Construction and dyeing application of recombinant escherichia coli for synthesizing indole acetate

Abstract

The invention relates to construction and dyeing application of recombinant Escherichia coli for synthesizing indole acetate, which is characterized in that recombinant Escherichia coli MG1655 delta pheA delta trpR delta tyrA delta yddG delta arsB: tac 70-MaFMO delta ldhA: tac 70-MaFMO delta yghWX: tac 70-tnaA delta poxB: tac 70-cav1 is taken as an initial strain, then endogenous acetylesterase is knocked out, and the recombinant Escherichia coli for synthesizing indole acetate is obtained by the steps of: preparing the recombinant Escherichia coli for synthesizing indole acetate. An endogenous tryptophan hydrolase gene tnaA and a flavin-containing monooxygenase gene maFMO are overexpressed, and meanwhile, an acetyltransferase gene is overexpressed, so that the tryptophan-containing flavin-containing monooxygenase gene is obtained; the yield of the indole acetate reaches up to 195.3 mg / L. When the indole acetate is used for dyeing, a reducing agent and an enzyme solution are not used, so that the dyeing cost is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the construction and dyeing application of a recombinant Escherichia coli for synthesizing indoleacetic acid ester. Background Art

[0002] Indigo is one of the most widely used dyes in the world and is widely used in the printing and dyeing of denim fabrics. However, the current indigo dyeing usually adopts a reduction dyeing process after synthesizing indigo by chemical or biological methods, which requires a large amount of reducing agent, increasing the treatment cost of dyeing wastewater. In order to solve the problem of using reducing agents for indigo dyeing, some people have also constructed a recombinant strain that produces indole glycosides, and proposed to soak cotton cloth with indole glycosides and then spray glycosidase for dyeing. However, the enzymatic hydrolysis time of this method is relatively long, and the cost of using enzyme solution on an industrial scale is still too high.

[0003] Therefore, a recombinant Escherichia coli that synthesizes indoleacetic acid ester is constructed, and indoleacetic acid ester is used for staining, which does not use reducing agents or enzyme solutions, greatly reducing the staining cost. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects in the prior art and provide a recombinant Escherichia coli construction and dyeing application for synthesizing indoleacetic acid esters, which does not use a reducing agent or an enzyme solution, thereby greatly reducing the dyeing cost.

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

[0006] A recombinant Escherichia coli for synthesizing indoleacetic acid ester, wherein indigo-producing recombinant Escherichia coli is used as an initial strain, and then endogenous acetylesterase is knocked out in sequence, and endogenous tryptophan hydrolase gene tnaA and flavin monooxygenase gene maFMO are overexpressed, and acetyltransferase gene is overexpressed at the same time, so as to obtain the recombinant Escherichia coli for synthesizing indoleacetic acid ester.

[0007] As a further technical solution, the flavin monooxygenase gene maFMO is derived from Methylophagaaminisulfidivorans;

[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 a further technical solution, 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 tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO were simultaneously overexpressed using the pCOLADuet-1 vector;

[0016] The pACYCDuet-1 vector was used to overexpress acetyltransferase genes CAT, ATF1 and ATF2 respectively.

[0017] As a further technical solution, the nucleotide sequence of the promoter tac is shown as 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 promoter tac is used to control the expression of the endogenous tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO;

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

[0022] As a further technical solution, the construction of indigo-producing recombinant Escherichia coli was carried out by taking Escherichia coli as the starting strain, knocking out the mycolate mutase (CM) / prephenate dehydratase (PD) bifunctional enzyme protein gene pheA (GeneID: 947081), tryptophan repressor protein gene trpR (Gene ID: 948917), prephenate dehydratase gene tyrA (GeneID: VWQ04783.1) and aromatic amino acid transporter gene yddG (Gene ID: 945942) of Escherichia coli in sequence, integrating the flavin monooxygenase gene maFMO at the arsB and ldhA sites, the tryptophan hydrolase gene tnaA at the yghWX site, and the caveolin gene cav1 (CAA79476.1) 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 for synthesizing indoleacetic acid ester is used in producing indoleacetic acid ester or dyeing.

[0027] A method for synthesizing indoleacetic acid esters, using tryptophan as a substrate, fermenting the recombinant Escherichia coli for synthesizing indoleacetic acid esters according to any one of claims 1 to 6 in a fermentation medium at 35 to 38°C until OD 600 After the pH value reaches 0.6-0.8, add IPTG with a final concentration of 0.05-1.0 mM and induce culture at 28-32° C. for at least 48 hours to generate indoleacetic acid ester, thereby obtaining a fermentation broth containing indoleacetic acid ester.

[0028] As a further technical solution, the fermentation medium is an 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 dyeing method using indoleacetic acid ester comprises the steps of centrifuging the fermentation liquid containing indoleacetic acid ester, taking the supernatant to soak the cotton cloth, air-drying the cotton cloth naturally, applying alkali solution, and air-drying the cotton cloth naturally by oxidation at room temperature.

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

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention utilizes genetic engineering technology, takes Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1 as the initial strain, knocks out endogenous acetylesterases aes, yjfP, tesA and nanS, overexpresses endogenous tryptophan hydrolase gene tnaA and flavin monooxygenase gene maFMO, and overexpresses acetyltransferase gene, to obtain recombinant Escherichia coli for synthesizing indoleacetic acid ester, which uses tryptophan as a substrate and can obtain 195.3 mg / L of indoleacetic acid ester in a shake flask fermentation for 48 hours.

[0034] The present invention soaks cotton cloth with the supernatant of fermentation liquid containing indoleacetic acid ester, and after air drying, indoleacetic acid ester is converted into indigo by spraying alkali solution and air oxidation under natural conditions, thereby achieving indigo dyeing. Compared with the traditional process, the process is simple and convenient, does not require the use of a reducing agent, and solves a series of defects caused by the use of reducing agent dyeing in the prior art. In addition, enzyme solution is not used, which greatly reduces the dyeing cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Synthesis pathway of indole acetate and synthesis diagram of indigo;

[0036] Figure 2 Schematic diagram of the construction of plasmid ACYC-CAT;

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

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

[0039] Figure 5 The figure shows the effect of different acetyltransferases on the production of indoleacetic acid ester;

[0040] Figure 6 This is a diagram showing the effect of knocking out acetyl esterase on indole acetate production;

[0041] Figure 7 This is the indoleacetic acid staining effect diagram;

[0042] exist Figure 7In the figure, A: 1 g / L indoleacetic acid ester solution; B: indoleacetic acid ester solution of IA1-2. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0045] In the present invention,

[0046] 1. All commercial products such as PCR amplification enzymes, endonucleases, plasmids, DNA gel recovery kits and column plasmid extraction kits used were operated according to the instructions of the kits. E. coli competent cell preparation, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation transformation, competent cell preparation, colony PCR and bacterial genome extraction and other routine molecular biology experimental operations were performed according to Molecular Cloning:

[0047] ALaboratory Manual (Fourth Edition). The sequencing of plasmids and PCR amplification products was completed by Suzhou Genewise Co., Ltd.

[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 was added to LB solid medium.

[0050] (2) M9 medium: 6.8 g / L Na 2 HPO 4 , 3g / L KH 2 PO 4 , 0.5g / LNaCl, 1g / LNH 4 Cl, 0.4929 g / LMgSO 4 7H 2 O, 0.02 g / L CaCl 2 , 10g / L glucose, 5g / L yeast extract.

[0051] The fermentation medium contains 50 mg / L kanamycin antibiotic and 100 mg / L ampicillin antibiotic according to different plasmid vector conditions, and 2 g / L tryptophan is added as a substrate.

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

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

[0054] During the fermentation process, pick a single colony with normal colony morphology on the corresponding LB solid plate and put it into a 4mL test tube LB culture liquid containing the corresponding antibiotic. After culturing for 10-12 hours, transfer the seed liquid in the test tube to a shake flask containing 25mL fermentation medium at a 2% (v / v) inoculation volume and culture at 37°C and 200rpm. When the bacterial OD 600 When the pH value 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 the fermentation, the fermentation liquid was collected and samples were prepared for the detection of indoleacetic acid ester.

[0055] 4. The sample preparation and detection method of indoleacetic acid ester are as follows:

[0056] Sample preparation: The fermentation broth obtained by fermentation was centrifuged at a centrifugal speed of 12000 rpm for 8 minutes, and the supernatant was collected. The supernatant was diluted a certain multiple with DMSO solution, filtered with 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); chromatographic column: Agilent ZORBAX Eclipse Plus C18 column, 4.6×250 mm, 5 μm; mobile phase: A pump with 0.1% formic acid in H 2 The O and B pumps were acetonitrile containing 0.1% formic acid; the gradient elution program was 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

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

[0059] 5. Dyeing performance test:

[0060] The dyed fabrics are tested for color fastness to abrasion and soap washing. The stained fabrics are evaluated with a standard gray scale, which is divided into 1-5 levels. The higher the level, the better the color fastness. The test method refers to GB / T 3920-2008 "Textiles - Tests for color fastness - Color fastness to abrasion"

[0061] And GB / T 3921-2008 "Textiles - Tests for colour fastness - Colour fastness to washing with soap".

[0062] (1) Abrasion color fastness test: Cut a 15cm×15cm sample and rub it with a dry and wet abrasion cloth respectively. After the abrasion cloth is stained, use the standard gray sample card to evaluate. For specific steps, refer to GB / T 3920-2008 "Textiles - Tests for color fastness - Abrasion color fastness".

[0063] (2) Soap-washing color fastness test: Cut a 5cm×10cm sample, sew the sample with one or two pieces of specified adjacent fabrics, put it in soap solution, stir it mechanically under specified time and temperature conditions, then rinse and dry it. Use a gray sample card to evaluate the color change of the sample and the staining of the adjacent fabric.

[0064] All the above samples are rated using GB / T 250-2008 Grey Scale for Assessing Color Change to assess the color fastness of the samples.

[0065] 251-2008 Grey Scale for Assessing Staining is used to assess the staining fastness of samples.

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

[0067] Table 1

[0068]

[0069]

[0070]

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

[0072] Table 2. Related primer sequence list

[0073]

[0074]

[0075]

[0076]

[0077] 8. In the present invention, the raw materials used are commercially available unless otherwise specified.

[0078] Example 1: Construction of indigo-producing recombinant Escherichia coli

[0079] Construction of indigo-producing recombinant Escherichia coli. Based on Escherichia coli MG1655, the mycolate mutase (CM) / prephenate dehydratase (PD) bifunctional enzyme protein gene pheA, tryptophan repressor protein gene trpR, prephenate dehydratase gene tyrA and aromatic amino acid transporter gene yddG of Escherichia coli MG1655 were knocked out in sequence, and the flavin monooxygenase gene maFMO, the tryptophan hydrolase gene tnaA were integrated at the arsB and ldhA sites, and the caveolin gene cav1 was integrated at the poxB site, respectively, to obtain the indigo-producing recombinant Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1.

[0080] Among them, 1. Taking the knockout of pheA gene as an example, the steps of gene knockout 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, and then the upstream and downstream homologous fragments of pheA were assembled into a linear fragment pheA-F.

[0082] (2) Using the pcrEG plasmid as a template, the primers pheA-N23-F / R were used to replace the N23 sequence on the pcrEG plasmid with the N23 sequence complementary to pheA by PCR amplification technology, and the plasmid pcrEG-pheA with targeted pheA was obtained. The PCR product was used to remove the template DNA using DpnⅠ enzyme, and was transformed into the competent Escherichia coli JM109 by heat shock transformation method, and then spread on LB plates containing spectinomycin and cultured at 37℃ to extract the plasmid and sequence it.

[0083] (3) The pEcCpf1 plasmid was transferred into the competent cell of E. coli MG1655. The transformed bacterial solution was spread on an LB plate containing kanamycin and cultured overnight in a 37°C incubator to obtain E. coli MG1655-pEcCpf1.

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

[0085] (5) The pcrEG-pheA plasmid and the linear fragment pheA-F were electroporated into E. coli MG1655-pEcCpf1 competent cells, spread 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) The positive clone colonies in the above (5) were picked into a 4 mL LB liquid test tube (containing rhamnose at a final concentration of 10 mM and 4 μL Kan), and cultured at 37°C for 12 h to remove the pcrEG-pheA plasmid, thereby obtaining recombinant E. coli MG1655ΔpheA containing the pEcCpf1 plasmid and using it 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 steps are introduced:

[0088] (1) Using the Escherichia coli MG1655 genome 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, respectively. Subsequently, 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 primers arsB-N23-F / R were used to replace the N23 sequence on the pcrEG plasmid with the N23 sequence complementary to arsB by PCR amplification technology, and the plasmid pcrEG-arsB with targeting arsB was obtained. The PCR product was used to remove the template DNA using DpnⅠ enzyme, and was transformed into the competent Escherichia coli JM109 by heat shock transformation method, and then spread on LB plates containing spectinomycin and cultured at 37℃ to extract the plasmid and sequence it.

[0090] (3) The pEcCpf1 plasmid was transformed into the competent cell of Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddG, and the transformed bacterial solution was spread on an LB plate containing kanamycin and cultured in a 37°C incubator overnight to obtain E. coliMG1655ΔpheAΔtrpRΔtyrAΔyddG-pEcCpf1.

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

[0092] (5) The pcrEG-arsB plasmid and the linear fragment arsB-UP-tac70-MaFMO-arsB-DH were electroporated into Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddG-pEcCpf1 competent cells, spread on LB 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) The positive clone colonies in the above (5) were picked into a 4 mL LB liquid test tube (containing rhamnose at a final concentration of 10 mM and 4 μL Kan), and cultured at 37°C for 12 h to remove the pcrEG-arsB plasmid, thereby obtaining recombinant E. coli E. coliMG1655ΔpheAΔtrpRΔtyrAΔyddG, arsB::tac70-MaFMO containing the pEcCpf1 plasmid and used for the next round of integration.

[0094] Example 2: Knockout of acetylesterase genome

[0095] Knockout of acetylesterase genes: The CRISPR / Cpf1 gene editing system was used to knock out the genes aes (Gene ID: 947514), yjfP (Gene ID: 948707), tesA (Gene ID: 945127) and nanS (Gene ID: 948835) encoding endogenous acetylesterase in the indigo-producing recombinant Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1, respectively, to obtain recombinant Escherichia coli IA1, IA2, IA3 and IA4, respectively. Taking the knockout gene aes as an example (the knockout steps of other genes are the same), the knockout steps are introduced:

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

[0097] (2) Using the pcrEG plasmid as a template, the primers aes-N23-F / R were used to replace the N23 sequence on the pcrEG plasmid with the N23 sequence complementary to aes by PCR amplification technology, and the plasmid pcrEG-aes with targeted aes was obtained. The PCR product was used to remove the template DNA using DpnⅠ enzyme, and was transformed into the competent Escherichia coli JM109 by heat shock transformation method, and then spread on LB plates containing spectinomycin and cultured at 37℃ to extract the plasmid and sequence it.

[0098] (3) The pEcCpf1 plasmid was transformed into the competent cell of Escherichia coli MG1655ΔpheAΔtrpRΔtyrAΔyddGΔarsB::tac70-MaFMOΔldhA::tac70-MaFMOΔyghWX::tac70-tnaAΔpoxB::tac70-cav1, and the transformed bacterial solution was spread on an LB plate containing kanamycin and cultured in a 37°C incubator overnight to form a

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

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

[0101] ΔyghWX::tac70-tnaAΔpoxB::tac70-cav1-pEcCpf1 was prepared into competent cells.

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

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

[0104] (6) The positive clones in (5) were transferred to a 4 mL LB liquid test tube (containing a final concentration of 10 mM rhamnose and 4 μL Kan), and cultured at 37°C for 12 h to remove the pcrEG-aes plasmid and obtain recombinant E. coli MG1655ΔpheAΔtrpRΔtyrAΔyddG

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

[0106] (7) The strain from which the pcrEG-aes plasmid was removed in step (6) was placed in a 4 mL LB test tube medium containing 5 g / L glucose at 37°C.

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

[0108] Example 3: Construction of recombinant plasmid

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

[0110] like Figure 2 As shown, pACYC-CAT was synthesized by Diying Biotechnology Co., Ltd., and the gene fragment of gene CAT (Uniprot Accession: O64988) derived from Clostridium scindens was connected between the restriction sites NcoI and BamHI 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, pACYCDuet1 empty vector was used as template and amplified using primer pair pACYC-F / R to obtain the linear vector pACYC;

[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, and then the two fragments were connected to the restriction sites NcoI and BamHI of pACYC by Gibson assembly to finally obtain plasmids pACYC-ATF1 and pACYC-ATF2.

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

[0115] First, the primers CO-tac-1F / R and CO-tac-2F / R were used for two whole plasmid amplifications to replace the original promoters at the first and second positions of the multiple cloning site of the pCOLADuet-1 vector with the promoter Ptac, respectively, to obtain the vector pCO-tac-tac; then, the gene tnaA was constructed between the NcoI and NotI sites at the first position of the multiple cloning site of the expression vector pCO-tac-tac (linear vector amplified by primers CO-V1-F / R), and MaFMO was constructed between the NdeI and MfeI sites at the second position of the multiple cloning site of the expression vector pCO-tac-tac (linear vector amplified by primers CO-V2-F / R), to form the recombinant plasmid pCO-Tac-tnaA-Tac-MaFMO.

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

[0117] The pCO-Tac-tnaA-Tac-MaFMO recombinant plasmid constructed in Example 3 was transferred into the 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 respectively transferred into the host IA-0 by chemical transformation to obtain the 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 seed solution. 500 μL of seed solution was inoculated into 25 mL of LB medium and cultured at 37°C and 200 rpm until OD 600The concentration of IPTG was 0.6-0.8, and the final concentration was 0.2 mM. The induction culture was continued at 30°C and 200 rpm for 48 hours. 1 mL of fermentation broth was taken, centrifuged at 12000 rpm for 8 minutes, the supernatant was taken, and diluted with DMSO for a certain multiple for HPLC determination. The results showed that the recombinant E. coli IA-1, IA-2 and IA-3 into which the acetyltransferase was transferred could successfully synthesize indoleacetic acid ester in M9 fermentation medium, with the highest yield of 170.5 mg / L ( Figure 5 ).

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

[0120] The pCO-Tac-tnaA-Tac-maFMO recombinant plasmid was respectively transferred into the strains IA1, IA2, IA3 and IA4 constructed in Example 2 by means of chemical transformation to obtain genetically engineered strains IA1-0, IA2-0, IA3-0 and IA4-0, and then the plasmid vector in Example 2 was respectively transferred into the hosts IA1-0, IA2-0, IA3-0 and IA4-0 by means of 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 seed solution. 500 μL of seed solution was inoculated into 25 mL of LB medium and cultured at 37°C and 200 rpm until OD 600 The concentration of IPTG was 0.6-0.8, and the final concentration was 0.2 mM. The induction culture was continued at 30°C and 200 rpm for 48 hours. 1 mL of fermentation broth was taken, centrifuged at 12000 rpm for 8 minutes, the supernatant was taken, and diluted with DMSO for a certain multiple for HPLC determination. The results showed that among the indole acetate-producing recombinant E. coli with acetyl esterase knocked out, IA1-2 had the highest indole acetate production, which was 195.3 mg / L ( Figure 6 ).

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

[0123] Method 1: Fermentation broth dyeing: Centrifuge the fermentation broth of the recombinant Escherichia coli strain IA1-2, take the supernatant, soak the cotton cloth with the supernatant, add 1mL of 750mM sodium hydroxide solution after the cotton cloth is naturally air-dried, and then place the cotton cloth at room temperature for natural oxidation and air-drying, and the dyeing can be successful. At the same time, prepare 1g / L indole acetate solution, and use 1g / L indole acetate solution for dyeing as a control to compare the dyeing effect. The results are shown in Figure 7 , the method of the present invention can achieve good dyeing effect.

[0124] The dyed cotton cloth was tested for color fastness according to GB / T 3920-2008 "Textiles - Tests for Color Fastness - Color Fastness to Rubbing" and GB / T 3921-2008 "Textiles - Tests for Color Fastness - Color Fastness to Washing with Soap". 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 with soap was grade 3-4.

[0125] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A recombinant Escherichia coli for synthesizing indoleacetic acid ester, characterized in that: Using indigo-producing recombinant Escherichia coli as the starting strain, the endogenous acetylesterase was knocked out, and the endogenous tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO were overexpressed, and the acetyltransferase gene was also overexpressed.

2. The recombinant Escherichia coli for synthesizing indoleacetic acid ester according to claim 1, characterized in that: The flavin monooxygenase gene maFMO is derived from Methylophaga aminisulfidivorans.

3. The recombinant Escherichia coli for synthesizing indoleacetic acid ester according to claim 1, characterized in that: The endogenous acetylesterase includes one or more of aes, yjfP, tesA, and nanS; The acetyltransferase gene includes one or more of CAT, ATF1 and ATF2.

4. The recombinant Escherichia coli for synthesizing indoleacetic acid ester according to claim 3, characterized in that: CAT is derived from Clostridium scindens, and 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.

5. The recombinant Escherichia coli for synthesizing indoleacetic acid ester according to claim 1, characterized in that: The pCOLADuet-1 vector was used to simultaneously overexpress the tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO; The pACYCDuet-1 vector was used to overexpress acetyltransferase genes CAT, ATF1 and ATF2 respectively.

6. The recombinant Escherichia coli for synthesizing indoleacetic acid ester according to claim 1, characterized in that: The promoter tac was used to control the expression of the endogenous tryptophan hydrolase gene tnaA and the flavin-containing monooxygenase gene maFMO; The nucleotide sequence of the promoter tac is shown in SEQ ID NO.

4.

7. The recombinant Escherichia coli for synthesizing indoleacetic acid ester according to claim 1, characterized in that: In order to construct indigo-producing recombinant Escherichia coli, Escherichia coli was used as the initial strain, and the mycolate mutase / prephenate dehydratase bifunctional enzyme protein gene pheA, tryptophan repressor protein gene trpR, prephenate dehydratase gene tyrA and aromatic amino acid transporter gene yddG of Escherichia coli were knocked out in sequence, and the flavin monooxygenase gene maFMO, the tryptophan hydrolase gene tnaA and the caveolin gene cav1 were integrated at the arsB site and ldhA site, respectively, to obtain indigo-producing recombinant Escherichia coli.

8. Use of the recombinant Escherichia coli for synthesizing indoleacetic acid ester according to any one of claims 1 to 7 in the production of indoleacetic acid ester or dyeing.

9. A method for synthesizing indoleacetic acid esters, characterized in that: In a fermentation medium containing tryptophan, the recombinant Escherichia coli for synthesizing indoleacetic acid ester according to any one of claims 1 to 6 is fermented and cultured at 35 to 38°C until the OD 600 After the pH value reaches 0.6-0.8, add IPTG with a final concentration of 0.05-1.0 mM and induce culture at 28-32° C. for at least 48 hours to generate indoleacetic acid ester, thereby obtaining a fermentation broth containing indoleacetic acid ester.

10. A method for dyeing using indoleacetic acid, characterized in that: The fermentation liquid containing indoleacetic acid ester according to claim 9 is centrifuged, and the supernatant is taken out, and the cotton cloth is soaked, and the mixture is naturally air-dried, and then an alkali solution is applied, and the mixture is naturally oxidized and air-dried at room temperature.

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