Recombinant EcN engineering bacterium for constitutive synthesis of valley blue pigment as well as construction method and synthesis method of recombinant EcN engineering bacterium

Abstract

The invention provides a recombinant EcN engineering bacterium for constitutive synthesis of valley blue as well as a construction method and a synthesis method of the recombinant EcN engineering bacterium, and belongs to the technical field of genetic engineering bacteria. The invention provides a construction method of recombinant EcN engineering bacteria for constitutive synthesis of valley blue pigment, EcN is taken as a chassis strain, endogenous hidden plasmids of EcN are modified, the recombinant engineering bacteria for stable constitutive expression of valley blue synthetase and PPTase are constructed, valley blue is generated, and the recombinant engineering bacteria have good genetic stability and environmental adaptability. The recombinant EcN engineering bacterium disclosed by the invention has the capability of realizing efficient co-expression of glutamine blue synthetase bpsA and phosphopantetheinyl transferase sfp protein without adding any exogenous inducer. In conclusion, the recombinant EcN engineering bacterium disclosed by the invention shows remarkable technical advantages and wide application prospects in sustainable industrial production and biological medicine application.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering bacteria technology, specifically relating to a recombinant EcN engineered bacterium that constitutively synthesizes glutenin, and its construction and synthesis methods. Background Technology

[0002] Escherichia coli Nissle 1917 (EcN) is a Gram-negative probiotic that can colonize the gastrointestinal (GI) system and has a growth advantage over other bacteria in the gut, inhibiting pathogenic bacteria. Later commercialized under the brand name Mutaflor, EcN has been used as a probiotic therapy to treat intestinal infections and various inflammatory diseases of the intestine in adults and infants.

[0003] EcN carries two cryptogenic plasmids, pMUT1 and pMUT2, with sizes of approximately 3.2 kb and 5.5 kb, respectively. Both plasmids have been fully sequenced and are stably inherited. EcN strains lacking pMUT1 and pMUT2 plasmids are functionally indistinguishable from wild-type EcN strains and can be used as vectors for recombinant plasmids. Further analysis of the EcN genome structure revealed that it lacks virulence factors and possesses an O6:K5:H1 serotype, an extracellular K5 capsule, and a semi-rough lipopolysaccharide phenotype. The ability to genetically engineer EcN's two endogenous plasmids to express endogenous proteins, and the stably inherited nature of these plasmids without antibiotic selection pressure, makes it a promising candidate for industrial applications.

[0004] Indigoidine is a natural pigment synthesized by certain microorganisms. It possesses excellent antioxidant and broad-spectrum antibacterial activities and belongs to bacterial secondary metabolites. Its unique molecular structure effectively scavenge free radicals and inhibit the growth of various common bacteria, thus holding potential application value in the pharmaceutical and food preservation fields. Furthermore, its bright and stable blue color, highly similar to the widely used synthetic dye indigo, makes indigoidine a promising environmentally friendly and renewable natural blue dye alternative for textile dyeing, food coloring, and other industrial applications, demonstrating significant development potential.

[0005] Studies have shown the discovery of indigoidine synthetase in microorganisms, which can condense two molecules of L-glutamine into one molecule of indigo. Indigoidine synthetase (bps) is a type of blue pigment synthetase, belonging to the nonribosomal peptide synthetase (NRPS) family. Activated by 4'-phosphopantetheinyl transferases (PPTases), it converts two molecules of L-glutamine within microorganisms into one molecule of indigo. The PPTase family is widely found in bacteria, archaea, and eukaryotes. It activates various synthases in primary metabolism (such as fatty acid synthesis) and secondary metabolism (such as the synthesis of polyketides, nonribosomal peptides, and other antibiotics) pathways by catalyzing the phosphopantetheinyl aminoylation modification of carrier proteins, which is a key step in its functional activation. Currently, the microorganisms that produce indigo that have been extensively studied include *Escherichia coli*, *Pseudomonas putida*, *Corynebacterium glutamicum*, and *Rhodotorula glutamicum*. However, in the process of industrial production, antibiotics are required to maintain plasmid stability or induce the expression of exogenous genes. This may not only lead to the spread of drug-resistant genes and environmental pollution, but also significantly increase production costs and management difficulties. Summary of the Invention

[0006] This invention provides a recombinant EcN engineered bacterium that can constitutively synthesize glutenin, as well as its construction and synthesis methods. It can constitutively overexpress related enzymes and constitutively synthesize and produce glutenin in EcN.

[0007] This invention provides a method for constructing a recombinant EcN engineered bacterium that synthesizes glutenin, comprising the following steps: inserting the encoding gene of glutenin synthase and the encoding gene of 4′-phosphoupanylthioethylaminotransferase into the cryptic plasmids pMUT1 and pMUT2 of the EcN strain, respectively, to construct the recombinant EcN engineered bacterium.

[0008] In one specific embodiment of the present invention, the GenBank address of the glutamic acid synthase is QTD71277.1. The encoding gene of the glutamic acid synthase is inserted into the expression vector as a template, and linearized amplification is performed using primer pairs bpsA-F and bpsA-R to obtain a linearized bpsA fragment. The nucleotide sequences of bpsA-F and bpsA-R are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0009] In one specific embodiment of the present invention, the linearized bpsA fragment is recombined and ligated with the linearized pMUT1 to construct the recombinant plasmid pMUT1-bpsA-Kan; The linearized pMUT1 was obtained by linearizing pMUT1 using pMUT1-Kan plasmid as template and primers pMUT1-F and pMUT1-R. The nucleotide sequences of pMUT1-F and pMUT1-R are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0010] In one specific embodiment of the present invention, the GenBank address of the 4′-phosphopanylthioethylamine transferase is WP_015382751.1. The encoding gene of the 4′-phosphopanylthioethylamine transferase is inserted into the expression vector as a template, and linearized amplification is performed using primer pairs sfp-F and sfp-R to obtain a linearized sfp fragment. The nucleotide sequences of sfp-F and sfp-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0011] In one specific embodiment of the present invention, the linearized sfp fragment is recombined and ligated with the linearized pMUT2 to construct the recombinant plasmid pMUT2-sfp-Amp; The linearized pMUT2 was obtained by linearizing pMUT2 using pMUT2-Amp plasmid as template and primer pairs pMUT2-F and pMUT2-R. The nucleotide sequences of pMUT2-F and pMUT2-R are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0012] In one specific embodiment of the present invention, the recombinant plasmid pMUT1-bpsA-Kan and the recombinant plasmid pMUT2-sfp-Amp are transformed into an EcNc strain that does not contain cryptic plasmids pMUT1 and pMUT2 to construct the recombinant EcN engineered strain.

[0013] In one specific embodiment of the present invention, the recombinant plasmid pMUT1-bpsA-Kan is transformed into an EcNc strain that does not contain the cryptic plasmids pMUT1 and pMUT2 to construct EcN-pMUT1-bpsA-Kan; After preparing competent cells from EcN-pMUT1-bpsA-Kan, the recombinant plasmid pMUT2-sfp-Amp was transformed into EcN-pMUT1-bpsA-Kan competent cells to construct the recombinant EcN engineered bacteria.

[0014] The present invention also provides recombinant EcN engineered bacteria constructed using the above-described construction method.

[0015] The present invention also provides a method for constitutively synthesizing glutenin using the above-mentioned recombinant EcN engineered bacteria, comprising inoculating the recombinant EcN engineered bacteria into LB liquid medium for culture, collecting the bacterial cells and centrifuging, and the precipitate containing the glutenin.

[0016] In one specific embodiment of the present invention, the culture temperature is 25°C and the time is 16~20h.

[0017] Beneficial Effects: This invention uses two endogenous cryptogenic plasmids, pMUT1 and pMUT2, of the EcN strain as expression vectors. Without altering the original functional structural elements of the endogenous cryptogenic plasmids, gene expression unit fragments containing a Tac promoter, a mutant lac operon, and an RBS sequence are inserted into appropriate positions on both plasmids for exogenous gene expression. Simultaneously, Amp and Kan resistance genes are inserted into appropriate positions on the pMUT1 and pMUT2 vectors, facilitating the construction of the two recombinant vectors and the screening of positive clones. No antibiotics are required after successful construction of the recombinant vectors. Using this method, the engineered bacteria EcN-pMUT1-bpsA, EcN-pMUT2-sfp, and EcN-pMUT1-bpsA / pMUT2-sfp are constructed, which can successfully express the glutenin-related enzyme proteins glutenin synthase and PPTase without the addition of antibiotics or inducers, and synthesize glutenin pigment. Attached Figure Description

[0018] Figure 1 Electrophoresis diagram of PCR nucleic acid constructed for plasmids. In the diagram, A: linearized pMUT1 vector amplified by PCR; B: bpsA gene fragment; C: linearized pMUT2 vector amplified by PCR; D: sfp gene fragment. Figure 2 Figure 1 shows the construction and protein expression identification results of EcN engineered bacteria expressing bpsA and sfp. Figure A: Schematic diagram of recombinant plasmids pMUT1-bpsA and pMUT2-sfp; B: SDS-PAGE identification of proteins expressed by EcN engineered strains (1. EcN-pMUT2-sfp; 2. EcN-pMUT1-bpsA). Figure 3 Figure showing the expression results of bpsA and sfp proteins in the engineered bacteria EcN-pMUT1-bpsA / pMUT2-sfp; Figure 4 The figure shows the results of plasmid genetic stability verification. In the figure, A: plasmid loss identification; B: SDS-PAGE identification of proteins expressed by EcN engineered strains at different generations under antibiotic-free conditions. Figure 5The figures show the results of the constitutive synthesis, extraction, and tolerance identification of *Indigofera tinctoria*. Figure A shows the results of *Indigofera tinctoria* dissolving in DMSO; Figure B shows the results of temperature tolerance; and Figure C shows the results of pH tolerance. Detailed Implementation

[0019] This invention provides a method for constructing a recombinant EcN engineered bacterium that synthesizes glutenin, comprising the following steps: inserting the encoding gene of glutenin synthase and the encoding gene of 4′-phosphoupanylthioethylaminotransferase into the cryptic plasmids pMUT1 and pMUT2 of the EcN strain, respectively, to construct the recombinant EcN engineered bacterium.

[0020] The GenBank database for the glutamic acid synthase bpsA described in this invention is QTD71277.1. The encoding gene for bpsA is inserted into an expression vector as a template, and linearized amplification is performed using primer pairs bpsA-F and bpsA-R to obtain linearized bpsA fragments. The nucleotide sequences of bpsA-F and bpsA-R are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. In one embodiment of this invention, the expression vector is pET-28a, and the bpsA gene is inserted six bases after the ribosome binding site RBS on the pET-28a plasmid.

[0021] This invention involves recombination and ligation of the linearized bpsA fragment with linearized pMUT1 to construct the recombinant plasmid pMUT1-bpsA-Kan. The linearized pMUT1 is then amplified using the pMUT1-Kan plasmid as a template and primers to linearize pMUT1-F and pMUT1-R, yielding linearized pMUT1. The nucleotide sequences of pMUT1-F and pMUT1-R are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively. The pMUT1-Kan plasmid of this invention is already patented in Chinese Patent CN119799750A (Application No. 2025100005520, Application Date January 2, 2025, Publication Date April 11, 2025).

[0022] The 4′-phosphoubiotinylthioethylamine transferase (sfp) described in this invention is indexed in GenBank: WP_015382751.1. The encoding gene of sfp is inserted into an expression vector as a template, and linearized sfp fragments are obtained using primer pairs sfp-F and sfp-R. The nucleotide sequences of sfp-F and sfp-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. In one embodiment of this invention, the expression vector is pET-28a, and the sfp gene is inserted six bases after the RBS site on the pET-28a plasmid ribosome binding site.

[0023] This invention involves recombination and ligation of the linearized SFP fragment with linearized pMUT2 to construct the recombinant plasmid pMUT2-sfp-Amp. The linearized pMUT2 is then linearized and amplified using primers with pMUT2-F and pMUT2-R as a template. The nucleotide sequences of pMUT2-F and pMUT2-R are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively. The pMUT2-Amp plasmid described in this invention is also disclosed in Chinese Patent CN119799750A.

[0024] In this invention, the recombinant plasmid pMUT1-bpsA-Kan and the recombinant plasmid pMUT2-sfp-Amp are transformed into an EcNc strain that does not contain the cryptic plasmids pMUT1 and pMUT2, thereby constructing the recombinant EcN engineered strain.

[0025] In this invention, the recombinant plasmid pMUT1-bpsA-Kan is transformed into an EcNc strain that does not contain the cryptic plasmids pMUT1 and pMUT2 to construct EcN-pMUT1-bpsA-Kan; after preparing competent cells from EcN-pMUT1-bpsA-Kan, the recombinant plasmid pMUT2-sfp-Amp is transformed into EcN-pMUT1-bpsA-Kan competent cells to construct the recombinant EcN engineered strain.

[0026] Table 1. Primers used and their target fragments amplified

[0027] In this embodiment of the invention, the primers bpsA-F, bpsA-R, sfp-F, and sfp-R include the RBS sequence of the pET28a vector, which can amplify the target gene fragments bpsA and sfp.

[0028] The present invention also provides recombinant EcN engineered bacteria constructed using the above-described construction method.

[0029] This invention uses EcN as the chassis strain and constructs a recombinant engineered bacterium that stably expresses glutathione synthase and PPTase by modifying its endogenous cryptic plasmid. The expressed exogenous protein PPTase can activate glutathione synthase, converting two molecules of L-glutamine into one molecule of glutathione, enabling experimental microbial synthesis of glutathione. The glutathione synthase engineered strain EcN-pMUT1-bpsA / pMUT2-sfp constructed in this invention has the ability to achieve efficient co-expression of glutathione synthase bpsA and phosphopanylthioethylamine transferase sfp protein without the addition of any exogenous inducers. Even after five generations of continuous culture without antibiotic selection pressure, the plasmid remains stable and is not lost, fully demonstrating its good genetic stability and environmental adaptability. The EcN-pMUT1-bpsA / pMUT2-sfp engineered strain developed in this invention shows significant technical advantages and broad application prospects in sustainable industrial production and biomedical applications. The present invention also provides a method for constitutively synthesizing glutenin using the above-mentioned recombinant EcN engineered bacteria, comprising inoculating the recombinant EcN engineered bacteria into LB liquid medium for culture, collecting the bacterial cells and centrifuging, and the precipitate containing the glutenin.

[0030] In this invention, the recombinant EcN engineered bacteria are inoculated into LB liquid medium for cultivation. After collecting the bacterial cells, centrifugation is performed. The precipitate contains both engineered bacteria and glutinous rice blue. The bacteria are lysed by sonication, and the precipitate obtained by centrifugation is glutinous rice blue. The supernatant is discarded, and the glutinous rice blue precipitate is washed with water and centrifuged again. The supernatant is discarded, and the precipitate is dissolved in DMSO, sonicated in a water bath, and centrifuged to collect the supernatant. The supernatant contains glutinous rice blue pigment. The cultivation temperature in this invention is 25℃, and the time is 16-20 hours.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a recombinant EcN engineered bacterium for constitutively synthesizing glutenin, its construction method, and its synthesis method, should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 1. Experimental materials The ORF gene of glutamic acid synthase bpsA (GenBank ID: QTD71277.1) was synthesized, and six bases after the RBS site of the pET-28a plasmid ribosome binding site were inserted into the bpsA gene to generate the recombinant plasmid pET28a-bpsA. The ORF gene of 4′-phosphopantointilimetin aminotransferase sfp (GenBank ID: WP_015382751.1) was synthesized, and six bases after the RBS site of the pET-28a plasmid sfp were inserted into the sfp gene to generate the recombinant plasmid pET28a-sfp, with BL21(DE3) as the host. This was accomplished by Suzhou Genewiz Biotechnology Co., Ltd. Wild-type EcN strains, EcNc strains without the cryptogenic plasmids pMUT1 / pMUT2, EcN-pMUT1-Kan, and EcN-pMUT2-Amp were preserved by the School of Life Sciences and Medicine, Zhejiang Sci-Tech University, and have been published in an article (doi: 10.1007 / s12010-025-05536-2.).

[0033] 2. Construction of engineered bacteria EcN-pMUT1-bpsA and EcN-pMUT2-sfp and constitutive expression of related enzymes Primers bpsA-F, bpsA-R, sfp-F, and sfp-R were designed to amplify the target gene fragments bpsA and sfp. Using existing pMUT1-Kan and pMUT2-Amp plasmids as templates, primers pMUT1-F, pMUT1-R, pMUT2-F, and pMUT2-R were designed to amplify the linearized pMUT1 and pMUT2 vectors via reverse PCR (Table 1). The PCR products were purified by gel extraction. The linearized pMUT1 and bpsA fragments, and pMUT2 and sfp fragments, were ligated using the ClonExpress Recombinant Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to construct recombinant plasmids. The ligation products were transformed into DH5α competent cells and verified by sequencing.

[0034] The constructed pMUT1-bpsA-Kan and pMUT2-sfp-Amp were added to the prepared EcNc electroporation competent cells at a final concentration of 10 μg / mL. After mixing, the mixture was transferred to a pre-chilled 0.2 cm electroporation cuvette and incubated on ice for 5 min. The electroporator was set to 2.5 kV, 25 μF, 200 Ω, and 5 ms for the pulse. Immediately after the pulse, 900 μL of room temperature LB medium was added, mixed, and transferred to a 2 mL sterile centrifuge tube. The mixture was incubated at 37 °C and 200 rpm for approximately 1 h 20 min. The culture product was centrifuged at 5000 rpm for 10 min, and 900 μL of supernatant was discarded in a clean bench. The remaining product was mixed and spread onto LB solid medium with the corresponding antibiotic resistance. The mixture was then incubated overnight at 37 °C with the plates inverted.

[0035] Single colonies were picked and inoculated into 5 mL of LB medium and cultured at 25°C for 16–18 h. The expression of pbsA and sfp proteins was analyzed by SDS-PAGE.

[0036] 3. Construction of the dual-plasmid engineered bacteria EcN-pMUT1-bpsA / pMUT2-sfp and constitutive co-expression of related enzymes EcN-pMUT1-bpsA-Kan was prepared as electrotransfer competent cells, and pMUT2-sfp-Amp was electrotransferred into EcN-pMUT1-bpsA-Kan. The culture products were spread onto LB agar medium containing both Kan and Amp and incubated upside down at 37°C for 12 h. Single colonies were picked and inoculated into 5 mL of LB medium and incubated at 25°C for 16–18 h. Protein expression was analyzed by SDS-PAGE.

[0037] 4. Plasmid stability verification The engineered bacterial strain EcN-pMUT1-bpsA-pMUT2-sfp was streaked onto LB agar medium containing the corresponding antibiotic and incubated overnight at 37°C. Single colonies were picked and transferred to 5 mL of LB medium containing the corresponding antibiotic, and incubated at 37°C and 220 rpm for 12 h. The activated bacterial culture was then transferred at a 1% inoculum to LB medium containing the corresponding antibiotic and incubated at 37°C and 220 rpm for 12 h, designated as generation 0 with antibiotic. Generation 0 with antibiotic was then transferred at a 1% inoculum to LB medium without antibiotic and incubated at 37°C and 220 rpm for 12 h, designated as generation 1 without antibiotic. Successive passages were performed to obtain generations 2, 3, 4, and 5 without antibiotic. A suitable amount of bacterial culture from each generation was prepared for SDS-PAGE analysis.

[0038] The antibiotic-free fifth-generation bacterial culture was appropriately diluted and spread onto antibiotic-free solid LB agar medium, and incubated upside down at 37°C for 12 hours. Single colonies were picked and inoculated onto plates without antibiotics and plates containing the corresponding antibiotics, and incubated upside down at 37°C for 12 hours. The number of colonies grown on each plate was compared, and the plasmid loss rate was calculated.

[0039] 5. Constitutive synthesis, extraction, and tolerance tests of *Indigofera tinctoria* The recombinant bacteria were inoculated into 100 mL LB medium containing the corresponding resistance, while wild-type EcN was inoculated into LB medium as a control. The cultures were incubated at 25°C for 16–20 h. The bacterial cells were collected, centrifuged, and the precipitate was collected to obtain a mixture of bacterial cells and *Gynostemma pentaphyllum* precipitate. This mixture was ultrasonically disrupted, washed three times with water, dissolved in DMSO, centrifuged, and the supernatant was collected. The supernatant was then ultrafiltered through a 3k ultrafiltration tube to obtain *Gynostemma pentaphyllum*. The obtained *Gynostemma pentaphyllum* solution was placed in a metal bath at room temperature, 30, 40, 50, 60, 70, 80, 90, and 100°C for 30 min, and its OD value was measured at 612 nm using a UV spectrophotometer to determine the temperature tolerance of *Gynostemma pentaphyllum*.

[0040] Similarly, the pH of the solution was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, and 10 using HCl and NaOH. After standing for 30 minutes, the absorbance at 612 nm was measured using the original solution as a control to determine the acid and alkali tolerance of *Gynostemma pentaphyllum*.

[0041] 6. Results Analysis 6.1 Construction of engineered bacteria EcN-pMUT1-bpsA and EcN-pMUT2-sfp and expression of bpsA and sfp proteins like Figure 1 As shown, linearized vectors pMUT1-Kan and pMUT2-Amp, containing the target gene fragments bpsA and sfp, were obtained by PCR amplification. Recombinant plasmids expressing bpsA and sfp were constructed using seamless cloning technology and named pMUT1-bpsA and pMUT2-sfp. The constructed pMUT1-bpsA and pMUT2-sfp recombinant plasmids were electroporated into EcNc competent cells to obtain EcN engineered strains EcN-pMUT1-bpsA and EcN-pMUT2-sfp. The results were detected by SDS-PAGE electrophoresis. Figure 2 As shown, both engineered bacteria can express the corresponding proteins.

[0042] 6.2 Construction of the dual-plasmid engineered strain EcN-pMUT1-bpsA / pMUT2-sfp and co-expression of bpsA and sfp proteins The results are as follows Figure 3 As shown, after the engineered bacteria EcN-pMUT1-bpsA / pMUT2-sfp were inoculated and cultured, the bacterial cells were detected by SDS-PAGE electrophoresis. It was found that both proteins could be expressed, and the addition of no inducer or antibiotic had no effect on the protein expression. This achieved constitutive expression of the two proteins bpsA and sfp required for the synthesis of glutamic acid.

[0043] 6.3 Plasmid stability verification To assess the genetic stability of recombinant plasmids expressing two proteins in the EcNc strain under antibiotic-free selection pressure, the engineered bacteria were first cultured at 37°C for 12 h under Kan and Amp pressure (recorded as generation 0). Then, the antibiotic pressure was removed, and the bacteria were cultured continuously for 5 generations under the same conditions. Samples from each generation were subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 4 The results showed that protein content did not decrease significantly with each generation. Fifth-generation antibiotic-free bacterial culture was diluted and plated. Single colonies were picked and inoculated onto LB agar plates containing both antibiotics (Kan and Amp) and cultured. The results are as follows: Figure 4 Figure A shows that all colonies could grow simultaneously on both antibiotic-free and antibiotic-treated LB media, with 67 effective colonies. This indicates that after 5 generations of continuous culture without antibiotic pressure, the loss rate of pMUT1-bpsA and pMUT2-sfp plasmids was 0, demonstrating the high genetic stability of the recombinant plasmids in EcN.

[0044] 6.4 Constitutive Synthesis, Extraction, and Tolerance Identification of *Gynostemma pentaphyllum* Studies have shown that *Vallisneria natans* is poorly soluble in water and most organic solvents, but has good solubility in DMSO. Most of the *Vallisneria natans* produced by recombinant bacterial fermentation exists as a precipitate. The bacterial culture is collected, centrifuged, and a mixture of bacterial cells and *Vallisneria natans* precipitate is obtained. The bacterial cells are then ultrasonically disrupted. After washing with water, the precipitate is dissolved in DMSO to obtain *Vallisneria natans*, as shown below. Figure 5 As shown in Figure A. Further temperature tolerance tests showed that heating the *Indigofera tinctoria* solution at different temperatures for 30 minutes, with the temperature increasing up to 100℃, had no significant effect on the color and OD value of the solution. Figure 5 (B). Meanwhile, acid-base tolerance tests showed that within the pH range of 2-10, the color and OD value of the gorgonian blue solution were not significantly affected. Figure 5 The presence of C indicates that *Indigofera tinctoria* has high acid and alkali tolerance.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a recombinant EcN engineered bacterium that constitutively synthesizes glutathione, characterized in that, Includes the following steps: The coding genes for glutamic acid synthase and 4′-phosphouinylthioethylamine transferase were inserted into the cryptic plasmids pMUT1 and pMUT2 of the EcN strain, respectively, to construct the recombinant EcN engineered strain.

2. The construction method according to claim 1, characterized in that, The GenBank entry for the glutenin synthase is QTD71277.

1. The encoding gene of the glutenin synthase is inserted into the expression vector as a template, and linearized amplification is performed using primer pairs bpsA-F and bpsA-R to obtain the linearized bpsA fragment. The nucleotide sequences of bpsA-F and bpsA-R are shown in SEQ ID No. 1 and SEQ ID No.

2.

3. The construction method according to claim 2, characterized in that, The linearized bpsA fragment was recombined and ligated with the linearized pMUT1 to construct the recombinant plasmid pMUT1-bpsA-Kan; The linearized pMUT1 was obtained by linearizing pMUT1 using pMUT1-Kan plasmid as template and primers pMUT1-F and pMUT1-R. The nucleotide sequences of pMUT1-F and pMUT1-R are shown in SEQ ID No. 5 and SEQ ID No.

6.

4. The construction method according to claim 1, characterized in that, The GenBank address of the 4′-phosphopanylthioethylamine transferase is WP_015382751.

1. The encoding gene of the 4′-phosphopanylthioethylamine transferase was inserted into the expression vector as a template, and linearized amplification was performed using primer pairs sfp-F and sfp-R to obtain the linearized sfp fragment. The nucleotide sequences of sfp-F and sfp-R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

5. The construction method according to claim 4, characterized in that, The linearized sfp fragment was recombined and ligated with the linearized pMUT2 to construct the recombinant plasmid pMUT2-sfp-Amp; The linearized pMUT2 was obtained by linearizing pMUT2 using pMUT2-Amp plasmid as template and primer pairs pMUT2-F and pMUT2-R. The nucleotide sequences of pMUT2-F and pMUT2-R are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

6. The construction method according to claim 3 or 5, characterized in that, The recombinant plasmid pMUT1-bpsA-Kan and recombinant plasmid pMUT2-sfp-Amp were transformed into an EcNc strain that does not contain cryptic plasmids pMUT1 and pMUT2 to construct the recombinant EcN engineered strain.

7. The construction method according to claim 6, characterized in that, The recombinant plasmid pMUT1-bpsA-Kan was transformed into an EcNc strain that does not contain the cryptic plasmids pMUT1 and pMUT2 to construct EcN-pMUT1-bpsA-Kan; After preparing competent cells from EcN-pMUT1-bpsA-Kan, the recombinant plasmid pMUT2-sfp-Amp was transformed into EcN-pMUT1-bpsA-Kan competent cells to construct the recombinant EcN engineered bacteria.

8. The recombinant EcN engineered bacteria constructed using the construction method according to any one of claims 1 to 7.

9. A method for constitutively synthesizing glutenin using the recombinant EcN engineered bacteria of claim 8, characterized in that, The process includes inoculating the recombinant EcN engineered bacteria into LB liquid medium for culture, collecting the bacterial cells and centrifuging them, and the precipitate containing glutathione.

10. The method according to claim 9, characterized in that, The culture temperature was 25℃ and the time was 16~20h.

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