Genetically engineered bacterium for synthesizing N-acetyl blue as well as construction method and application of genetically engineered bacterium

Abstract

The invention relates to the technical field of dye synthesis, and particularly discloses a genetically engineered bacterium for synthesizing N-acetyl blue as well as a construction method and application of the genetically engineered bacterium. The supply capacity of precursor substances glutamic acid and glutamine of the genetically engineered bacterium is high, the fermentation process is simple, the production intensity of the precursor substances glutamine and N-acetylglutamine is improved by cutting off a consumption path, relieving negative feedback inhibition and enhancing the expression level of key genes through the genetically engineered bacterium, and finally the yield of N-acetylblue reaches 41.2 g / L.

Description

Technical Field

[0001] The present application relates to the technical field of dye synthesis, and in particular to a genetically engineered bacterium for synthesizing N-acetyl indigo, a construction method thereof, and an application thereof. Background Art

[0002] N-acetyl indigoidine is a new type of natural blue pigment, an acetylated derivative of indigoidine, synthesized by microbial fermentation technology. The molecular formula of N-acetyl indigoidine is C 12 H 10 N4O5. The acetyl group in the structure of N-acetyl indigo replaces the amino group of indigo, making the pH of the dyeing solution closer to neutral (pH 7.0-8.0), thereby significantly reducing the amount of strong alkali (such as caustic soda) used in traditional dyeing processes, while improving color fastness and dyeing effects on protein fabrics (such as silk and wool). With its unique structural advantages and environmentally friendly properties, N-acetyl indigo has become a powerful alternative to traditional synthetic blue pigments. With the optimization of fermentation technology and the expansion of application scenarios, its potential in sustainable industry will be further unleashed.

[0003] Patent publication number CN 118562910 A developed a two-stage fermentation method. By introducing the indigo synthase gene and glutamine synthetase gene into Escherichia coli BL21(DE3), the recombinant strain BL21(DE3) / pRSFDuet-EcglnA-EcargA+pCDFDuet-bpsA-entD was obtained, achieving efficient synthesis of N-acetyl indigo using glucose as the substrate. By regulating conditions such as pH, temperature, and dissolved oxygen in stages, the final N-acetyl indigo production reached 27.3g / L.

[0004] However, this method has the following defects: (1) Low yield: The yield of 27.3 g / L is difficult to meet the needs of industrial production; (2) The dual-plasmid system is unstable: The plasmid system is easily lost during the fermentation process, affecting production efficiency; (3) The cost of industrial production is high: The complex process and unstable plasmid system lead to high production costs. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the above-mentioned prior art and provide a genetically engineered bacterium for synthesizing N-acetyl indigo, its construction method, and its application. The present invention provides a genetically engineered bacterium with a strong supply capacity of the precursor substances glutamate and glutamine, a simple fermentation process, and a high yield of N-acetyl indigo.

[0006] To achieve the above objectives, the technical solutions adopted in this application are:

[0007] The present application provides a genetically engineered bacterium for synthesizing N-acetyl indigo, wherein the genetically engineered bacterium is:

[0008] The glutamate efflux transporter yggB gene, N-acetylglutamate kinase argB gene, glutaminase glsK gene and nitrogen global transcriptional regulator amtR gene of Corynebacterium glutamicum were knocked out to obtain a genetically engineered bacterium.

[0009] The Corynebacterium glutamicum knocks out the glutamate transporter yggB gene and inserts the cyanine synthase bpsA gene at the knockout site;

[0010] The N-acetylglutamate kinase argB gene is knocked out in the Corynebacterium glutamicum, and the N-acetylglutamate synthetase argA gene is inserted into the knockout site;

[0011] The Corynebacterium glutamicum knocks out the glutaminase glsK gene and inserts the phosphopantetheinyl transferase entD gene at the knockout site;

[0012] The Corynebacterium glutamicum knocks out the nitrogen global transcription regulatory factor amtR gene, and inserts the glutamine synthetase glnA gene into the knockout site.

[0013] In the technical solution of the present application, the present application knocks out the glutamate extracellular transporter yggB gene in Corynebacterium glutamicum, and inserts the indigo synthase bpsA gene at this knockout site. Knocking out the glutamate extracellular transporter encoded by the yggB gene can reduce the transport of L-glutamate to the extracellular space and increase the intracellular glutamate concentration; overexpressing the indigo synthase encoded by bpsA can catalyze the synthesis of N-acetyl indigo from glutamine and N-acetylglutamine.

[0014] The present application knocks out the N-acetylglutamate kinase argB gene in Corynebacterium glutamicum and inserts the N-acetylglutamate synthetase argA gene at this knockout site. Knocking out the N-acetylglutamate kinase encoded by argB can block the synthesis pathway of L-arginine and relieve the feedback inhibition of L-arginine on argA; overexpressing the N-acetylglutamate synthetase encoded by argA can enhance the strain's ability to synthesize N-acetylglutamine.

[0015] The present application knocks out the glutaminase glsK gene in Corynebacterium glutamicum and inserts the phosphopantetheinyl transferase entD gene at this knockout site. Knocking out the glutaminase encoded by glsK can reduce the consumption of the precursor substance glutamine; overexpressing the phosphopantetheinyl transferase encoded by entD can activate the indigo synthase encoded by bpsA to catalyze the synthesis of N-acetyl indigo.

[0016] The present invention knocks out the nitrogen global transcriptional regulatory factor amtR gene in Corynebacterium glutamicum and inserts the glutamine synthetase glnA gene at this knockout site. Knocking out the nitrogen global transcriptional regulatory factor amtR can release the transcriptional inhibition of nitrogen metabolism-related genes and promote the NH4 + uptake; overexpression of glutamine synthetase encoded by glnA can improve the glutamine synthesis ability of the strain.

[0017] The genetically engineered bacteria of the present application interrupt the consumption pathway, relieve negative feedback inhibition, enhance the expression level of key genes, and increase the production intensity of the precursor substances glutamine and N-acetylglutamine. The final N-acetylglutamine production reaches 41.2g / L, which is 1.5 times higher than the 27.3g / L production of the recombinant engineered bacteria in the prior art CN 118562910 A, and is the highest level to date.

[0018] The genetically engineered bacteria provided in this application utilize Corynebacterium glutamicum as a starting strain, have a strong glutamate production capacity, and can provide sufficient precursors for N-acetyl indole.

[0019] Preferably, the Corynebacterium glutamicum includes Corynebacterium glutamicum ATCC14067 (hereinafter referred to as Corynebacterium glutamicum 14067).

[0020] This application uses Corynebacterium glutamicum ATCC14067 as the starting strain. Corynebacterium glutamicum 14067 is a high-yield glutamate production strain. Glucose is used as a carbon source to provide a sufficient supply of precursor glutamate, and the production process is mature.

[0021] As a preferred embodiment of the genetically engineered bacteria for synthesizing N-acetyl indigo as described in the present application, the indigo synthase bpsA gene is derived from Streptomyces lavendulae; the N-acetylglutamate synthase argA gene is derived from Corynebacterium glutamicum; the phosphopantetheinyl transferase entD gene is derived from Corynebacterium glutamicum; and the glutamine synthetase glnA gene is derived from Saccharomyces cerevisiae.

[0022] As a preferred embodiment of the genetically engineered bacteria for synthesizing N-acetyl indigo as described in the present application, the gene encoding the indigo synthase bpsA gene is shown in SEQ ID NO: 1; the gene encoding the N-acetylglutamate synthetase argA gene is shown in SEQ ID NO: 2; the gene encoding the phosphopantetheinyl transferase entD gene is shown in SEQ ID NO: 3; and the gene encoding the glutamine synthetase glnA gene is shown in SEQ ID NO: 4.

[0023] The present application introduces glutamine synthetase glnA from Saccharomyces cerevisiae, which can relieve the negative feedback regulation of glutamine synthesis by the genetically engineered bacteria itself and significantly improve the glutamine synthesis capacity of the genetically engineered bacteria.

[0024] The present application also provides primers for detecting the coding genes in the above-mentioned genetically engineered bacteria. The nucleotide sequences of the primers are shown in SEQ ID NO: 9 to SEQ ID NO: 10, SEQ ID NO: 13 to SEQ ID NO: 16, SEQ ID NO: 19 to SEQ ID NO: 20, SEQ ID NO: 23 to SEQ ID NO: 24, SEQ ID NO: 27 to SEQ ID NO: 34, and SEQ ID NO: 39 to SEQ ID NO: 52.

[0025] The present application provides a vector or recombinant vector comprising the coding gene in the genetically engineered bacteria.

[0026] This application also provides a method for constructing the above-mentioned genetically engineered bacteria, comprising the following steps:

[0027] S1. Using gene editing methods, the glutamate ectotransporter yggB gene in the genome of Corynebacterium glutamicum 14067 was replaced with the cyanobacterium synthase bpsA gene to obtain the genetically engineered bacterium HG-Nidg-Cg-01.

[0028] S2, replacing the N-acetylglutamate kinase argB gene in the genome of HG-Nidg-Cg-01 with the N-acetylglutamate synthetase argA gene to obtain the genetically engineered bacterium HG-Nidg-Cg-02;

[0029] S3. Replace the glutaminase glsK gene in the HG-Nidg-Cg-02 genome with the phosphopantetheinyltransferase entD gene to obtain the genetically engineered strain HG-Nidg-Cg-03;

[0030] S4. Replace the nitrogen global transcriptional regulatory factor amtR gene in the HG-Nidg-Cg-03 genome with the glutamine synthetase glnA gene to obtain the genetically engineered bacterium HG-Nidg-Cg-04.

[0031] The present application performs modifications on the genome by cutting off the consumption pathway, relieving negative feedback inhibition, and enhancing the expression level of key genes, and the genetically engineered strain does not contain a plasmid.

[0032] The present application directly biosynthesizes N-acetyl indigo in the genetically engineered bacterium HG-Nidg-Cg-04. During the production process, no antibiotics are needed to maintain genetic characteristics or expensive inducers are needed to induce protein expression. The production cost is low and the product is green and environmentally friendly.

[0033] The genetically engineered bacteria of the present application do not contain a plasmid expression system and are obtained by performing a series of gene knockouts and gene knockins on the genome of Corynebacterium glutamicum 14067 using CRISPR-Cpf1 gene editing technology.

[0034] Preferably, the 5' end of the knock-in gene is introduced into the endogenous constitutive promoter P tuf , which not only enhances the gene expression level, but also the constitutive promoter P tuf It works without adding inducement.

[0035] The present application also provides the use of the above-mentioned genetically engineered bacteria in the production of N-acetyl indigo.

[0036] The present application also provides a method for biosynthesizing N-acetyl indigo, comprising the following steps:

[0037] The genetically engineered bacteria HG-Nidg-Cg-04 was activated, and a single colony was picked and inoculated into a culture medium for shaking culture to obtain an N-acetyl indigo fermentation broth.

[0038] The specific steps are:

[0039] 1) The genetically engineered strain HG-Nidg-Cg-04 was cultured in BHISG solid medium at 30°C for 48 h;

[0040] 2) Pick a single colony and inoculate it into 5 mL of BHISG medium. Cultivate at 30°C and 220 rpm for 12-16 hours to obtain the primary seed solution.

[0041] 3) Inoculate 25 mL of GAP medium at a 2% inoculum concentration and culture at 30° C. and 220 rpm for 72 h to obtain N-acetyl indigo fermentation broth.

[0042] The genetically engineered bacteria HG-Nidg-Cg-04 of the present application can successfully synthesize N-acetyl indigo plant using glucose through shake flask fermentation, and the yield of N-acetyl indigo plant is 10.78 g / L.

[0043] HG-Nidg-Cg-04 uses glucose as a substrate to produce a large amount of glutamate through the glycolysis pathway, the tricarboxylic acid cycle, and the glutamate synthesis pathway. Glutamate is converted into N-acetyl pantothenate by the synergistic action of four enzymes: N-acetylglutamate synthetase encoded by argA, glutamine synthetase encoded by glnA, pantothenate synthase encoded by bpsA, and phosphopantetheinyltransferase encoded by entD.

[0044] As a preferred embodiment of the method for biosynthesizing N-acetyl indigo described in the present application, the culture medium includes GAP culture medium.

[0045] As a preferred embodiment of the method for biosynthesis of N-acetyl indigo plant described in the present application, the inoculation amount of the genetically engineered bacteria HG-Nidg-Cg-04 is 1%.

[0046] As a preferred embodiment of the method for biosynthesizing N-acetyl indigo described in this application, the conditions for shaking culture are:

[0047] The shaking temperature is 30°C-37°C, the rotation speed is 220 rpm, and the incubation time is 24h-72h.

[0048] The present application also provides a fermentation method for N-acetyl indigo, comprising the following steps:

[0049] S1. Activate the genetically engineered bacteria HG-Nidg-Cg-04, inoculate it into a culture medium, and culture it on a shaking platform to obtain a seed solution;

[0050] S2. The seed solution obtained in step S1 is inoculated into a fermentation medium for fermentation to obtain a fermentation liquid, and the dissolved oxygen parameter is maintained at 20-30%. The pH of the seed solution is controlled at 7.0-7.5 24 hours before fermentation, and the pH of the fermentation liquid is controlled at 6.0-6.5 after 24 hours of fermentation;

[0051] S3. When the culture is continued for 72 h, the fermentation ends and the N-acetyl indigo fermentation culture fluid is obtained.

[0052] In the technical solution of the present application, the fermentation conditions of step S2 are adopted, the genetically engineered bacteria grow vigorously, and have a strong ability to synthesize the precursor substance glutamate.

[0053] As a preferred embodiment of the fermentation method of N-acetyl indigo plant described in the present application, in step S1, the culture medium includes BHISG culture medium.

[0054] As a preferred embodiment of the fermentation method of N-acetyl indigo plant described in the present application, in step S1, the conditions for shaking culture are:

[0055] The shaking temperature was 30°C, the rotation speed was 220 rpm, and the culture time was 8 to 10 h. 600 5 to 7.

[0056] As a preferred embodiment of the fermentation method of N-acetyl indigo described in the present application, in step S2, when the residual sugar in the fermentation medium is less than 15 g / L, feed medium is started to be added to maintain the glucose concentration in the fermentation medium at 15 g / L to 20 g / L.

[0057] The fermentation method of N-acetyl indigo in the present application adopts a two-stage pH control strategy (the pH is controlled at 7.0-7.5 by ammonia water 24 hours before fermentation; and the pH is controlled at 6.0-6.5 after 24 hours of fermentation), so that the growth of genetically engineered bacteria, the synthesis of precursor substances, and the synthesis of N-acetyl indigo are respectively carried out under different suitable pH conditions, and the yield of N-acetyl indigo is high.

[0058] This application utilizes glucose as a carbon source and corn steep liquor as an organic nitrogen source. The genetically engineered strain HG-Nidg-Cg-04 uses glucose as a substrate to generate large amounts of glutamate through the glycolysis pathway, the tricarboxylic acid cycle, and the glutamate biosynthesis pathway. Glutamate is then efficiently synthesized into N-acetyl indigo through the synergistic action of four enzymes: bpsA, argA, entD, and glnA. Through shake flask and fermentation tank cultures, the yields of N-acetyl indigo reached 10.78 g / L and 41.2 g / L, respectively.

[0059] This application adopts a two-stage pH control strategy, allowing the growth of genetically engineered strains, the synthesis of precursor substances, and the synthesis of N-acetyl indigo to be carried out under different suitable pH conditions. The yield of N-acetyl indigo is the highest, with the yield in a 5L fermentor increasing by 1.5 times compared to the 27.3g / L yield of recombinant engineered bacteria in the prior art (CN 118562910 A), the highest level to date. Without the need to add antibiotics to maintain genetic characteristics or expensive inducers to induce protein expression, production costs are significantly reduced. It has the advantages of being environmentally friendly and free of chemical residues, making it suitable for industrial production.

[0060] During the production process, there is no need to add antibiotics to maintain genetic characteristics or expensive inducers to induce protein expression, so the production cost is low and it is green and environmentally friendly.

[0061] Compared with the prior art, this application has the following beneficial effects:

[0062] The present application provides a genetically engineered bacterium for synthesizing N-acetyl indigo, a construction method, and an application thereof. The genetically engineered bacterium of the present application uses Corynebacterium glutamicum ATCC14067 as a starting strain. Corynebacterium glutamicum 14067 is a high-yield glutamate-producing strain. Glucose is used as a carbon source to provide a sufficient supply of the precursor glutamate, and the production process is mature. In addition, the genetically engineered bacterium of the present application interrupts the consumption pathway, relieves negative feedback inhibition, enhances the expression level of key genes, and improves the production intensity of the precursor substances glutamine and N-acetylglutamine. The final yield of N-acetyl indigo reaches 41.2 g / L, which is 1.5 times higher than the yield of 27.3 g / L of the recombinant engineered bacteria in the prior art CN 118562910 A, which is the highest level currently. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The synthetic pathway of the genetically engineered bacteria for synthesizing N-acetyl indigo in this application;

[0064] Figure 2 This is a curve diagram of the N-acetyl indigo fermentation process of genetically engineered bacteria. DETAILED DESCRIPTION

[0065] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below with reference to the accompanying drawings and specific embodiments.

[0066] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are all the same.

[0067] The Escherichia coli DH5α (hereinafter referred to as DH5α) mentioned in this application was used for vector construction, and Corynebacterium glutamicum ATCC14067, plasmids pTargetF, pUC57, and pJYS3-ΔcrtYf were purchased from Biowind.

[0068] High-fidelity DNA polymerase, restriction endonucleases, and In-Snap Assembly Master Mix were purchased from TAKARA.

[0069] Plasmid extraction kit, DNA purification kit, gel recovery kit, and bacterial genomic DNA extraction kit were purchased from OMEGA.

[0070] LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, add water to a total volume of 1 L, and sterilize at 121°C for 20 min. If preparing as a solid medium, add 15-20 g / L agar powder.

[0071] BHISG medium: 37 g / L brain heart infusion, 10 g / L glucose, add water to a total volume of 1 L, and sterilize at 121°C for 20 min. If prepared as a solid medium, add 15-20 g / L agar powder. Brain heart infusion was purchased from Nanjing Unojia Laboratory Equipment Technology Co., Ltd.

[0072] GAP medium: 70 g / L glucose, 20 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.01 g / L ferrous sulfate, 0.01 g / L manganese sulfate, 4 μg / L biotin, 20 μg / L thiamine hydrochloride, 15 mL / L commercial soy sauce, 50 g / L calcium carbonate. Add water to a total volume of 1 L and sterilize at 121°C for 20 min. The commercial soy sauce can be any commercially available soy sauce.

[0073] Fermentation medium: 140 g / L glucose, 50 g / L ammonium sulfate, 20 g / L corn steep liquor, 30 g / L KH2PO4, 0.6 g / LMgSO4·7H2O, 0.01 g / L MnSO4·H2O, 0.01 g / L ZnSO4·7H2O, 0.01 g / L CuSO4·5H2O, 5 μg / L biotin.

[0074] Feed medium: 500 g / L glucose, 2 g / LMgSO4·7H2O.

[0075] In the following examples, the cyanine synthase bpsA gene is derived from Streptomyces lavendulae; the N-acetylglutamate synthase argA gene is derived from Corynebacterium glutamicum; the phosphopantetheinyltransferase entD gene is derived from Corynebacterium glutamicum; and the glutamine synthetase glnA gene is derived from Saccharomyces cerevisiae.

[0076] The coding gene of the cyanobacterium synthase bpsA gene is shown in SEQ ID NO: 1; the coding gene of the N-acetylglutamate synthetase argA gene is shown in SEQ ID NO: 2; the coding gene of the phosphopantetheinyl transferase entD gene is shown in SEQ ID NO: 3; and the coding gene of the glutamine synthetase glnA gene is shown in SEQ ID NO: 4.

[0077] The primers involved in the examples of this application are shown in Table 1.

[0078] Table 1

[0079]

[0080]

[0081] In the following examples, the HPLC method for detecting the concentration of N-acetyl indigo plant samples is as follows.

[0082] Chromatographic conditions:

[0083] Mobile phase: Gradient elution of methanol and pure water. Gradient Table 2 is as follows:

[0084] Table 2

[0085] Time min A (pure water)% B (methanol)% 0 80 20 9 50 50 13 80 20 18 80 20

[0086] Wavelength: 600 nm, flow rate: 1.0 ml / min, sample preparation solution: DMSO, injection volume: 10 μL, column temperature: 35°C, running time: 18 min.

[0087] Chromatographic column: EF-C18M 4.6mm id×250mm L(SN B06211801).

[0088] Example 1. Construction of genetically engineered bacteria HG-Nidg-Cg-01

[0089] This embodiment provides an engineered bacterium HG-Nidg-Cg-01 for synthesizing N-acetyl indigo and a method for constructing the same. The method comprises the following steps:

[0090] 1.1 Construction of pTarget-N24-yggB plasmid:

[0091] (1) Using plasmid pTargetF as a template, which was purchased from Biowind, the pTarget-N24 plasmid backbone was obtained by PCR amplification using primers pT-N24-F and pT-N24-R;

[0092] (2) Using plasmid pJYS3-ΔcrtYf as a template, plasmid pJYS3-ΔcrtYf was purchased from Biowind Company, and PCR amplification was performed using primers 23119-crRNA-F and 23119-crRNA-R to obtain the crRNA-crtYf fragment; the pTarget-N24 plasmid backbone and crRNA-crtYf fragment were assembled by Gibson and transformed into DH5α to obtain the pTarget-N24-crtYf plasmid;

[0093] (3) Using pTarget-N24-crtYf as a template, PCR amplification was performed using primers pT-N24-yggB-F and pT-N24-yggB-R, and the resulting plasmid was digested with Dpn I and transformed into DH5α to obtain the pTarget-N24-yggB plasmid.

[0094] 1.2pUC-P tuf -bpsA plasmid construction:

[0095] (1) Using pUC57 plasmid as template, PCR amplification was performed using primers pUC-IF and pUC-IR to obtain the pUC plasmid backbone;

[0096] (2)bpsA expression vector P tuf -bpsA includes P tuf The promoter, bpsA gene, and stop codon are represented by SEQ ID NO: 53 and were synthesized by Qingke Biotechnology. The bpsA gene (SEQ ID NO: 1) derived from the blue synthase gene of Streptomyces lavendulae was codon-optimized for expression in Corynebacterium glutamicum.

[0097] (3) Assemble the pUC plasmid backbone and expression vector P by Gibson tuf -bpsA, transferred into DH5α to obtain pUC-P tuf -bpsA plasmid;

[0098] 1.3 Construction of ΔyggB::bpsA donor fusion fragment:

[0099] (1) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification was performed using primers yggB-U1200-F and yggB-U1200-R to obtain the ΔyggB up fragment;

[0100] (2) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification was performed using primers yggB-D1200-F and yggB-D1200-R to obtain the ΔyggB down fragment;

[0101] (3) Using the pUC-P obtained in step 1.2 tuf -bpsA plasmid as a template, using P tuf -F and rrnB-R1 primers to obtain P tuf -bpsA fragment;

[0102] (4) Using ΔyggB-up, P tuf -bpsA and ΔyggB-down were used as templates, and the ΔyggB::bpsA donor fusion fragment was amplified by overlap extension PCR using primers yggB-U1000-F and yggB-D1000-R;

[0103] 1.4 Construction of editing plasmid pJYS3-ΔyggB::bpsA:

[0104] (1) Using pTarget-N24-ΔyggB obtained in step 1.1 as a template, 23119-crRNA-F and 23119-crRNA-R primers were used to PCR amplify the crRNA-ΔyggB fragment;

[0105] (2) Using pJYS3-ΔcrtYf as a template, PCR amplification was performed using primers pJYS3-vector-F and pJYS3-vector-R to obtain the pJYS3 plasmid backbone;

[0106] (3) The crRNA-ΔyggB, ΔyggB::bpsA donor, and pJYS3 plasmid backbone were assembled by Gibson and transformed into DH5α to obtain the pJYS3-ΔyggB::bpsA plasmid.

[0107] 1.5 Construction of HG-Nidg-Cg01 strain:

[0108] The pJYS3-ΔyggB::bpsA plasmid obtained in step 1.4 was transformed into Corynebacterium glutamicum ATCC14067 by electroporation. The target strain expressing ΔyggB::bpsA was obtained by colony PCR and named HG-Nidg-Cg-01.

[0109] Example 2: Construction of genetically engineered bacteria HG-Nidg-Cg-02

[0110] This embodiment provides a genetically engineered bacterium HG-Nidg-Cg-02 for synthesizing N-acetyl indigo and a method for constructing the same. The method comprises the following steps:

[0111] 2.1 Construction of pTarget-N24-argB plasmid:

[0112] Using pTarget-N24-ΔyggB obtained in step 1.1 of Example 1 as a template, PCR amplification was performed using primers pT-N24-argB-F and pT-N24-argB-R, followed by digestion with Dpn I and transfer into DH5α to obtain the pTarget-N24-argB plasmid.

[0113] 2.2pUC-P tuf -argA plasmid construction:

[0114] (1) pUC57-P obtained in step 1.2 of Example 1 tuf -bpsA plasmid as a template, using pUC-P tuf -IF and pUC-P tuf -IR primers PCR amplification to obtain pUC-p tuf plasmid backbone;

[0115] (2) The genome of Corynebacterium glutamicum ATCC14067 was used as a template and the argA fragment was amplified by PCR using primers pUC-argA-F and pUC-argA-R. The fragment was synthesized by Qingke Biotechnology Co., Ltd.

[0116] (3) Assembly of pUC-P by Gibson tuf The plasmid backbone and argA fragment (SEQ ID NO: 2) were transformed into DH5α to obtain pUC-P tuf -argA plasmid.

[0117] 2.3 Construction of ΔargB::argA donor fusion fragment:

[0118] (1) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification was performed using primers argB-U1200-F and argB-U1200-R to obtain the ΔargB::argA up fragment;

[0119] (2) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification was performed using primers argB-D1200-F- and argB-D1200-R to obtain the ΔargB::argA down fragment;

[0120] (3) Using the pUC-P obtained in step 2.2 tuf -argA plasmid as a template, using P tuf -F and rrnB-R1 primers to obtain P tuf -argA fragment;

[0121] (4) With ΔargB::argA up, P tuf-argA and ΔargB::argA down were used as templates, and the ΔargB::argA donor fusion fragment was amplified by overlap extension PCR using primers argB-up1000-F and argB-D1000-R.

[0122] 2.4 Construction of editing plasmid pJYS3-ΔargB::argA:

[0123] (1) Using pTarget-N24-ΔargB obtained in step 2.1 as a template, 23119-crRNA-F and 23119-crRNA-R primers were used for PCR amplification to obtain the crRNA-ΔargB fragment;

[0124] (2) Using pJYS3-ΔcrtYf as a template, PCR amplification was performed using primers pJYS3-vector-F and pJYS3-vector-R to obtain the pJYS3 plasmid backbone;

[0125] (3) The crRNA-ΔargB, ΔargB::argA donor, and pJYS3 plasmid backbone were assembled by Gibson and transformed into DH5α to obtain the pJYS3-ΔargB::argA plasmid.

[0126] 2.5 Construction of HG-Nidg-Cg02 strain:

[0127] The pJYS3-ΔargB::argA plasmid obtained in step 2.4 was transformed into HG-Nidg-Cg-01 by electroporation. The target strain expressing ΔargB::argA was obtained by colony PCR and named HG-Nidg-Cg-02.

[0128] Example 3: Construction of genetically engineered bacteria HG-Nidg-Cg-03

[0129] This embodiment provides an engineered bacterium HG-Nidg-Cg-03 for synthesizing N-acetyl indigo and a method for constructing the same. The method comprises the following steps:

[0130] 3.1 Construction of pTarget-N24-glsK plasmid:

[0131] Using pTarget-N24-ΔyggB obtained in step 1.1 of Example 1 as a template, PCR amplification was performed using primers pT-N24-glsK-F and pT-N24-glsK-R, followed by digestion with Dpn I and transfer into DH5α to obtain the pTarget-N24-glsK plasmid.

[0132] 3.2pUC-P tuf -entD plasmid construction:

[0133] (1) pUC57-P obtained in step 1.2 of Example 1 tuf -bpsA plasmid as a template, using pUC-P tuf -IF and pUC-P tuf -IR primers PCR amplification to obtain pUC-P tuf plasmid backbone;

[0134] (2) The genome of Corynebacterium glutamicum ATCC14067 was used as a template and the entD fragment was amplified by PCR using primers pUC-entD-F and pUC-entD-R. The fragment was synthesized by Qingke Biotechnology Co., Ltd.

[0135] (3) Assembly of pUC-P by Gibson tuf The plasmid backbone and entD fragment (SEQ ID NO: 3) were transformed into DH5α to obtain pUC-P tuf -entD plasmid.

[0136] 3.3 Construction of ΔglsK::entD donor fusion fragment:

[0137] (1) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification with primers glsK-U1200-F and glsK-U1200-R yielded the ΔglsK::entD up fragment;

[0138] (2) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification with primers glsK-D1200-F and glsK-D1200-R yielded the ΔglsK::entD down fragment;

[0139] (3) Using the pUC-P obtained in step 3.2 tuf -entD plasmid as a template, using P tuf -F and rrnB-R1 primers to obtain P tuf -entD fragment;

[0140] (4) With ΔglsK::entD up, P tuf -entD and ΔglsK::entD down were used as templates, and the ΔglsK::entD donor fusion fragment was amplified by overlap extension PCR using primers glsK-U1000-F and glsK-D1000-R.

[0141] 3.4 Construction of editing plasmid pJYS3-ΔglsK::entD:

[0142] (1) Using pTarget-N24-ΔglsK obtained in step 3.1 as a template, 23119-crRNA-F and 23119-crRNA-R primers were used to PCR amplify the crRNA-ΔglsK fragment;

[0143] (2) Using pJYS3-ΔcrtYf as a template, PCR amplification was performed using primers pJYS3-vector-F and pJYS3-vector-R to obtain the pJYS3 plasmid backbone;

[0144] (3) The crRNA-ΔglsK, ΔglsK::entD donor, and pJYS3 plasmid backbone were assembled by Gibson and transformed into DH5α to obtain the pJYS3-ΔglsK::entD plasmid.

[0145] 3.5 Construction of HG-Nidg-Cg03 strain:

[0146] The pJYS3-ΔglsK::entD plasmid obtained in step 3.4 was transformed into HG-Nidg-Cg-02 by electroporation. The target strain expressing ΔglsK::entD was obtained by colony PCR and named HG-Nidg-Cg-03.

[0147] Example 4. Construction of genetically engineered bacteria HG-Nidg-Cg-04

[0148] This embodiment provides an engineered bacterium HG-Nidg-Cg-04 for synthesizing N-acetyl indigo and a method for constructing the same. The method comprises the following steps:

[0149] 4.1 Construction of pTarget-N24-amtR plasmid:

[0150] Using pTarget-N24-ΔyggB obtained in step 1.1 of Example 1 as a template, PCR amplification was performed using primers pT-N24-amtR-F and pT-N24-amtR-R, followed by digestion with Dpn I and transfer into DH5α to obtain the pTarget-N24-amtR plasmid.

[0151] 4.2pUC-P tuf -glnA plasmid construction:

[0152] (1) pUC57-P obtained in step 1.2 of Example 1 tuf -bpsA plasmid as a template, using pUC-P tuf -F and pPtuf -IR primers were used to amplify pUC-P tuf plasmid backbone;

[0153] (2) The sequence of the glutamine synthetase gene glnA from Saccharomyces cerevisiae was codon-optimized and synthesized by Qingke Biotechnology Co., Ltd. The sequence of the glnA fragment is shown in SEQ ID NO: 4;

[0154] (3) Assembly of pUC-p by Gibson tuf The plasmid backbone and glnA fragment were transformed into DH5α to obtain pUC-P tuf -glnA plasmid.

[0155] 4.3 Construction of ΔamtR::glnA donor fusion fragment:

[0156] (1) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification was performed using primers amtR-up1200-F and amtR::glnA-up500-R to obtain the ΔamtR::glnA up fragment;

[0157] (2) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification was performed using primers amtR::glnA-down500-F-- and amtR-down1200-R to obtain the ΔamtR::glnA down fragment;

[0158] (3) Using the pUC-P obtained in step 4.2 tuf -glnA plasmid as a template, using P tuf -F and rrnB-R1 primers to obtain P tuf -glnA fragment;

[0159] (4) Using ΔamtR::glnA up, P tuf -glnA and ΔamtR::glnA down were used as templates, and the ΔamtR::glnA donor fusion fragment was amplified by overlap extension PCR using primers amtR-U1000-F and amtR-D1000-R.

[0160] 4.4 Construction of editing plasmid pJYS3-ΔamtR::glnA:

[0161] (1) Using pTarget-N24-ΔamtR obtained in step 4.1 as a template, 23119-crRNA-F and 23119-crRNA-R primers were used for PCR amplification to obtain the crRNA-ΔamtR fragment;

[0162] (2) Using pJYS3-ΔcrtYf as a template, PCR amplification was performed using primers pJYS3-vector-F and pJYS3-vector-R to obtain the pJYS3 plasmid backbone;

[0163] (3) The crRNA-ΔamtR, ΔamtR::glnA donor, and pJYS3 plasmid backbone were assembled by Gibson and transformed into DH5α to obtain the pJYS3-ΔamtR::glnA plasmid.

[0164] 4.5 Construction of HG-Nidg-Cg04 strain:

[0165] The pJYS3-ΔamtR::glnA plasmid obtained in step 4.4 was transformed into HG-Nidg-Cg-03 by electroporation. The target strain expressing ΔamtR::glnA was obtained by colony PCR and named HG-Nidg-Cg-04.

[0166] The synthetic pathway of HG-Nidg-Cg04 using glucose to synthesize N-acetyl indigo blue is as follows Figure 1 shown.

[0167] Example 5: A fermentation method for N-acetyl indigo

[0168] This embodiment provides a fermentation method for N-acetyl indigo, comprising the following steps:

[0169] 1) The genetically engineered bacteria HG-Nidg-Cg-04 in Example 4 was cultured in BHISG solid medium at 30° C. for 48 h.

[0170] 2) Pick a single colony and inoculate it into 5 mL of BHISG medium. Cultivate at 30°C and 220 rpm for 16 h to obtain the primary seed solution.

[0171] 3) The primary seed solution obtained in step 1) was inoculated into 25 mL of GAP medium at a rate of 2%, and cultured at 30° C. and 220 rpm for 72 h to obtain N-acetyl indigo fermentation culture medium.

[0172] Example 6: A fermentation method for N-acetyl indigo

[0173] This embodiment provides a fermentation method of N-acetyl indigo, which comprises the following steps:

[0174] (1) Take out the frozen bacterial solution of genetically engineered bacteria HG-Nidg-Cg-04, wait for it to thaw, take 2 mL and inoculate it into a 1000 mL shaking flask containing 200 mL BHIS medium, and culture it in a shaking incubator at 30°C and 220 rpm. After culturing for 10 h, the OD value of the bacterial solution is 600 =5 or above, obtain seed solution;

[0175] (2) 200 mL of seed liquid was transferred to a 5 L fermenter containing 2 L of fermentation medium for the first stage of fermentation culture. The pH was controlled at 7.2, the temperature was controlled at 30 °C, the initial ventilation ratio was 1 vvm, the initial rotation speed was 300 rpm, and the stirring was linked to the dissolved oxygen (DO). The dissolved oxygen (DO) was controlled at 20%. Under these conditions, the culture was carried out for 24 h.

[0176] (3) After 24 hours, the fermentation parameters were adjusted to maintain the dissolved oxygen (DO) at 30% and the pH at 6.2. When the glucose concentration in the fermentation broth was lower than 15 g / L, feed medium was added to control the glucose concentration within the range of 15-20 g / L. The fermentation was terminated after 72 hours to obtain the N-acetyl indigo fermentation broth.

[0177] The maximum biomass OD during fermentation was determined. 600 is 255, the N-acetyl indigo production is 41.2 g / L, the indigo production and biomass OD corresponding to the fermentation culture time 600 like Figure 2 shown.

[0178] Comparative Example 1

[0179] Corynebacterium glutamicum ATCC14067 and the genetically engineered bacteria HG-Nidg-Cg-01, HG-Nidg-Cg-02, HG-Nidg-Cg-03, and HG-Nidg-Cg-04 constructed in Examples 1 to 4 were used to synthesize N-acetyl indigo plant according to the method of Example 5.

[0180] The yield of N-acetyl indigo in the fermentation broth was determined by HPLC, as shown in Table 3.

[0181] Table 3

[0182]

[0183] As shown in Table 3, the genetically engineered bacteria HG-Nidg-Cg-04 of the present application can successfully synthesize N-acetyl indigo plant from glucose through shake flask fermentation, and the yield of N-acetyl indigo plant is 10.78 g / L.

[0184] HG-Nidg-Cg-04 uses glucose as a substrate to produce a large amount of glutamate through the glycolysis pathway, the tricarboxylic acid cycle, and the glutamate synthesis pathway. Glutamate is converted into N-acetyl pantothenate by the synergistic action of four enzymes: N-acetylglutamate synthetase encoded by argA, glutamine synthetase encoded by glnA, pantothenate synthase encoded by bpsA, and phosphopantetheinyltransferase encoded by entD.

[0185] Comparative Example 2

[0186] Compared with Example 6, the difference is that the pH of the first stage fermentation culture in step (2) is controlled to 6.2, and the other conditions are the same as those in Example 6.

[0187] Fermentation was completed after 72h, and the maximum biomass OD 600 The yield of N-acetyl indigo was 25.5 g / L.

[0188] Compared with Example 6, the growth of the strain was affected under the conditions of Comparative Example 2, and the yield of N-acetyl indigo was reduced by 40.5%.

[0189] Comparative Example 3

[0190] Compared with Example 6, the difference is that the pH of the fermentation culture in step (3) is controlled to 7.2, and the other conditions are the same as those in Example 6.

[0191] Fermentation was completed after 72h, and the maximum biomass OD 600 The yield of N-acetyl indigo was 33.8 g / L.

[0192] Compared with Example 6, the strain grew better under the conditions of Comparative Example 3, but the yield of N-acetyl indigo decreased by 17.9%.

[0193] The genetically engineered strain HG-Nidg-Cg-04 of the present application uses glucose as a substrate to produce a large amount of glutamate through the glycolysis pathway, the tricarboxylic acid cycle, and the glutamate synthesis pathway. Glutamate is efficiently synthesized into N-acetyl guanidine through the synergistic action of four enzymes: bpsA, argA, entD, and glnA.

[0194] By shaking flask culture and fermentation tank culture, the output of N-acetyl indigo plant reaches 10.78g / L and 41.2g / L respectively. Adopting dual-stage pH control strategy, the synthesis of the growth of genetically engineered strain, precursor material and the synthesis of N-acetyl indigo plant are carried out respectively under its different suitable pH conditions, the output of N-acetyl indigo plant is the highest, wherein 5L fermentation tank output is compared with recombinant engineering bacteria output 27.3g / L in prior art (CN 118562910 A), improves 1.5 times, is current highest level. Maintain genetic characteristics and expensive inducing agent induced protein expression without adding antibiotics, significantly reduce production cost, have environmental protection, no advantages such as chemical residues, are applicable to industrialized production.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A genetically engineered bacterium for synthesizing N-acetyl indigo, characterized in that: The genetically engineered bacteria are: The glutamate efflux transporter yggB gene, N-acetylglutamate kinase argB gene, glutaminase glsK gene and nitrogen global transcriptional regulator amtR gene of Corynebacterium glutamicum were knocked out to obtain a genetically engineered bacterium. The Corynebacterium glutamicum knocks out the glutamate transporter yggB gene and inserts the cyanine synthase bpsA gene at the knockout site; The N-acetylglutamate kinase argB gene is knocked out in the Corynebacterium glutamicum, and the N-acetylglutamate synthetase argA gene is inserted into the knockout site; The Corynebacterium glutamicum knocks out the glutaminase glsK gene and inserts the phosphopantetheinyl transferase entD gene at the knockout site; The Corynebacterium glutamicum knocks out the nitrogen global transcription regulatory factor amtR gene, and inserts the glutamine synthetase glnA gene into the knockout site.

2. The genetically engineered bacterium according to claim 1, wherein The cyan synthase bpsA gene is derived from Streptomyces lavendulae; the N-acetylglutamate synthase argA gene is derived from Corynebacterium glutamicum; the phosphopantetheinyl transferase entD gene is derived from Corynebacterium glutamicum; and the glutamine synthetase glnA gene is derived from Saccharomyces cerevisiae.

3. The genetically engineered bacterium according to claim 2, wherein The coding gene of the cyanobacterium synthase bpsA gene is shown in SEQ ID NO: 1; the coding gene of the N-acetylglutamate synthetase argA gene is shown in SEQ ID NO: 2; the coding gene of the phosphopantetheinyl transferase entD gene is shown in SEQ ID NO: 3; and the coding gene of the glutamine synthetase glnA gene is shown in SEQ ID NO:

4.

4. A primer for detecting a gene encoding a gene in a genetically engineered bacterium according to claim 3, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO: 9 to SEQ ID NO: 10, SEQ ID NO: 13 to SEQ ID NO: 16, SEQ ID NO: 19 to SEQ ID NO: 20, SEQ ID NO: 23 to SEQ ID NO: 24, SEQ ID NO: 27 to SEQ ID NO: 34, and SEQ ID NO: 39 to SEQ ID NO:

52.

5. A vector or recombinant vector comprising the gene encoding the genetically engineered bacterium as claimed in claim 3.

6. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 3, wherein: The following steps are involved: S1. Using gene editing methods, the glutamate transporter yggB gene in the genome of Corynebacterium glutamicum was replaced with the cyanobacterium synthase bpsA gene to obtain the genetically engineered bacterium HG-Nidg-Cg-01. S2, replacing the N-acetylglutamate kinase argB gene in the genome of HG-Nidg-Cg-0 with the N-acetylglutamate synthetase argA gene to obtain the genetically engineered bacterium HG-Nidg-Cg-02; S3. Replace the glutaminase glsK gene in the HG-Nidg-Cg-02 genome with the phosphopantetheinyltransferase entD gene to obtain the genetically engineered strain HG-Nidg-Cg-03; S4. Replace the nitrogen global transcriptional regulatory factor amtR gene in the HG-Nidg-Cg-03 genome with the glutamine synthetase glnA gene to obtain the genetically engineered bacterium HG-Nidg-Cg-04.

7. Use of the genetically engineered bacteria according to any one of claims 1 to 3 in the production of N-acetyl indigo.

8. A method for biosynthesizing N-acetyl indigo, characterized in that: The following steps are involved: Activate the genetically engineered bacteria HG-Nidg-Cg-04 as claimed in claim 6, pick a single colony and inoculate it into the culture medium for shaking culture to obtain N-acetyl indigo fermentation liquid.

9. A fermentation method for N-acetyl indigo, characterized in that: The following steps are involved: S1. Activate the genetically engineered bacterium HG-Nidg-Cg-04 according to claim 6, inoculate it into a culture medium, and culture it on a shaking platform to obtain a seed solution; S2. The seed solution obtained in step S1 is inoculated into a fermentation medium for fermentation to obtain a fermentation liquid, and the dissolved oxygen parameter is maintained at 20-30%. The pH of the seed solution is controlled at 7.0-7.5 24 hours before fermentation, and the pH of the fermentation liquid is controlled at 6.0-6.5 after 24 hours of fermentation; S3. When the culture is continued for 72 h, the fermentation ends and the N-acetyl indigo fermentation culture fluid is obtained.

10. The fermentation method of N-acetyl indigo plant according to claim 9, wherein: In step S2, when the residual sugar in the fermentation medium is less than 15 g / L, feed medium is started to be fed to maintain the glucose concentration in the fermentation medium at 15 g / L to 20 g / L.

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