Recombinant escherichia coli for synthesizing melanin and application thereof in fabric dyeing

Abstract

The application relates to the technical field of biological genetic engineering, in particular to a recombinant escherichia coli for synthesizing melanin and application of the recombinant escherichia coli in fabric dyeing. The recombinant escherichia coli is obtained by integrating and inserting a tyrosinase encoding gene melA into three sites of feaB, pheA and tyrR in an escherichia coli genome, and introducing a molecular chaperone protein expression plasmid expressing dnaK, dnaJ and grpE into the escherichia coli. The recombinant escherichia coli obtained by the application can efficiently convert tyrosine into melanin, and the yield reaches 4.3 g / L after 48 h of reaction; the extracted and purified melanin can be successfully applied to cotton fabric dyeing.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to a recombinant Escherichia coli that synthesizes melanin and its application in fabric dyeing. Background Technology

[0002] Melanin is a class of polymers, a unique biomolecule formed by the polymerization of indole and phenolic monomers, and is widely found in living organisms. Melanin possesses properties such as free radical scavenging, metal ion chelation, antioxidant, and antibacterial effects, and has enormous application potential in green technology, materials, biomedicine, cosmetics, and environmental remediation.

[0003] Melanin, as a safe, biodegradable, and sustainable natural dye, can successfully replace synthetic dyes in the textile industry because synthetic dyes are harmful to the environment. On the one hand, synthetic melanin is mainly synthesized through chemical reagents, which are expensive. On the other hand, the large amount of wastewater generated during the dyeing process of synthetic dyes will also cause environmental pollution and ecosystem damage to aquatic ecosystems. Biosynthetic melanin has the advantages of low cost, high efficiency, and eco-friendliness. Currently, the strains that have been reported to produce melanin mainly include Streptomyces, Pseudomonas, Bacillus, and certain fungi. Fungal cells have a natural metabolic pathway for melanin synthesis, and their yield can reach a high level, but the fermentation cycle is long. Currently, the most studied method is to use the isolated fungal strain Armillaria mellea as the fermentation object, and the yield can reach about 28 g / L in 125 days (Javier Ribera. Scalable Biosynthesis of Melanin by the Basidiomycete Armillaria cepistipes. J Agric Food Chem. 2019 Jan 9; 67(1):132-139.). High yields can also be achieved using bacterial strains such as Streptomyces. After optimization of the culture medium, Streptomyces can achieve a yield of 13.7 g / L in 128 h (Guo J. High-level production of melanin by a novel isolate of Streptomyces kathirae. FEMS Microbiol Lett. 2014 Aug; 357(1):85-91.). However, the excessively long fermentation cycle is not only costly but also prone to contamination. Therefore, the large-scale production and utilization of melanin synthesized by the natural metabolism of fungi and bacteria cannot be achieved.

[0004] Metabolic engineering-based deep fermentation catalysis by microorganisms offers advantages such as low cost, high yield, and environmental friendliness, making it an ideal method for melanin synthesis. Currently, by integrating the melA gene into the non-critical gene lac site of the *E. coli* genome using key gene integration technology, the resulting engineered strain exhibits low melanin production after 72 hours of culture, at only about 0.8 g / L (Sabido A. Anovel plasmid vector designed for chromosomal gene integration and expression: use for developing a genetically stable *Escherichia coli* melanin production strain. Plasmid. 2013 Jan; 69(1):16-23.). Furthermore, exogenous proteins in *E. coli* suffer from low enzyme expression levels and incorrect enzyme folding. Therefore, promoting protein expression and improving correct protein folding are crucial for effectively increasing enzyme activity and enabling the application of *E. coli* in the biosynthesis of high-yield melanin.

[0005] Therefore, there is an urgent need for a recombinant Escherichia coli strain with high melanin production for use in fabric dyeing. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a recombinant Escherichia coli that synthesizes melanin and its application in fabric dyeing. The recombinant Escherichia coli constructed using this application can efficiently convert tyrosine into melanin, with a yield of 4.3 g / L after 48 hours of reaction; the extracted and purified melanin has been successfully applied to the dyeing of cotton fabrics.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0008] This application provides a recombinant Escherichia coli that synthesizes melanin, wherein the recombinant Escherichia coli is:

[0009] The tyrosinase-encoding gene melA was integrated into three sites in the E. coli genome: feaB, pheA, and tyrR. Then, expression plasmids for molecular chaperone proteins dnaK, dnaJ, and grpE were introduced into E. coli to obtain recombinant E. coli that synthesizes melanin.

[0010] In the technical solution of this application, the tyrosinase encoding gene melA is integrated and inserted into the key enzyme gene sites of the three branching metabolic pathways feaB, pheA, and tyrR in the Escherichia coli genome to knock them out. This increases the copy number of the melA gene while blocking the flow of the branching metabolic pathways, allowing more of the precursor metabolite tyrosine to flow to the synthesis pathway of the target product melanin. At the same time, expression plasmids expressing molecular chaperone proteins dnaK, dnaJ, and grpE are introduced to promote the correct folding of the expressed tyrosinase and improve its activity.

[0011] On the one hand, gene expression levels determine protein synthesis rates, thus affecting the yield of heterologous proteins. Increasing the copy number of the target gene through genome integration technology can effectively improve gene expression levels and strain genetic stability. This application selects genes related to the tyrosine bifurcation metabolic pathway as insertion sites, which can block the tyrosine bifurcation metabolic flow and promote the accumulation of tyrosine towards melanin synthesis. On the other hand, if heterologous proteins in *E. coli* cannot fold correctly, they will form insoluble aggregates and remain within the cell, eventually being cleared by cell wall-bound proteases. This application, combined with molecular chaperones, can assist proteases in correctly folding proteins during protein secretion or refold and assemble denatured proteins, preventing degradation by proteases, promoting protein expression, and increasing enzyme activity.

[0012] This application co-expresses the tyrosinase-encoding gene melA and a molecular chaperone protein in Escherichia coli. After 48 hours, the melanin yield can be increased to 4.3 g / L. Compared with existing engineered strains, this can effectively increase the melanin yield, while also shortening the fermentation cycle and improving the production stability of the strain.

[0013] As a preferred embodiment of the recombinant Escherichia coli that synthesizes melanin described in this application, the Genebank accession numbers of feaB, pheA, and tyrR are ECK1087, ECK2596, and ECK1319, respectively.

[0014] feaB encodes the phenylacetaldehyde dehydrogenase gene, which promotes the conversion of 4-hydroxyphenylacetaldehyde to 4-hydroxyphenylacetic acid. pheA encodes the prephenylacetic acid dehydratase gene, which converts prephenylpropionic acid to phenylpyruvate. Deletion of either of these genes leads to the accumulation of the tyrosine precursor 4-hydroxyphenylpyruvate. tyrR is a global regulatory protein of aromatic amino acids that inhibits transaminases; deletion of the tyrR gene relieves feedback inhibition and increases transaminase activity. Knocking out feaB, pheA, and tyrR blocks the tyrosine bifurcation pathway, allowing tyrosine to accumulate and thus increasing melanin synthesis.

[0015] As a preferred embodiment of the recombinant Escherichia coli that synthesizes melanin described in this application, the sequence of the tyrosinase encoding gene melA is shown in SEQ ID NO: 1.

[0016] Preferably, the tyrosinase-encoding gene melA is derived from rhizobia. The Genebank accession number for the tyrosinase-encoding gene melA is M59289.1.

[0017] The tyrosinase encoding gene melA is the rate-limiting enzyme in melanin synthesis, catalyzing the synthesis of melanin from tyrosine.

[0018] As a preferred embodiment of the recombinant Escherichia coli that synthesizes melanin described in this application, the tyrosinase encoding gene melA is integrated using a gene integration expression cassette.

[0019] The method for constructing the gene integration expression cassette includes the following steps:

[0020] Will have P J23119 The promoter tyrosinase encoding gene melA was seamlessly cloned into the plasmid to obtain a recombinant plasmid. The upstream 800bp fragment, the downstream 800bp fragment of the three integration sites, and the melA gene fragment in the recombinant plasmid were fused by PCR amplification to obtain the linear integration expression cassettes of the melA gene ΔfeaB::melA Donor, ΔpheA::melA Donor, and ΔtyrR::melA Donor.

[0021] Preferably, the plasmid includes the pS95s plasmid.

[0022] As a preferred embodiment of the recombinant Escherichia coli that synthesizes melanin described in this application, the linear integration expression cassettes ΔfeaB::melA Donor, ΔpheA::melA Donor, and ΔtyrR::melADonor are sequentially transfected into competent cells using the CRISPR-Cas9 method, ultimately forming a recombinant Escherichia coli that synthesizes melanin.

[0023] As a preferred embodiment of the recombinant Escherichia coli that synthesizes melanin described in this application, the gene sequence encoding dnaK is shown in SEQ ID NO: 2, the gene sequence encoding dnaJ is shown in SEQ ID NO: 3, and the gene sequence encoding grpE is shown in SEQ ID NO: 4.

[0024] The Genebank accession numbers for the dnaK, dnaJ, and grpE genes are ECK0014, ECK0015, and ECK2610, respectively.

[0025] dnaK is an ATP-dependent Hsp70 chaperone protein, while danJ and grpE are nucleotide exchange factors that co-mediate protein folding.

[0026] The gene encoding the above sequence was co-expressed in Escherichia coli BL21(DE3). It is a molecular chaperone protein that helps protease folding or refolds denatured proteins, promotes protein expression, enhances protease expression activity, and thus increases melanin production.

[0027] Molecular chaperone proteins are derived from Escherichia coli. The plasmids in the expression plasmids of molecular chaperone proteins include the pKJE7 plasmid, and can also be plasmids commonly used in this field.

[0028] This application also provides the application of the aforementioned recombinant Escherichia coli for melanin synthesis in melanin synthesis.

[0029] This application also provides a method for synthesizing melanin, which uses the above-mentioned recombinant Escherichia coli to catalyze the conversion of tyrosine into melanin.

[0030] A preferred embodiment of the method for synthesizing melanin described in this application includes the following steps:

[0031] 1) The recombinant Escherichia coli cells that synthesize melanin above catalyze tyrosine synthesis;

[0032] 2) After catalysis, trichloroacetic acid was added dropwise, the precipitate was collected by filtration, and crude melanin product was obtained;

[0033] 3) Wash the crude melanin product until neutral, then add acid solution for reflux, filter, and obtain melanin.

[0034] Specifically:

[0035] The recombinant E. coli that synthesized melanin was activated and transferred to ZYM medium, induced and cultured at 30°C for 20 h, and the OD was measured. 600 After fermentation, the cells were collected by centrifugation at 4000 rpm for 10 min at 4℃. The cells were resuspended in 50 mM PB buffer (30 OD / mL). The cells were then disrupted using a cell disruptor. 10 g / L of tyrosine was added to the buffer solution after cell disruption. Sufficient oxygen was maintained during the reaction. The reaction conditions were 30℃ and 220 rpm. The biosynthesis of melanin was then catalyzed. After the catalytic biosynthesis reaction was complete, 10% trichloroacetic acid was added dropwise to the reaction solution. The enzyme protein and melanin precipitated to the bottom of the flask. The precipitate was collected by filtration, yielding crude melanin. This was then washed with dilute acid and dilute alkali, followed by washing with water until neutral. The neutralized crude melanin was placed in a flask, and refluxed with an appropriate amount of HCl for 8-20 hours to hydrolyze the proteins. After cooling, the melanin was filtered out, washed with dilute alkali, and finally washed with water until neutral. The mixture was then dried at 60℃ to obtain melanin.

[0036] This application also provides the application of the above-mentioned recombinant Escherichia coli that synthesizes melanin in fabric dyeing.

[0037] Soak cotton or linen fabric pieces in distilled water. Prepare dyeing solutions of varying concentrations using the previously obtained melanin. The dye-to-fabric mass ratio is 20-50%, the dyeing temperature is 60-85℃, and the pH is 1.5-4.5. After dyeing, remove the fabric, air dry, and wash with water to obtain pre-dyed cotton or linen. Prepare a fixing solution of a certain concentration, immerse the pre-dyed cotton or linen in it to fix the color, wash with water, and air dry.

[0038] This application describes a recombinant *E. coli* strain that efficiently produces melanin using L-tyrosine as a substrate. This recombinant *E. coli* strain integrates the tyrosinase gene into three sites in the *E. coli* genome: feaB, pheA, and tyrR. This integration serves two purposes: firstly, it intercepts and relieves negative feedback inhibition, increasing the supply of the precursor L-tyrosine; secondly, it integrates three copies of melA into the genome, increasing gene expression and enhancing melanin synthesis. Simultaneously, a molecular chaperone protein is introduced to promote the correct folding of the expressed tyrosinase and improve enzyme activity.

[0039] Compared with the prior art, this application has the following beneficial effects:

[0040] This application provides a recombinant Escherichia coli that synthesizes melanin and its application in fabric dyeing. The application integrates the tyrosinase encoding gene melA from rhizobia into the E. coli genome, knocking out the key enzyme gene sites of the three branching metabolic pathways: feaB, pheA, and tyrR. This increases the copy number of the melA gene while blocking the flow of the branching metabolic pathways, allowing more of the precursor metabolite tyrosine to flow into the target product, melanin. Simultaneously, it introduces expression plasmids of molecular chaperone proteins dnaK, dnaJ, and grpE to promote the correct folding of the expressed tyrosinase and improve enzyme activity. The resulting recombinant E. coli cell lysate can efficiently convert tyrosine into melanin, achieving a yield of 4.3 g / L after 48 hours. The extracted and purified melanin can be used for dyeing cotton fabrics. Attached Figure Description

[0041] Figure 1 The main metabolic pathway for melanin synthesis in recombinant Escherichia coli provided in this application, and a schematic diagram showing the integration of tyrosinase into different sites in the genome (melA: tyrosinase encoding gene; feaB: phenylacetaldehyde dehydrogenase encoding gene; pheA: phenolic acid ester dehydratase encoding gene; tryR: DNA-binding transcription dual regulator; pJ23119 promoter: melA gene uses pJ23119 promoter);

[0042] Figure 2 Electrophoresis gel images of molecular chaperone proteins and tyrosinase-encoding gene melA co-expressed proteins in Comparative Examples 2-3 (lanes 1 and 2: BL21 / pET28a-melA cell fragments, pellets, and supernatants; lanes 5 and 6: BL21 / pET28a-melA+pKJE7-dnaK-dnaJ-grpE cell fragments, pellets, and supernatants; lanes 7 and 8: BL21 / pET28a-melA+pKJE8-dnaK-dnaJ-grpE cell fragments, pellets, and supernatants).

[0043] Figure 3 The image shows the results of dyeing cotton fabric with melanin purified using HG-MEL05-1 catalysis.

[0044] Figure 4 The image shows the appearance of the melanin product extracted and purified using HG-MEL05-1 catalysis. Detailed Implementation

[0045] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0046] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0047] The pKJE7-dnaK-dnaJ-grpE plasmid was commercially purchased from TaKaRa.

[0048] The culture medium components involved in the embodiments and comparative examples of this application are as follows:

[0049] LB (Luria-Bertani) liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, sterilized at 121℃ for 20 min.

[0050] LB (Luria-Bertani) solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder. Sterilize at 121°C for 20 min. Cool the solid medium to about 50°C, add the required antibiotics, pour the plate, and let it solidify. Then, place it at 4°C for later use.

[0051] ZYM fermentation medium: 96mL ZY medium + 2mL 50×M salts + 2mL 50×5052 + 200μL 1mol / L magnesium sulfate + 100μL trace elements, 20g / L glucose.

[0052] ZY medium: 10 g / L peptone, 5 g / L yeast extract, sterilized at 121℃ for 20 min before use.

[0053] 50×M salts: 1.25 mol / L Na2HPO4, 1.25 mol / L KH2PO4, 2.5 mol / L NH4Cl and 0.25 mol / L Na2SO4;

[0054] 50×5052: 250 g / L glycerol, 25 g / L glucose; 1 mol / L MgSO4;

[0055] Trace elements: 50 mmol / L FeCl3, 20 mmol / L CaCl2, 10 mmol / L MnCl2, 10 mmol / L ZnSO4, and 2 mmol / L each of CoCl2, NiCl2, Na2Mo4, Na2SeO3 and H3BO3.

[0056] The PCR reaction system and amplification conditions involved in the examples and comparative examples of this application are as follows: 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 10–50 ng of template DNA, 25 μL of 2×Primer Star Mix, and ddH2O added to 50 μL. Amplification conditions: 98℃ for 5 min; 30 cycles (98℃ for 10 s, 50℃ for 10 s, 72℃ for 30 s), followed by extension at 72℃ for 10 min.

[0057] In the following examples and comparative examples, the sequences of the tyrosinase-encoding gene melA, the gene encoding dnaK are shown in SEQ ID NO: 2, the gene encoding dnaJ is shown in SEQ ID NO: 3, and the gene encoding grpE is shown in SEQ ID NO: 4. See Table 1 for details.

[0058] The forward and reverse primers used below are shown in Table 2.

[0059] Table 1 Gene Sequences

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] Table 2 Primer Table

[0066]

[0067]

[0068] The specific steps for preparing competent Escherichia coli cells in this application are shown in (1)-(4):

[0069] (1) Pick a single Escherichia coli DH5α colony from the plate and put it into 5 mL of LB liquid medium. Incubate overnight at 37°C and 220 rpm for 16 h.

[0070] (2) Transfer 1 / 100 of the culture to 50 mL of liquid LB medium and continue culturing at 37 °C and 220 rpm until OD. 600 =0.6-0.8;

[0071] (3) Transfer to a 50mL centrifuge tube, incubate on ice for 15min, centrifuge at 4000rpm and 4℃ for 10min and discard the supernatant;

[0072] (4) Add 5 mL of 0.1 M CaCl2 to resuspend the bacterial cells, place on ice for 30 min; centrifuge at 4000 rpm and 4℃ for 10 min to remove the supernatant;

[0073] (5) Add 5 mL of 0.1 M CaCl2 glycerol solution to resuspend the bacterial cells to obtain competent Escherichia coli cells. Dispense 100 μL / part and use immediately or store at -80℃.

[0074] The steps for transforming E. coli are as follows:

[0075] Take 10 μL of the recombinant product and add it to 100 μL of competent E. coli cells. Gently mix and incubate on ice for 30 min. Heat shock in a 42°C water bath for 60 s, then quickly transfer to ice and incubate for 2 min. Add 900 μL of ILB medium and revive at 37°C and 220 rpm for 60 min. Centrifuge at 4500 rpm for 2 min, remove about 900 μL of supernatant, resuspend the cells in the remaining medium, and spread on plates with the corresponding antibiotics.

[0076] The method for sample catalysis in this embodiment is as follows:

[0077] Bacterial culture:

[0078] Single clones from the plates were picked and activated in LB medium for 16 hours, then transferred to ZYM medium at a 1 / 100 inoculum and cultured at 30°C and 220 rpm for 20 hours. After culture, samples were taken and OD was measured using a spectrophotometer. 600Biomass was collected, and the cells were centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was discarded, and the cells were resuspended in PB buffer at pH 7.0. The PB volume was calculated to 30 OD / mL. The cells were sonicated for 20 min to disrupt the cell mass. After disruption, the disrupted liquid was transferred to a shake flask, and tyrosine and dopa were added separately. The flask was sealed with gauze and incubated on a shaker at 30 °C and 220 rpm. For example, if the OD600 of 300 mL of fermentation broth was measured to be 4 before centrifugation, the cells were resuspended in 300 * 4 / 30 = 40 mL of PB buffer after centrifugation.

[0079] Example 1: A recombinant Escherichia coli for synthesizing melanin and its construction method

[0080] This application integrates the tyrosinase-encoding gene melA into three sites in the Escherichia coli BL21(DE3) genome: glucose-specific transport membrane permeabilization enzyme (feaB), branched acid mutase / prebenzoic acid dehydratase (pheA), and transcription regulatory factor (tyrR).

[0081] The main metabolic pathway for melanin synthesis in recombinant Escherichia coli provided in this application, and a schematic diagram of the integration of tyrosinase into different sites in the genome are shown below. Figure 1 As shown.

[0082] This embodiment provides a method for constructing recombinant Escherichia coli HG-MEL03 strain, which specifically includes the following steps:

[0083] Step 1: Construction of recombinant plasmid pS95s-melA, the specific process is as follows:

[0084] Step 1.1: Using the Escherichia coli genome as a template, primers 95s-melA-F and 95s-melA-R were used to amplify the melA fragment (SEQ ID NO: 1) which encodes the tyrosinase gene. The melA fragment has homologous arms at both ends that are linked to the pS95s plasmid.

[0085] Step 1.2: Using pS95s plasmid as a template, wherein the pS95s sequence is shown in SEQ ID NO: 48, PCR amplification was performed using primers pS95s-iF and pS95s-iR (the above PCR system), and the product was purified to obtain the pS95s linearized vector.

[0086] The pS95s linearized vector and tyrosinase melA fragment were ligated using Takara's Seamless Cloning Kit and then transformed into E. coli DH5α via chemical transformation. After resuscitation and culture, the mixture was plated on LB agar plates containing 50 μg / L streptomycin resistance and incubated at 37°C for approximately 16 h.

[0087] Colony PCR verification was performed using primers J23119-F (SEQ ID NO: 43) and rrnB-R (SEQ ID NO: 44). Strains that passed PCR verification were cultured, and recombinant plasmids were extracted and sequenced. The correctly sequenced plasmid was identified as pS95s-melA. The extracted plasmids were then sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0088] Step 2: Construct the ΔfeaB::melA linear integration expression box;

[0089] Using the Escherichia coli genome as a template, PCR amplification was performed using middle primers feaB-U800-F and feaB-U800-R, feaB-D800-F and feaB-D800-R, respectively, to obtain feaB-Up and feaB-Down fragments, respectively.

[0090] Using recombinant plasmid pS95s-melA as a template, PCR amplification was performed using primers J23119-F (SEQ ID NO: 43) and rrnB-R (SEQ ID NO: 44) to obtain the J23119-melA-rrnB fragment (SEQ ID NO: 49), as shown in SEQ ID NO: 5;

[0091] Using feaB-U500-F and feaB-D500-R as primers, the three fragments feaB-Up, feaB-Down, and J23119-melA-rrnB were ligated by fusion PCR to obtain the ΔfeaB::melA linear integration expression cassette.

[0092] Step 3: Construction of HG-MEL03 strain, the specific process is as follows:

[0093] Step 3.1: Prepare competent cells from Escherichia coli BL21(DE3), plasmidize pCas9 containing Cas9 protein into competent cells, plate them on LB solid medium plates containing 50 μg / L kanamycin resistance, and incubate overnight at 30°C. The single colony that grows is BL21(DE3) / pCas9.

[0094] Step 3.2: Using the website CHOPCHOP, primers were designed to introduce N20 at the 5' end of the knockout site. Using pTargetF as a template, PCR amplification was performed using pT-feaB-F and pT-feaB-F. The PCR product was digested with DpnI enzyme and transformed into DH5α competent cells. After resuscitation and culture, the cells were plated on streptomycin-resistant plates and incubated overnight at 37°C. One to two single clones were picked, and plasmids were extracted in 5 mL of streptomycin-resistant LB broth. The plasmids were then sequenced using primer pTarget-cexu-F (SEQ ID NO: 45). The correctly sequenced plasmid was identified as pTarget-feaB plasmid.

[0095] Step 3.3: The pTarget-feaB plasmid and the above-mentioned ΔfeaB::melA integration expression cassette were electroporated into BL21(DE3) / pCas9 competent cells. After revival culture, the cells were plated on LB solid medium plates containing 50 μg / L kanamycin and 50 μg / L streptomycin, and cultured at 30°C for 24 h. The resulting single colonies were verified by colony PCR using primers feaB-U800-F and feaB-D800-R. The strain BL21(DE3) / pCas9 was used as a control.

[0096] Step 3.4: Select colonies that have been verified by PCR and transfer them to 5 mL of LB liquid medium containing 0.1 mM IPTG and 50 μg / L kanamycin resistance. Incubate at 30°C and 220 rpm for 4-5 h. Then, plate the culture onto LB solid medium plates containing 50 μg / L kanamycin resistance and incubate overnight at 30°C. Select single colonies for resistance verification. Colonies that grow only on kanamycin-resistant plates and do not grow on dual-resistance LB solid medium plates are considered strains that have eliminated the pT-feaB plasmid.

[0097] The strain was transferred to antibiotic-free LB liquid medium and incubated at 42°C and 220 rpm for 4-5 hours. Then, it was streaked to antibiotic-free LB solid medium and incubated overnight at 37°C. Single colonies were picked for resistance verification. Colonies that grew only on antibiotic-free plates and not on the kanamycin-resistant plates were considered strains that had eliminated the pCas9 plasmid, and were thus considered antibiotic-free stable strain HG-MEL03.

[0098] The pKJE7-dnaK-dnaJ-grpE plasmid was transformed into HG-MEL03 to obtain the recombinant strain HG-MEL03-1. Shake-flask tests showed that the melanin production of HG-MEL03-1 was significantly increased, reaching 3.08 g / L, which was 23% higher than that without the addition of molecular chaperone protein. This indicates that the addition of molecular chaperone protein helps to enhance the expression of protease activity and increase melanin production.

[0099] Example 2: A recombinant Escherichia coli for synthesizing melanin and its construction method.

[0100] This embodiment describes the construction of recombinant Escherichia coli strain HG-MEL04. The specific implementation method is the same as in Example 1, except that the starting strain is Escherichia coli HG-MEL03 obtained in Example 1, the integration gene is melA, and the integration site is pheA.

[0101] The pT-pheA plasmid was constructed using primers pT-pheA-F and pT-pheA-R. PCR amplification was performed using primers pheA-U800-F and pheA-U800-R, and pheA-D800-F and pheA-D800-R, respectively, yielding pheA-Up and pheA-Down fragments.

[0102] Using pheA-U500-F and pheA-D500-R as primers, the three fragments J23119-melA-rrnB, pheA-Up, and pheA-Down obtained by PCR amplification in step 2 of Example 1 were ligated to obtain the ΔpheA::melA linear expression cassette. Escherichia coli HG-MEL04 was prepared according to the method described in step 3 of Example 1. The pKJE7-dnaK-dnaJ-grpE plasmid was transformed into HG-MEL04 to obtain the recombinant strain HG-MEL04-1.

[0103] Shake-flask tests showed that HG-MEL04 melanin production increased by 20% compared to HG-MEL03. After expressing the chaperone protein, the melanin production of HG-MEL04-1 reached 3.6 g / L, which was 24% higher than that without the addition of the chaperone protein. Knocking out pheA and increasing the copy number of the melA gene also increased melanin production.

[0104] Example 3: A recombinant Escherichia coli for synthesizing melanin and its construction method.

[0105] This embodiment describes the construction of recombinant Escherichia coli HG-MEL05 strain. The specific implementation method is the same as in Example 1. The starting strain is Escherichia coli HG-MEL-04 obtained in Example 2. The integration gene is melA and the integration site is tyrR.

[0106] The pT-tyrR plasmid was constructed using primers pT-tyrR-F and pT-tyrR-R. PCR amplification was performed using primers tyrR-U800-F and tyrR-U800-R, and tyrR-D800-F and tyrR-D800-R, respectively, yielding tyrR-Up and tyrR-Down fragments.

[0107] Using tyrR-U500-F and tyrR-D500-R as primers, the three fragments J23119-melA-rrnB, tyrR-Up, and tyrR-Down obtained by PCR amplification in step 2 of Example 1 were ligated to obtain the ΔtyrR::melA linear expression cassette. Following the method described in step 3 of Example 1, Escherichia coli HG-MEL05 was prepared. The pKJE7-dnaK-dnaJ-grpE plasmid was transformed into HG-MEL05 to obtain the recombinant strain HG-MEL05-1.

[0108] After shake-flask fermentation, the melanin production of strain HG-MEL05 increased by 10% compared to HG-MEL04. After expressing the chaperone protein, the production of HG-MEL05-1 increased by 34%. This may be due to the fact that the absence of tyrR promotes the accumulation of tyrosine, and that increasing the copy number of melA can also effectively increase melanin production.

[0109] Comparative Example 1: Construction of Recombinant Escherichia coli HG-MEL01 strain

[0110] This comparative example provides the recombinant Escherichia coli HG-MEL01 strain, including the following steps:

[0111] Step 1): Using the commercially available expression plasmid pET28a as a template, PCR amplification was performed using primers pET28a-iF / pET28a-iR (using the PCR system described above), and the product was purified to obtain the pET28a linearized vector. The pET28a linearized vector and the melA gene (homological arms were introduced during synthesis; the melA gene is from rhizobium, and its sequence is shown in SEQ ID NO: 1) were ligated using a seamless cloning kit from Takara. The recombinant product was then transformed into the prepared *E. coli* DH5α using chemical transformation. After resuscitation and culture, the product was plated onto LB agar plates containing 50 μg / L kanamycin-resistant medium and incubated at 37°C for approximately 16 hours. Colony PCR verification was performed using primers T7 / T7T (as shown in SEQ ID NO: 46 and SEQ ID NO: 47, respectively). Strains that were correctly verified by PCR were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing detection. The correctly sequenced plasmid was pET28a-melA.

[0112] Step 2): Transform the pET28a-melA plasmid into E. coli BL21(DE3) competent cells to obtain recombinant E. coli HG-MEL01, namely BL21(DE3) / pET28a-melA.

[0113] Step 3): Pick the recombinant E. coli and inoculate it into LB medium. After activation culture at 37°C for 16 h, transfer it to ZYM medium. Add IPTG to BL21(DE3) / pET28a-melA to a final concentration of 0.1 mM, and then induce expression culture at 30°C for 20 h.

[0114] Step 4): Centrifuge at 4000 rpm for 10 min to collect the bacterial cells. The catalytic system contains 30 OD / mL of bacterial cells, 10 g / L of tyrosine, and 50 mM PB buffer at pH 7.0. The catalytic conditions are 30℃.

[0115] The final melanin yield of strain HG-MEL-01 was measured to be 1.24 g / L, while no melanin was detected in strain BL21(DE3). This indicates that melA in strain HG-MEL-01 possesses tyrosine activity and can synthesize melanin.

[0116] Comparative Example 2: Construction of Recombinant Escherichia coli HG-MEL02

[0117] This comparative example provides a method for constructing recombinant Escherichia coli HG-MEL02 expressed with a molecular chaperone protein, including the following steps:

[0118] Using HG-MEL01, prepared in Comparative Example 1, as the starting strain, the pKJE7-dnaK-dnaJ-grpE plasmid was chemically transformed into HG-MEL01 to construct strain HG-MEL02, in order to promote the correct folding of the melA gene.

[0119] After shaking flask testing, the melanin yield was 2.8 g / L, indicating that the pKJE7-dnaK-dnaJ-grpE chaperone protein can effectively promote the expression of melA protease and increase melanin yield. Compared with the control strain without added chaperone protein, the yield increased by 125.8%.

[0120] The tyrosinase-encoding gene melA, as shown in SEQ ID NO: 1, was cloned into the pET28a vector for heterologous expression. The tyrosinase gene was ligated into plasmid PET28a to obtain recombinant plasmid PET28a-melA, which was then transformed into Escherichia coli BL21(DE3) by chemical transformation to obtain recombinant Escherichia coli HG-MEL01. Protein gel electrophoresis showed that the expression level of melA protein was low.

[0121] The melA gene originates from rhizobia. Heterologous expression in *E. coli* suffers from folding errors and low enzyme activity. This application, based on recombinant *E. coli* HG-MEL01, introduces molecular chaperone protein expression plasmids expressing dnaK, dnaJ, and grpE into HG-MEL01, obtaining recombinant *E. coli* HG-MEL02. This utilizes the molecular chaperones dnaK, dnaJ, and grpE to promote correct protease folding, increasing protein expression levels and thus enhancing melanin biosynthesis. However, it was found that *E. coli* BL21(DE3) itself can consume the substrate tyrosine, producing byproducts. Subsequent attempts were made to knock out some genes to block tyrosine consumption. To increase the accumulation of tyrosine precursors and thus improve melanin production, this application discovered that knocking out the feaB, pheA, and tyrR sites leads to tyrosine shunting, resulting in tyrosine accumulation and increased melanin synthesis.

[0122] Comparative Example 3: Construction of Recombinant Escherichia coli HG-MEL06

[0123] This comparative example provides a method for constructing recombinant Escherichia coli HG-MEL06 expressed with a molecular chaperone protein, including the following steps:

[0124] Using HG-MEL01 as the starting strain, the pG-KJE8-dnaK-dnaJ-grpE plasmid (commercially purchased from TaKaRa) was chemically transformed into HG-MEL01 to construct strain HG-MEL06, in order to promote the correct folding of the melA gene.

[0125] After shaking flask testing, the melanin yield was 2.4 g / L, indicating that the pG-KJE8-dnaK-dnaJ-grpE plasmid can effectively promote the expression of melA protease and increase melanin yield. Compared with the control strain without added chaperone protein, the yield increased by 93.5%.

[0126] This application compares the yield and extraction rate of HG-MEL02 and HG-MEL06, and compares the effects of different chaperone proteins. After shaking flask testing, strain HG-MEL02 produced 2.8 g / L of melanin with an extraction rate of 77.4%; strain HG-MEL06 produced 2.4 g / L of melanin with an extraction rate of 58.1%. The yield results indicate that pKJE7-dnaK-dnaJ-grpE is more effective than pG-KJE8-dnaK-dnaJ-grpE in promoting the correct folding of the melA gene, thereby increasing melanin production.

[0127] Depend on Figure 2As shown in the protein gel electrophoresis image, after the pG-KJE8-dnaK-dnaJ-grpE plasmid promotes melA protease expression, the target protein is mostly present in the precipitate and is an intracellular protein, which increases the difficulty of extraction. However, after the chaperone protein pKJE7-dnaK-dnaJ-grpE promotes melA protease expression, the melanin is mostly present in the supernatant and is an extracellular protein, which is easier to extract and separate in the subsequent process.

[0128] Comparative Example 4: Construction of Recombinant Escherichia coli HG-MEL07

[0129] Comparative Example 4 describes the construction of the recombinant Escherichia coli HG-MEL07 strain, following the same implementation method as in Example 1. The starting strain was Escherichia coli BL21(DE3), the integrated gene was melA, and the integration site was pyruvate kinase pykA.

[0130] The pT-pykA plasmid was constructed using primers pT-pykA-F and pT-pykA-R. PCR amplification was performed using primers pykA-U800-F and pykA-U800-R, and pykA-D800-F and pykA-D800-R, respectively, yielding pykA-Up and pykA-Down fragments. Using primers pykA-U500-F and pykA-D500-R, the three fragments J23119-melA-rrnB and pykA-Up and pykA-Down obtained from PCR amplification in step 2 of Example 1 were ligated using a fusion amplification method to obtain the ΔpykA::melA linear expression cassette. Following the method described in step 3 of Example 1, E. coli HG-MEL07 was prepared. Shake-flask testing showed that HG-MEL07 grew slowly; knocking out pheA may affect the tricarboxylic acid cycle, leading to slow growth and almost no melanin production.

[0131] Example 4: Fermentation production and product separation and purification of melanin using the above-mentioned recombinant Escherichia coli.

[0132] This embodiment uses the recombinant Escherichia coli from Examples 1-3 and Comparative Examples 1-4 to ferment and produce melanin, and the product is separated and purified. The steps include:

[0133] Step 1: Pick single clones from the plate and activate them in LB medium for 16 hours. Then, transfer them to ZYM medium at a 1 / 100 inoculum and incubate at 30°C and 220 rpm for 20 hours. After incubation, take samples and measure the OD using a spectrophotometer. 600Biomass was collected, and the cells were centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was discarded, and the cells were resuspended in PB buffer at pH 7.0. The PB volume was calculated to 30 OD / mL. The cells were sonicated for 20 min to disrupt the cell mass. After disruption, the disrupted liquid was transferred to a shake flask, and tyrosine and dopa were added separately. The flask was sealed with gauze and incubated on a shaker at 30 °C and 220 rpm. For example, if the OD600 of 300 mL of fermentation broth was measured to be 4 before centrifugation, the cells were resuspended in 300 * 4 / 30 = 40 mL of PB buffer after centrifugation.

[0134] Step 2: Add 100 ml of trichloroacetic acid to 1 L of fermentation broth obtained by recombinant Escherichia coli catalysis. After centrifugation at 4000 rpm for 10 min in a refrigerated centrifuge, the enzyme protein and melanin settle to the bottom of the bottle together. After discarding the supernatant, filter and collect the precipitate to obtain crude melanin product.

[0135] Step 3: After washing the precipitate with dilute acid and dilute alkali, wash it with water until neutral.

[0136] Step 4: Place the neutralized crude melanin product in a flask, add an appropriate amount of HCl and reflux for 8-20 hours to hydrolyze the proteins, let it cool, and filter out the melanin.

[0137] Step 5: Wash with dilute alkali, then wash with water until neutral, and dry at 60℃ to obtain pure melanin.

[0138] The results of the yield and extraction rate of melanin synthesized by the recombinant Escherichia coli constructed above are shown in Table 3.

[0139] Table 3. Results of melanin synthesis yield and extraction rate from different recombinant Escherichia coli strains.

[0140]

[0141]

[0142] The recombinant Escherichia coli strains HG-MEL01, HG-MEL02, HG-MEL03, HG-MEL03-1, HG-MEL04, HG-MEL04-1, HG-MEL05, HG-MEL05-1, and HG-MEL06 of this application can all produce melanin, can all be purified, and can all be used to dye cotton fabric.

[0143] Example 5: Dyeing cotton fabric with isolated and purified melanin.

[0144] This embodiment uses isolated and purified melanin to dye cotton fabric, including the following steps:

[0145] Step 1: Mix 1g of melanin with 100ml of distilled water until homogeneous to prepare a dye solution with a concentration of 10g / L.

[0146] Step 2: Cut the cotton fabric into 4×4cm squares, soak them in distilled water for 10 minutes, then wring them out.

[0147] Step 3: Place the staining solution in an 80°C water bath and adjust the staining pH to 3.5 using 1 mol / L hydrochloric acid and sodium hydroxide.

[0148] Step 4: Immerse a 2g piece of cotton fabric cut into 4×4cm pieces in the dye solution and dye for 80 minutes, then remove it.

[0149] Step 5: Dry the cotton fabric, then wash it 5 times to obtain the preliminarily dyed cotton fabric.

[0150] Step 6: Use SG-50 formaldehyde-free fixing agent (purchased from Qingdao Dayin Chemical Co., Ltd.) to fix the color of cotton or linen. Prepare a 1g / L fixing solution, immerse the pre-dyed cotton in it for 60 minutes, and fix at a temperature of 40℃. After fixing, wash three times with water and air dry.

[0151] The results of dyeing cotton fabric with the purified melanin obtained after HG-MEL05-1 catalysis are as follows. Figure 3 As shown, the appearance of the extracted and purified melanin product is as follows. Figure 4 As shown.

[0152] The melanin staining results and product appearance obtained by catalysis using HG-MEL01, HG-MEL02, HG-MEL03, HG-MEL03-1, HG-MEL04, HG-MEL04-1, HG-MEL05, and HG-MEL06 are similar to those of HG-MEL05-1.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A recombinant Escherichia coli that synthesizes melanin, characterized in that, The recombinant Escherichia coli is: The tyrosinase-encoding gene melA was integrated into the three sites feaB, pheA, and tyrR in the E. coli genome, and molecular chaperone protein expression plasmids expressing dnaK, dnaJ, and grpE were introduced into E. coli to obtain recombinant E. coli that synthesizes melanin. The sequence of the tyrosinase encoding gene melA is shown in SEQ ID NO: 1; The gene sequence encoding dnaK is shown in SEQ ID NO: 2, the gene sequence encoding dnaJ is shown in SEQ ID NO: 3, and the gene sequence encoding grpE is shown in SEQ ID NO:

4.

2. The recombinant Escherichia coli for synthesizing melanin as described in claim 1, characterized in that, The Genebank accession numbers for feaB, pheA, and tyrR are ECK1087, ECK2596, and ECK1319, respectively.

3. The recombinant Escherichia coli for synthesizing melanin as described in claim 1, characterized in that, The tyrosinase-encoding gene melA was integrated using a gene integration expression cassette. The method for constructing the gene integration expression cassette includes the following steps: Will have P J23119 The promoter tyrosinase encoding gene melA was seamlessly cloned into the plasmid to obtain a recombinant plasmid. The upstream 800bp fragment, the downstream 800bp fragment of the three integration sites, and the melA gene fragment in the recombinant plasmid were fused by PCR amplification to obtain the linear integration expression cassettes of the melA gene ΔfeaB::melA Donor, ΔpheA::melA Donor, and ΔtyrR::melA Donor.

4. The recombinant Escherichia coli for synthesizing melanin as described in claim 3, characterized in that, Using the CRISPR-Cas9 method, the linear integrated expression cassettes ΔfeaB::melA Donor, ΔpheA::melA Donor, and ΔtyrR::melADonor were sequentially transfected into competent cells, ultimately forming recombinant E. coli that synthesize melanin.

5. The use of the recombinant Escherichia coli for melanin synthesis as described in any one of claims 1 to 4 in melanin synthesis.

6. A method for synthesizing melanin, characterized in that, The recombinant Escherichia coli described in any one of claims 1 to 4 is used to catalyze the conversion of tyrosine into melanin.

7. The method for synthesizing melanin as described in claim 6, characterized in that, Includes the following steps: 1) Using the cells of recombinant Escherichia coli that synthesize melanin as described in any one of claims 1 to 4 to catalyze tyrosine synthesis; 2) After catalysis, trichloroacetic acid was added dropwise, the precipitate was collected by filtration, and crude melanin product was obtained; 3) Wash the crude melanin product until neutral, then add acid solution for reflux, filter, and obtain melanin.

8. The application of the recombinant Escherichia coli that synthesizes melanin as described in any one of claims 1 to 4 in fabric dyeing.

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