Construction method of indigo blue producing strain and application of indigo blue producing strain in indigo blue production

Abstract

The invention belongs to the technical field of synthetic biology and fermentation engineering, and discloses an engineering bacterium for producing Indigoidine based on membrane vesicle engineering reinforced corynebacterium glutamicum and application of the engineering bacterium. According to the invention, the ncp1 gene of corynebacterium glutamicum is knocked out through a CRISPR-Cas12a gene editing technology, connection between cell walls and cell membranes is relieved, and a chassis strain of high-yield extracellular membrane vesicles (OMVs) is constructed; meanwhile, a recombinant expression vector is introduced, indigo synthetase BpsA is positioned and displayed on membrane vesicles by utilizing PorB anchoring protein, and activating enzyme Sfp is co-expressed. In fermentation production, a staged feeding and IPTG (isopropyl-beta-d-thiogalactoside) and Tween-80 dual induction strategy is adopted to promote thalli to express zymoprotein and release a large number of membrane vesicles at the same time. According to the method, the membrane vesicles are used as an extracellular microreactor, so that the'simultaneous synthesis and secretion 'of the indissolvable indigo is realized, the problems of cytotoxicity and metabolic inhibition caused by intracellular precipitation of the product are effectively solved, and the yield and extraction efficiency of the indigo are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and synthetic biology technology, specifically relating to an engineered bacterium based on membrane vesicle engineering of Corynebacterium glutamicum, which achieves efficient extracellular synthesis and secretion of the insoluble pigment indigoidine by constructing a membrane vesicle reactor, and its production method. Background Technology

[0002] Indigoidine is a natural pyridine-based blue pigment with a vibrant blue hue similar to indigo. Besides its use as a natural colorant in food, cosmetics, and textile dyeing, indigoidine also possesses antioxidant and antibacterial activities due to its unique chemical structure, and shows potential application value in organic semiconductors and bioelectronic materials. Compared to traditional synthetic dyes, indigoidine is natural, environmentally friendly, and highly safe, meeting the urgent global demand for green and sustainable products.

[0003] Currently, the biosynthesis of indigo mainly relies on microbial fermentation. Its core synthetic mechanism utilizes non-ribosomal peptide synthases (NRPS), typically such as BpsA enzyme derived from *Streptomyces lavendulae*. This enzyme is activated with the assistance of 4'-phosphate pantothenic acid thioethylamine transferases (PPTases, such as Sfp enzymes), catalyzing the condensation and cyclization of two molecules of L-glutamine to generate one molecule of indigo, releasing ammonium ions. However, existing indigo biosynthesis technologies face the following bottlenecks:

[0004] (1) Insoluble nature and intracellular accumulation of the product: Indigofera tinctoria is an insoluble hydrophobic compound. During fermentation, the synthesized indigofera tinctoria is very easy to precipitate in the cytoplasm, forming insoluble pigment particles. This intracellular accumulation not only causes physical congestion, interfering with normal physiological metabolism and division of cells, but also has feedback inhibition or steric hindrance on the synthase BpsA, making it difficult to increase the yield per cell.

[0005] (2) Lack of an effective secretion mechanism: Indigofera tinctoria lacks a transmembrane transport signal peptide, and its poor solubility prevents it from being efficiently expelled from the cell through conventional diffusion or transport proteins. This causes the product to remain mainly inside the cell, which not only limits the final concentration of the product in the fermentation broth, but also greatly increases the difficulty and cost of subsequent separation and purification (which requires cell disruption).

[0006] (3) Competition between precursor supply and metabolic flux: The direct precursor of indigo synthesized is L-glutamine, which is a core node in cellular nitrogen metabolism. Due to substrate competition, a balance often needs to be found between cell growth and pigment synthesis.

[0007] (4) Host bacterial suitability: Previous studies have often used Escherichia coli (E. coli) as a heterologous expression host, but E. coli is not a high-yield strain of glutamine and is susceptible to inclusion bodies and metabolic byproducts. In contrast, Corynebacterium glutamicum, as the main industrial producer of L-glutamic acid and L-glutamine, has a strong amino acid synthesis metabolic flux and is an ideal substrate for producing indigo, which uses glutamine as a precursor. Corynebacterium glutamicum is a widely used "generally considered safe" (GRAS) microorganism in industrial biotechnology and has the following advantages: (1) clear genetic background and mature gene manipulation tools; (2) high tolerance to a variety of organic solvents and aromatic compounds; (3) unique mycobacterial outer membrane structure, capable of secreting extracellular membrane vesicles (EMVs); (4) suitable for industrial high-density fermentation, OD 600 It can reach over 100.

[0008] In recent years, vesicle engineering has provided a new approach to solving the problem of "intracellular retention" of hydrophobic products. If the indigo synthase system can be localized to membrane vesicles, and the vesicles can be used as independent "microreactors" and "transport carriers" to achieve "simultaneous synthesis, encapsulation, and secretion" of indigo, the inhibition of intracellular products can be effectively relieved, and the downstream extraction process can be simplified.

[0009] However, no research has been reported on using genetically engineered Corynebacterium glutamicum to enhance membrane vesicle formation and utilize these vesicles to load and secrete indigo. Therefore, developing a novel method for the efficient production of indigo from Corynebacterium glutamicum based on membrane vesicle engineering is of great significance for promoting the industrial application of this natural blue pigment. Summary of the Invention

[0010] 1. Purpose of the invention

[0011] The present invention aims to solve the technical problems existing in the current biosynthesis of indigoidine, such as the poor solubility of the product, its easy accumulation of intracellular precipitation leading to cytotoxicity and metabolic inhibition, as well as the difficulty of extracellular secretion and the complexity of the extraction process.

[0012] This invention provides a method and engineered strain for enhancing the production of indigo from Corynebacterium glutamicum using membrane vesicle engineering. This method utilizes genetic engineering to modify Corynebacterium glutamicum, enabling it to secrete large quantities of extracellular membrane vesicles (OMVs) loaded with indigo synthase systems. This shifts the site of indigo synthesis from the cytoplasm to the extracellular vesicles, achieving simultaneous synthesis and secretion of the insoluble pigment, significantly improving fermentation yield and cell survival rate.

[0013] 2. Technical Solution

[0014] In a first aspect, the present invention provides a recombinant Corynebacterium glutamicum engineered strain that produces extracellular membrane vesicles.

[0015] The engineered bacteria were developed based on the starting strain Corynebacterium glutamicum, with the ncp1 gene (locus cg1977) knocked out or inactivated, thereby breaking the tight connection between the outer membrane and the cell wall and enhancing the ability to form membrane vesicles.

[0016] Meanwhile, the engineered bacteria contain a recombinant expression vector containing a fusion gene expression cassette, which can express a fusion protein of membrane anchoring protein PorB and indigo synthase BpsA, and independently express the activator enzyme Sfp; PorB anchors BpsA onto the cell membrane or the surface of secreted membrane vesicles.

[0017] Secondly, the present invention provides an expression vector and editing plasmid for constructing the above-mentioned engineered bacteria.

[0018] Expression vector: Contains the PorB-Linker-BpsA fusion gene and the Sfp gene; wherein the Linker is a flexible linker peptide, with the preferred amino acid sequence being (Gly-Gly-Gly-Gly-Ser)3.

[0019] Editing plasmid: Constructed based on the CRISPR-Cas12a system, it contains crRNA targeting the ncp1 gene and homologous arms for mediating homologous recombination repair, enabling scarless knockout of the ncp1 gene.

[0020] Thirdly, the present invention provides a fermentation method for producing indigo dye using the above-mentioned engineered bacteria.

[0021] This method employs a high-density fermentation process, adding an inducer (such as IPTG) during the mid-fermentation stage to initiate the efficient expression and assembly of BpsA and Sfp enzyme proteins, and then supplementing the fermentation process with L-glutamine feed solution to maintain substrate supply.

[0022] 3. Beneficial effects

[0023] Compared with the prior art, the present invention has the following significant advantages:

[0024] Detoxification of Product Toxicity and Metabolic Inhibition: This invention innovatively utilizes membrane vesicles as "extracellular microreactors." Since the BpsA enzyme is anchored to the vesicles, indigo synthesis mainly occurs on or inside the vesicles. The resulting insoluble pigment detaches directly from the cell along with the vesicles, avoiding physical damage and metabolic feedback inhibition caused by excessive pigment precipitation in the cytoplasm, thereby significantly extending the enzyme production cycle of the bacteria.

[0025] Significantly improved secretion efficiency: Through genetic modification (knockout of ncp1), the ability of Corynebacterium glutamicum to secrete membrane vesicles was greatly enhanced, solving the bottleneck of indigo secretion in traditional fermentation.

[0026] Simplified downstream extraction process: The product indigo is mainly found in the vesicles of the fermentation supernatant, eliminating the need for energy-intensive cell disruption steps, thus reducing the difficulty of separation and purification and production costs.

[0027] The chassis has obvious advantages: The selected Corynebacterium glutamicum is the dominant production bacterium for L-glutamine (direct precursor of indigo) in industry. Compared with Escherichia coli, it has a stronger precursor supply capacity and a clearer genetic background, and is free of endotoxins, making it more suitable for large-scale industrial production.

[0028] 4. Description of the accompanying drawings in the instruction manual

[0029] Figure 1 The pEKEx-porB-BpsA-Sfp plasmid map;

[0030] Figure 2 Comparison of fermentation curves for 10L of indigo dye. Detailed Implementation

[0031] Example 1: Construction of the recombinant expression vector pEKEx-porB-BpsA-Sfp

[0032] This embodiment describes a recombinant expression vector constructed based on the shuttle plasmid pEKEx2. The vector can be shuttle-expressed in Escherichia coli-Corynebacterium glutamate and uses the outer membrane porin PorB to anchor the indigo synthase BpsA to the cell membrane vesicles, while independently expressing the activator enzyme Sfp.

[0033] The specific construction steps are as follows:

[0034] 1. Design and synthesis of fusion gene expression cassettes

[0035] Based on the codon preference of Corynebacterium glutamicum, a polycistronic expression cassette sequence was designed and optimized. The sequence contains the following elements sequentially from the 5' end to the 3' end:

[0036] (1) Anchor protein coding sequence: porB gene (GenBank Accession No: NC_003450, locus cg1109) from C. glutamicum ATCC 13032, with its original stop codon removed.

[0037] (2) Linker sequence: The linker encodes a flexible linker with the amino acid sequence (Gly-Gly-Gly-Gly-Ser)3, which is used to maintain the spatial conformational flexibility of the fusion protein.

[0038] (3) Target enzyme coding sequence: bpsA, derived from the indigo synthase gene of Streptomyces lavendulae.

[0039] (4) Ribosome binding site (RBS): The sequence is AGGAGG, which is used to independently initiate the translation of downstream genes.

[0040] (5) Activation enzyme coding sequence: derived from the 4'-phosphoubiotinyl thioethylamine transferase gene sfp of Bacillus subtilis, retaining its own stop codon.

[0041] To facilitate subsequent cloning, an EcoRI restriction endonuclease recognition site was added to the 5' end of the above sequence, and a PstI restriction endonuclease recognition site was added to the 3' end. The designed full-length DNA fragment was then commissioned to a biotechnology company for whole-genome synthesis, and the synthesized fragment was cloned into the universal vector pUC57. Sequencing confirmed the sequence was correct.

[0042] 2. Preparation of the carrier skeleton

[0043] Plasmid pEKEx2 (containing the tac promoter, lacI repressor gene, and kanamycin resistance gene) was extracted and double-digested with restriction endonucleases EcoRI and PstI at 37°C for 2 hours. The digestion products were separated by agarose gel electrophoresis, and the approximately 7.6 kb linearized vector fragment was recovered by gel excision.

[0044] 3. Ligation and transformation of recombinant plasmids

[0045] The DNA fragment containing the porB-Linker-bpsA-RBS-sfp structure synthesized in step 1 was also recovered after double digestion with EcoRI and PstI. The recovered target fragment was mixed with the linearized pEKEx2 vector prepared in step 2 at a molar ratio of 3:1, and T4 DNA ligase was added. The mixture was then ligated overnight at 16°C.

[0046] The ligation product was transformed into E. coli DH5α competent cells, plated on LB solid medium containing 50 µg / mL kanamycin, and incubated at 37°C for 12–16 hours.

[0047] 4. Screening and identification of positive clones

[0048] Transformant single colonies were randomly selected and preliminarily screened using colony PCR. Plasmid DNA was extracted from suspected positive clones and verified by double digestion with EcoRI and PstI. The restriction enzyme pattern showed an insert fragment of approximately 5.5 kb and a vector backbone band of 7.6 kb (see appendix). Figure 1 The recombinant plasmid that was ultimately verified was named pEKEx-porB-BpsA-Sfp.

[0049] Example 2: Construction and Induced Expression of Engineered Bacteria

[0050] 1. Electroconversion

[0051] Prepare C. glutamicum ATCC 13032 electrotransformation competent cells. Add 1 µg of the recombinant plasmid pEKEx-porB-BpsA-Sfp constructed in Example 1 to the competent cells and perform electrotransformation under conditions of 2500 V, 25 µF, and 200 Ω. After recovery, plate the cells on BHI plates containing 25 µg / mL kanamycin.

[0052] 2. Validation of Induced Expression

[0053] Transformants were picked and inoculated into liquid culture medium, and OD was collected. 600 When the cell growth reaches 0.6-0.8, IPTG inducer at a final concentration of 0.5 mM is added. After culturing for another 12 hours, the cells and fermentation broth turn blue, indicating that the BpsA enzyme has been successfully expressed and activated by Sfp, catalyzing the production of indigo from the substrate.

[0054] Example 3: Construction of ncp1 gene knockout Corynebacterium glutamicum engineered strain using the CRISPR-Cas12a system

[0055] 1. Using CRISPR-Cas12a genome editing technology, the ncp1 gene (locus cg1977) was specifically knocked out in Corynebacterium glutamicum ATCC 13032 to construct a chassis strain that produces high-yield outer membrane vesicles (OMVs).

[0056] 2. Construct a "two-in-one" editing plasmid pK18-Cas12a-ncp1-sgRNA for gene knockout. This plasmid integrates the following three core modules:

[0057] (1) Cas protein expression module: Francisella novicidaCpf1 (FnCpf1) gene driven by constitutive promoter P119.

[0058] (2) Guide RNA (crRNA) module: a crRNA targeting the ncp1 gene driven by the constitutive strong promoter P119.

[0059] (3) Repair templates (Homologous Arms): The left homologous arm (LHA) and right homologous arm (RHA) used to mediate homologous recombination repair (HDR).

[0060] 3. Specific construction steps

[0061] (1) Target Design (crRNA Design)

[0062] A suitable PAM site was identified within the coding region of the ncp1 gene (cg1977) (the PAM for Cas12a is TTTV, V=A / G / C). The selected target spacer was: 5'-GCT...(20-23nt specific sequence) -3'. The PAM sequence was: TTTC or TTTA located upstream of the target site.

[0063] crRNA transcription unit design: P119 promoter - Direct Repeat (DR) - Spacer (target) - Terminator.

[0064] (2) Repair template design

[0065] Designed for the complete deletion of the homologous arm of the ncp1 gene:

[0066] Left homologous arm (LHA): A segment approximately 1000 bp upstream of the ncp1 start codon ATG.

[0067] Right homologous arm (RHA): A segment approximately 1000 bp downstream of the ncp1 stop codon.

[0068] LHA and RHA were fused by PCR, without retaining the coding sequence of ncp1 (in-frame deletion) to ensure that the expression of downstream genes was not affected.

[0069] (3) Editing plasmid assembly

[0070] Using Gibson Assembly or one-step cloning techniques:

[0071] Vector backbone: Select the suicide plasmid pK18mobsacB (containing KanR resistance and the sacB sucrose lethal gene), which is not capable of replicating against Corynebacterium glutamicum, or use a temperature-sensitive replication plasmid. Note: If using Cas12a, it is usually paired with a replication-capable plasmid such as the pJYSW-Cpf1 series. For simplicity, the universal pJYSW-Cpf1 derived vector is used here.

[0072] The synthesized crRNA transcription unit and LHA-RHA repair template were inserted into the corresponding sites of the pJYSW-Cpf1 vector.

[0073] The plasmid pEdit-ncp1 was constructed and obtained.

[0074] 4. Gene knockout procedure

[0075] (1) Electroconversion

[0076] Prepare competent cells of C. glutamicum ATCC 13032.

[0077] 1 µg of pEdit-ncp1 plasmid was electroconverted (2.5 kV, 25 µF, 200 Ω).

[0078] After transformation and thawing for 2 hours, the samples were plated on BHI plates containing 25 µg / mL kanamycin and incubated at 30°C for 2-3 days.

[0079] (2) Mutant screening

[0080] Cas12a cuts DNA, creating double-strand breaks. Cells repair these breaks using homologous recombination on the plasmid's homologous arms, thereby deleting the ncp1 gene. Cells that fail to repair the breakage die due to DNA fragmentation (selective pressure).

[0081] Pick a single colony of transformants that has grown on the plate.

[0082] (3) Plasmid Curing

[0083] If a temperature-sensitive plasmid is used, the positive strain is continuously passaged at 37°C (not an allowable temperature) to cause the editing plasmid to be lost. Finally, the bacterial culture is plated on antibiotic-free plates and then copied onto kanamycin-containing plates to screen for kanamycin-sensitive strains (with the plasmid lost).

[0084] 5. Identification and Verification

[0085] (1) Colony PCR identification

[0086] Design a pair of validation primers (Primer-F / R) located on the outer side of the homologous arms:

[0087] Primer-F: Located in the upstream genomic region of LHA.

[0088] Primer-R: Located in the downstream genomic region of RHA.

[0089] Expected results:

[0090] Wild type (WT): The amplified band size is 1000bp (LHA) + ncp1 gene length + 1000bp (RHA).

[0091] Knockout strain (Δncp1): The amplification band size was significantly reduced, only 1000bp + 1000bp (approximately 2 kb).

[0092] (2) Sequencing verification

[0093] The PCR products were sent for Sanger sequencing. The sequencing results showed that the ncp1 coding region was completely excised, and the upstream and downstream sequences were consistent with the design, with no frameshift mutations.

[0094] (3) Preliminary phenotypic observation

[0095] Knockout strains were observed using transmission electron microscopy (TEM) or scanning electron microscopy (SEM).

[0096] Expected results: Compared with the wild type, the cell surface of the Δncp1 strain showed a large number of spherical protrusions or free vesicle structures, proving that the knockout successfully induced high vesicle production.

[0097] Example 4: High-density fermentation of engineered bacteria in a 10L fermenter for indigo production

[0098] This embodiment details the use of constructed Corynebacterium glutamicum engineered strain (Δ ncp1A process flow for 10L high-density fermentation using pEKEx-PorB-BpsA-Sfp was described. This process employs a staged fed-batch strategy, inducing protein expression through IPTG and utilizing the physiological characteristics of genetically modified strains at high cell densities to achieve spontaneous and massive release of membrane vesicles and efficient extracellular secretion of indigo.

[0099] 1. Culture medium and feed solution formulation

[0100] Seed culture medium (TB+SUC): Used for strain activation and propagation. Components: yeast extract 24 g / L, tryptone 12 g / L, KH₂PO₄ 2.2 g / L, K₂HPO₄ 9.4 g / L, glycerol 4 mL / L, sucrose 20 g / L; after sterilization, kanamycin 50 mg / L and chloramphenicol 10 mg / L are added. Basal fermentation medium: Used in the initial stage of fermentation. Components: glucose 40 g / L, (NH₄)₂SO₄ 20 g / L, KH₂PO₄ 1 g / L, MgSO₄ 0.2 g / L, MnSO₄ 0.01 g / L, corn steep liquor powder 20 g / L, L-glutamine 10 g / L, and vitamins (thiamine 200 μg / L, biotin 50 μg / L); antibiotic concentration is the same as above. High-concentration fed-batch solution: Used for fed-batch feeding. The components are: glucose 600 g / L, (NH4)2SO4 80 g / L, MgSO4·7H2O 2 g / L, L-glutamine 100 g / L.

[0101] 2. Seed liquid preparation

[0102] First, streak glycerol bacteria stored at -80℃ onto TB+SUC plates and activate at 30℃ for 24-48 hours. Then, pick single colonies and inoculate them into 50 mL of seed culture medium (250 mL Erlenmeyer flask), and incubate at 30℃ and 200 rpm for 12 hours to obtain primary seed culture. Next, transfer 10% of the inoculum to a 500 mL baffle bottle containing seed culture medium and incubate under the same conditions until OD... 600 Approximately 10-15, yielding a secondary seed solution.

[0103] 3. Fermentation process control

[0104] The secondary seed culture was inoculated at a rate of 10% into a 10L fully automated fermenter containing 7L of basal fermentation medium. During fermentation, the temperature was maintained at 30℃, and the pH was maintained at 7.0 ± 0.2 by adding 25% ammonia. Dissolved oxygen (DO) was maintained above 30% by adjusting the turbine rotation speed (400-900 rpm) and aeration rate (1-2 vvm) in conjunction with dissolved oxygen. A staged control strategy was employed during the fermentation process.

[0105] Phase 1 (0-12 h, biomass accumulation period): The main goal of this phase is rapid cell growth. After fermentation starts, when the residual sugar in the fermentation broth drops below 10 g / L, fed-batch feeding is initiated to maintain the glucose concentration in the fermentation broth at 5-10 g / L.

[0106] Phase Two (12-32 h, protein induction and synthesis period): When fermentation reaches 12 hours (OD) 600 At approximately 30°C, IPTG was added to a final concentration of 0.5 mM to induce efficient expression of the BpsA and Sfp genes. During this stage, the cells continued to grow, and pigments began to accumulate intracellularly and on membrane vesicles, gradually deepening the color of the fermentation broth.

[0107] Phase 3 (32-44 h, spontaneous vesicle release and product secretion): As fermentation enters the high-density phase (OD)... 600 >100), utilizing the engineered bacteria of this invention ncp1 The instability of cell wall-membrane connections caused by gene deletion, under the synergistic effect of shear force generated by high-speed stirring in the fermenter, causes the accumulated pigmented membrane vesicles to break through the cell wall binding and undergo large-scale spontaneous shedding.

[0108] 4. Results Analysis

[0109] Samples were taken periodically throughout the fermentation process, and the yield was measured after dissolving the released vesicles and indigo within the cells using dimethyl sulfoxide (DMSO). Data showed that in the early fermentation stage (0-32 hours), the yield steadily increased with cell growth, reaching 2.27 g / L at 12 hours and 5.36 g / L at 32 hours. Notably, a significant jump in indigo yield occurred between 32 and 34 hours, surging from 5.36 g / L to 10.87 g / L. This phenomenon confirms that under the combined effects of high cell density and mechanical shear force, the genetically engineered strain achieved a concentrated and explosive release of membrane vesicles. The yield continued to climb rapidly, finally reaching a peak of 17.63 g / L at the end of fermentation (44 hours). These results demonstrate that the engineered strain of this invention can achieve highly efficient extracellular production of insoluble pigments without relying on additional chemical inducers, solely through genetic defects and fermentation process control, possessing extremely high industrial application value.

Claims

1. A recombinant Corynebacterium glutamicum engineered bacterium that produces extracellular membrane vesicles (OMVs), characterized in that: The engineered bacteria are based on the starting strain Corynebacterium glutamicum, with the ncp1 gene (locus cg1977) knocked out or inactivated, thereby breaking the tight junction between the outer membrane and the cell wall and significantly enhancing the ability of the engineered bacteria to secrete extracellular membrane vesicles. Preferably, the engineered bacteria are also introduced with an exogenous expression vector containing a fusion gene expression cassette, which can display the indigo synthase BpsA on the extracellular membrane vesicles via the membrane anchoring protein PorB, and co-express the activator enzyme Sfp.

2. The recombinant Corynebacterium glutamicum engineered strain according to claim 1, characterized in that: The nucleotide sequence of the fusion gene expression cassette, from the 5' end to the 3' end, includes, in sequence: the porin gene porB from C. glutamicum, the sequence encoding the flexible linker peptide (Gly-Gly-Gly-Gly-Ser)3, the indigo synthase gene bpsA from Streptomyces lavendulae, the ribosome binding site (RBS), and the 4'-phosphoubiotinyl thioethylamine transferase gene sfp from Bacillus subtilis.

3. A gene-editing plasmid for constructing the engineered bacteria of claim 1, characterized in that: The plasmid is a suicide plasmid constructed based on the CRISPR-Cas12a system, which integrates the following elements: (1) The constitutive promoter P119 drives the Francisella novicida Cpf1 (FnCpf1) gene; (2) A crRNA transcription unit targeting the ncp1 gene, containing a Direct Repeat (DR) sequence and a spacer sequence specific to the ncp1 coding region; (3) The left homologous arm (LHA) and right homologous arm (RHA) for mediating homologous recombination repair, wherein the homologous arms are located 1000 bp upstream and downstream of the ncp1 gene, respectively.

4. The gene-editing plasmid according to claim 3, characterized in that: The Spacer sequence is a 20-23 bp nucleotide sequence designed based on the 5'-TTTV-3' PAM site in the ncp1 gene; preferably, the gene editing completely removes the coding region of ncp1 through in-frame deletion.

5. A fermentation method for producing indigo using the engineered bacteria of claim 1, characterized in that: The method employs a high-density fermentation process, including a seed culture stage and a fermenter culture stage; during the fermenter culture process, a staged feeding and dual induction strategy are adopted. The dual induction strategy includes: (a) adding an inducer (such as IPTG) to induce the expression of BpsA and Sfp proteins; (b) Adding chemical vesicle inducers to promote the release of extracellular membrane vesicles.

6. The fermentation method according to claim 5, characterized in that: The chemical vesicle inducer comprises glycine and / or Tween-80; preferably, it is administered during the late logarithmic growth phase (e.g., OD). 600 When the concentration reaches 80-100, add glycine to the fermentation system at a final concentration of 1%-3% (w / v) and Tween-80 to a final concentration of 0.05%-0.2% (v / v).

7. The fermentation method according to claim 5, characterized in that: The temperature control strategy for the fermentation process is as follows: (1) Adaptation period for bacterial growth (0-6 hours): The temperature should be controlled at 28-32℃; (2) Protein induction and product synthesis period (6 hours to end of fermentation): Reduce the temperature to 23-27℃ to facilitate the correct folding of enzyme proteins and the stability of vesicles.

8. The fermentation method according to claim 5, characterized in that: During fermentation, the concentration of the substrate L-glutamine in the fermentation broth is maintained at 5-15 g / L by feeding a high-concentration feed solution containing L-glutamine; the high-concentration feed solution also contains glucose, ammonium sulfate and the inducer IPTG.

9. A recombinant expression vector pEKEx-porB-BpsA-Sfp, characterized in that: The vector uses pEKEx2 as a backbone and inserts the fusion gene expression cassette as described in claim 2; the vector can be shuttle-expressed in Escherichia coli-Corynebacterium glutamicum and uses the Tac promoter to regulate the transcription of the target gene.

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