Column temperature: 30℃; Injection volume: 10 μL.

[0059] 5. Staining performance test:

[0060] The dyed fabrics were tested for color fastness to rubbing and color fastness to washing. Stained fabrics were assessed using a standard gray scale, graded from 1 to 5, with higher grades indicating better color fastness. The testing methods followed GB / T 3920—2008 "Textiles—Tests for Color Fastness—Color Fastness to Rubbing".

[0061] And GB / T 3921—2008 "Textiles - Tests for color fastness - Color fastness to washing".

[0062] (1) Test for color fastness to rubbing: Cut a 15cm×15cm sample and rub it with a dry rubbing cloth and a wet rubbing cloth respectively. After the rubbing cloth is stained, evaluate it with a standard gray scale. Refer to GB / T 3920—2008 "Textiles - Tests for color fastness to rubbing" for specific steps.

[0063] (2) Test for color fastness to soap washing: Cut a 5cm×10cm sample, sew the sample to one or two pieces of specified lining fabric, place it in soap solution, and mechanically stir it under specified time and temperature conditions, followed by rinsing and drying. Use a gray scale to evaluate the color change of the sample and the staining of the lining fabric.

[0064] All the above sample ratings were conducted using GB / T 250-2008 "Grey Chart for Assessing Color Change" to evaluate the color fastness of the samples.

[0065] 251-2008 "Grey Standard for Staining" is used to assess the staining fastness of a sample.

[0066] 6. The strains involved in this invention are shown in Table 1.

[0067] Table 1

[0068]

[0069]

[0070]

[0071] 7. The nucleotide sequences of the primers involved in this invention are shown in Table 2.

[0072] Table 2. List of relevant primer sequences

[0073]

[0074]

[0075]

[0076]

[0077] 8. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0078] Example 1: Construction of Indigo-producing Recombinant Escherichia coli

[0079] The construction of indigo-producing recombinant *E. coli* involved sequentially knocking out the mycolate mutase (CM) / prebenzoic acid dehydratase (PD) bifunctional enzyme protein gene pheA, tryptophan repressor protein gene trpR, prebenzoic acid dehydratase gene tyrA, and aromatic amino acid transporter gene yddG from *E. coli* MG1655. Then, the flavin-containing monooxygenase gene maFMO was integrated at the arsB and ldhA sites, the tryptophan hydrolase gene tnaA was integrated at the yghWX site, and the caveolin gene cav1 was integrated at the poxB site, resulting in the indigo-producing recombinant *E. coli* MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1.

[0080] 1. Taking the knockout of the pheA gene as an example, the gene knockout steps are introduced:

[0081] (1) Using the genome of Escherichia coli MG1655 as a template, the upstream and downstream homologous fragments of pheA were amplified by PCR using primer pairs pheA-UP-F / R and pheA-DH-F / R. The upstream and downstream homologous fragments of pheA were then assembled into a linear fragment pheA-F.

[0082] (2) Using pcrEG plasmid as a template, PCR amplification was performed using primers pheA-N23-F / R to replace the N23 sequence on the pcrEG plasmid with an N23 sequence complementary to pheA, resulting in the plasmid pcrEG-pheA targeting pheA. The PCR product was used to remove template DNA with DpnI enzyme, and then transformed into E. coli JM109 competent cells using heat shock transformation. The cells were then plated on LB plates containing spectinomycin and cultured at 37°C to extract plasmids and sequence them.

[0083] (3) Take the pEcCpf1 plasmid and transform it into the competent cells of E. coli MG1655. Spread the transformed bacterial solution onto LB agar containing kanamycin and incubate overnight at 37°C to become E. coli MG1655-pEcCpf1.

[0084] (4) E.coli MG1655-pEcCpf1 was prepared into competent cells.

[0085] (5) The pcrEG-pheA plasmid and the linear fragment pheA-F were electroporated into E. coli MG1655-pEcCpf1 competent cells, plated on LB plates containing kanamycin and spectinomycin, and cultured at 37°C for 24 h. PCR colony verification was performed using primers pheA-F / R.

[0086] (6) Pick the positive clones from (5) above into 4 mL LB liquid test tubes (containing 10 mM rhamnose and 4 μL Kan), incubate at 37 °C for 12 h to remove pcrEG-pheA plasmid, and obtain recombinant E. coli MG1655ΔpheA containing pEcCpf1 plasmid for the next round of integration or knockout.

[0087] 2. Taking the integration of tac70-MaFMO at the arsB site as an example, the integration operation steps are introduced:

[0088] (1) Using the genome of Escherichia coli MG1655 as a template, the upstream and downstream fragments of arsB and the tac70-MaFMO fragment were amplified by PCR using primer pairs arsB-UP-F / R, arsB-DH-F / R and B-tac70-MaFMO-F / R. Then, the upstream and downstream fragments of arsB and tac70-MaFMO were assembled into a linear fragment arsB-UP-tac70-MaFMO-arsB-DH.

[0089] (2) Using the pcrEG plasmid as a template, the N23 sequence on the pcrEG plasmid was replaced with an N23 sequence complementary to arsB using PCR amplification with primers arsB-N23-F / R, resulting in the pcrEG-arsB plasmid targeting arsB. The PCR product was used to remove the template DNA with DpnI enzyme, and then transformed into E. coli JM109 competent cells using heat shock transformation. The cells were then plated on LB plates containing spectinomycin and cultured at 37°C to extract the plasmid and sequence it.

[0090] (3) The pEcCpf1 plasmid was transformed into the competent cells of E. coli MG1655ΔpheAΔtrpRΔtyrAΔyddG. The transformed bacterial culture was spread on LB agar plates containing kanamycin and cultured overnight at 37°C to become E. coli MG1655ΔpheAΔtrpRΔtyrAΔyddG-pEcCpf1.

[0091] (4) E. coli MG1655ΔpheAΔtrpRΔtyrAΔyddG-pEcCpf1 was prepared into competent cells.

[0092] (5) The pcrEG-arsB plasmid and the linear fragment arsB-UP-tac70-MaFMO-arsB-DH were electroporated into E. coli MG1655ΔpheAΔtrpRΔtyrAΔyddG-pEcCpf1 competent cells, plated on LB agar plates containing kanamycin and spectinomycin, and cultured at 37°C for 24 h. PCR colony verification was performed using primers arsB-F / R.

[0093] (6) Pick the positive clones from (5) above into 4 mL LB liquid tubes (containing 10 mM rhamnose and 4 μL Kan), incubate at 37 °C for 12 h to remove pcrEG-arsB plasmid, and obtain recombinant E. coli E. coli MG1655ΔpheAΔtrpRΔtyrAΔyddG,arsB::tac70-MaFMO containing pEcCpf1 plasmid for the next round of integration.

[0094] Example 2: Knockout of the acetylesterase genome

[0095] Acetylesterase gene knockout: Using the CRISPR / Cpf1 gene editing system, the genes encoding endogenous acetylesterase, aes (Gene ID: 947514), yjfP (Gene ID: 948707), tesA (Gene ID: 945127), and nanS (Gene ID: 948835), were sequentially knocked out from recombinant Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1, respectively, to obtain recombinant Escherichia coli IA1, IA2, IA3, and IA4. The knockout procedure is described using the aes gene knockout as an example (the knockout steps for other genes are the same):

[0096] (1) Using the genome of Escherichia coli MG1655 as a template, the upstream and downstream homologous fragments of aes were amplified by PCR using primer pairs aes-UP-F / R and aes-DH-F / R. The upstream and downstream homologous fragments of aes were then assembled into a linear fragment aes-F.

[0097] (2) Using the pcrEG plasmid as a template, the N23 sequence on the pcrEG plasmid was replaced with an N23 sequence complementary to aes using PCR amplification with primers aes-N23-F / R, resulting in the plasmid pcrEG-aes targeting aes. The PCR product was used to remove the template DNA with DpnI enzyme, and then transformed into E. coli JM109 competent cells using the heat shock transformation method. The cells were then plated on LB plates containing spectinomycin and cultured at 37°C to extract the plasmid and sequence it.

[0098] (3) The pEcCpf1 plasmid was transformed into competent cells of Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1. The transformed bacterial culture was plated onto LB agar plates containing kanamycin and incubated overnight at 37°C to become...

[0099] MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1-pEcCpf1.

[0100] (4) MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMO

[0101] ΔyghWX::tac70-tnaAΔpoxB::tac70-cav1-pEcCpf1 was used to prepare competent cells.

[0102] (5) Electroporate the pcrEG-aes plasmid and the linear fragment aes-F into Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddG

[0103] Competent cells of ΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1-pEcCpf1 were plated on LB agar plates containing kanamycin and spectinomycin and incubated at 37°C for 24 h. PCR colony verification was performed using primers aes-F / R.

[0104] (6) Pick the positive clones from (5) above into 4 mL LB liquid tubes (containing 10 mM rhamnose and 4 μL Kan), incubate at 37°C for 12 h to remove the pcrEG-aes plasmid, and obtain recombinant Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddG

[0105] ΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1Δaes contains pEcCpf1 plasmid and is used for the next round of integration.

[0106] (7) Pick the strain from (6) above that has had the pcrEG-aes plasmid removed and transfer it to a 4 mL LB broth containing 5 g / L glucose. Incubate at 37°C.

[0107] After culturing at 200 rpm for 12 h, the pEcCpf1 plasmid was removed by streaking on a sucrose-glucose plate (containing 25 g / L sucrose and 5 g / L glucose) to obtain recombinant Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1Δaes, which was named IA1.

[0108] Example 3: Construction of recombinant plasmids

[0109] 1) Construction of plasmid pACYC-CAT:

[0110] like Figure 2 As shown, pACYC-CAT was synthesized by Diwin Biotechnology Co., Ltd. The gene fragment of CAT (UniprotAccession: O64988) from Clostridium scindens was ligated between the NcoI and BamHI restriction sites of pACYCDuet1 to obtain the plasmid pACYC-CAT.

[0111] 2) Construction of plasmids pACYC-ATF1 and pACYC-ATF2:

[0112] like Figure 3-4 As shown, using the empty pACYCDuet1 vector as a template, the linear vector pACYC was obtained by amplification using the primer pair pACYC-F / R.

[0113] Using the genome of Saccharomyces cerevisiae as a template, PCR amplification was performed using primer pairs ATF1-F / R and ATF2-F / R to obtain linear vectors ATF1 and ATF2. Then, Gibson assembly was used to ligate the two fragments between the NcoI and BamHI restriction sites of pACYC, respectively, to obtain plasmids pACYC-ATF1 and pACYC-ATF2.

[0114] 3. Construction of plasmid pCO-tac-tnaA-tac-MaFMO:

[0115] First, using primers CO-tac-1F / R and CO-tac-2F / R, the original promoters at positions 1 and 2 of the multiple cloning site of the pCOLADuet-1 vector were amplified twice to obtain the vector pCO-tac-tac. Then, the gene tnaA was constructed between the NcoI and NotI sites at position 1 of the multiple cloning site of the expression vector pCO-tac-tac (the linear vector amplified by primers CO-V1-F / R), and MaFMO was constructed between the NdeI and MfeI sites at position 2 of the multiple cloning site of the expression vector pCO-tac-tac (the linear vector amplified by primers CO-V2-F / R), forming the recombinant plasmid pCO-Tac-tnaA-Tac-MaFMO.

[0116] Example 4: Construction and fermentation verification of recombinant Escherichia coli producing indoleacetate (without knockout of acetylesterase)

[0117] The pCO-Tac-tnaA-Tac-MaFMO recombinant plasmid constructed in Example 3 was transformed into host IA in Example 1 by chemical transformation to obtain the genetically engineered strain IA-0. Then, the plasmid vectors pACYC-ATF1, pACYC-ATF2, and pACYC-CAT in Example 3 were transformed into host IA-0 by chemical transformation to obtain genetically engineered strains IA-1, IA-2, and IA-3.

[0118] The recombinant strain was inoculated into M9 liquid medium containing kanamycin and cultured overnight at 37°C and 200 rpm for 12 h to obtain a seed culture. 500 μL of the seed culture was then inoculated into 25 mL of LB medium and cultured at 37°C and 200 rpm until OD reached the target value. 600The concentration was 0.6–0.8, and IPTG was added to a final concentration of 0.2 mM. The culture was induced at 30°C and 200 rpm for 48 h. 1 mL of fermentation broth was centrifuged at 12000 rpm for 8 min, the supernatant was collected, and diluted with DMSO for HPLC analysis. Results showed that recombinant E. coli IA-1, IA-2, and IA-3 transformed with acetyltransferase could successfully synthesize indoleacetic acid ester in M9 fermentation medium, with a maximum yield of 170.5 mg / L. Figure 5 ).

[0119] Example 5: Construction and fermentation verification of recombinant Escherichia coli producing indoleacetate (acetylesterase knocked out)

[0120] The pCO-Tac-tnaA-Tac-maFMO recombinant plasmid was transformed into strains IA1, IA2, IA3, and IA4 constructed in Example 2 via chemical transformation to obtain genetically engineered strains IA1-0, IA2-0, IA3-0, and IA4-0. The plasmid vector from Example 2 was then transformed into hosts IA1-0, IA2-0, IA3-0, and IA4-0 via chemical transformation to obtain genetically engineered strains IA1-1, IA1-2, IA1-3, IA2-1, IA2-2, IA2-3, IA3-1, IA3-2, IA3-3, IA4-1, IA4-2, and IA4-3.

[0121] The recombinant strain was inoculated into M9 liquid medium containing kanamycin and cultured overnight at 37°C and 200 rpm for 12 h to obtain a seed culture. 500 μL of the seed culture was then inoculated into 25 mL of LB medium and cultured at 37°C and 200 rpm until OD reached the target value. 600 The concentration was 0.6–0.8, and IPTG was added to a final concentration of 0.2 mM. The culture was incubated at 30°C and 200 rpm for 48 h. 1 mL of fermentation broth was centrifuged at 12000 rpm for 8 min, the supernatant was collected, and diluted with DMSO for HPLC analysis. The results showed that among the indoleacetate-producing recombinant *E. coli* with acetylesterase knockout, IA1-2 produced the highest indoleacetate yield, at 195.3 mg / L. Figure 6 ).

[0122] Example 6: Fabric dyeing method and dyeing performance test using indoleacetic acid recombinant Escherichia coli fermentation broth

[0123] Method 1: Fermentation broth staining: Centrifuge the fermentation broth of recombinant Escherichia coli strain IA1-2, collect the supernatant, soak a cotton cloth in the supernatant, and after the cloth has air-dried naturally, add 1 mL of 750 mM sodium hydroxide solution. Then, allow the cloth to air-dry naturally at room temperature. Simultaneously, prepare a 1 g / L indoleacetic acid solution and use it as a control for staining. Compare the staining effects. Results are shown below. Figure 7 The method of the present invention can achieve good staining effect.

[0124] The dyed cotton fabric was tested for color fastness according to GB / T 3920—2008 "Textiles - Tests for Color Fastness to Rubbing" and GB / T 3921—2008 "Textiles - Tests for Color Fastness to Rubbing". The results showed that the color fastness to dry and wet rubbing was grade 4 and grade 3-4 respectively, and the color fastness to washing was grade 3-4.

[0125] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A recombinant *Escherichia coli* strain for synthesizing indoleacetic acid ester, characterized in that, Construction of indigo-producing recombinant Escherichia coli: Using Escherichia coli as the initial strain, the mycolate mutase / prebenzoic acid dehydratase bifunctional enzyme protein gene pheA, tryptophan repressor protein gene trpR, prebenzoic acid dehydratase gene tyrA, and aromatic amino acid transporter gene yddG of Escherichia coli were knocked out sequentially. The flavin monooxygenase gene maFMO was integrated at the arsB site and ldhA site, the tryptophan hydrolase gene tnaA was integrated at the yghWX site, and the caveolin gene cav1 was integrated at the poxB site, respectively, to obtain indigo-producing recombinant Escherichia coli; The strain was obtained by knocking out endogenous acetylesterase and overexpressing the endogenous tryptophan hydrolase gene tnaA and the flavin monooxygenase gene maFMO, while also overexpressing the acetyltransferase gene. The endogenous acetylesterases include any combination of aes, or aes and yjfP, aes, yjfP and tesA, or aes, yjfP, tesA and nanS. The acetyltransferase gene includes any one of CAT, ATF1, and ATF2; The flavin-containing monooxygenase gene maFMO is derived from Methylophaga aminisulfidivorans; CAT is derived from Clostridium scindens, while ATF1 and ATF2 are derived from Saccharomyces cerevisiae; The nucleotide sequence of CAT is shown in SEQ ID No. 1; The nucleotide sequence of ATF1 is shown in SEQ ID No. 2; The nucleotide sequence of ATF2 is shown in SEQ ID No.

3.

2. The recombinant E. coli for synthesizing indoleacetate according to claim 1, characterized in that, The tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO were simultaneously overexpressed using the pCOLADuet-1 vector. The acetyltransferase genes CAT, ATF1, or ATF2 were overexpressed using the pACYCDuet-1 vector.

3. The recombinant E. coli for synthesizing indoleacetate according to claim 1, wherein, The expression of the endogenous tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO was controlled using the promoter tac; The nucleotide sequence of the promoter tac is shown in SEQ ID NO.

4.

4. The use of the recombinant Escherichia coli that synthesizes indole acetate as described in any one of claims 1-3 in the production of indole acetate or in staining.

5. A method of synthesizing an indoleacetate, characterized by, The recombinant E. coli synthesizing indoleacetate of any one of claims 1-3 is fermented in a tryptophan-containing fermentation medium at 35-38°C to an OD 600 0.6-0.8, and then a final concentration of 0.05-1.0 mM IPTG is added, and the culture is induced at 28-32°C for at least 48 h to produce indoleacetate, to obtain a fermentation broth containing indoleacetate.

6. A method of dyeing with an indoleacetate, characterized in that, Centrifuge the fermentation broth containing indoleacetic acid ester as described in claim 5, take the supernatant, soak a cotton cloth in it, let it air dry naturally, then apply an alkaline solution and let it air dry naturally at room temperature.

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