A high-glutathione-producing engineered Escherichia coli and its application

Abstract

This invention discloses a high-glutathione-producing engineered Escherichia coli strain and its applications, belonging to the field of biotechnology; the engineered Escherichia coli strain is named ZMGSH01F, with accession number CCTCC M 20252236; the engineered Escherichia coli strain is based on Escherichia coli BL21(DE3) as the starting strain, with gene clusters knocked out. ptsH‐ ptsI‐crr And integrate at this site glf‐glk Fusion genes, knockout gene clusters gsiA‐gsiB‐gsiC Integrate the endogenous glutathione transporter gene at this site. cycDC Knockout of γ-glutamyltransferase gene ggt Simultaneously expressing the recombinant plasmid pRSFDuet- gshAB K177E‑E275K‑V513A - ppk2 The engineered Escherichia coli strain can efficiently produce glutathione through fermentation culture using glucose, monosodium glutamate, cysteine, and glycine as substrates.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered Escherichia coli strain that produces high levels of glutathione and its applications. Background Technology

[0002] Glutathione (GSH) is a tripeptide molecule formed by glutamic acid, cysteine, and glycine linked by peptide bonds. As the most abundant non-protein thiol compound in living organisms, GSH is widely distributed in animal, plant, and microbial cells, existing in two dynamic equilibrium forms: reduced GSH and oxidized GSH (GSSG), with the reduced form accounting for over 90%. This balance is jointly regulated by glutathione reductase and NADPH.

[0003] Glutathione (GSH), a key intracellular antioxidant, possesses powerful reducing capabilities. It not only efficiently scavenge reactive oxygen species such as hydroxyl radicals and superoxide anions, maintaining intracellular redox homeostasis, but also promotes metabolic excretion by binding to exogenous toxic substances. Simultaneously, it regulates immune cell activity and enhances the body's defense capabilities. These core physiological functions make it an important active substance used in the pharmaceutical, cosmetic, and food industries. This multi-field demand is driving the continuous optimization of GSH production technologies such as fermentation and enzymatic methods to improve yield and purity.

[0004] The production processes of GSH are mainly divided into three categories: chemical synthesis, plant and animal extraction, and microbial fermentation. Chemical synthesis is gradually being phased out due to its high pollution and cost. Plant and animal extraction is limited by raw material sources and inefficient extraction processes. Microbial fermentation, with its environmental friendliness, high production efficiency, and ease of scaling, has become the preferred solution for industrial production. However, traditional production relies on yeast extraction, which suffers from low yield, high cost, and complex extraction processes. For example, Chinese patent CN109929869A, filed on December 11, 2018, discloses a genetically engineered bacterium for synthesizing glutathione, its preparation method, and its application. This invention uses integrative expression vectors pδGAP′g and pδGAPh and a strong promoter, either the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) or the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene promoter GAP′, to construct a fusion expression plasmid. Through transformation, the target gene is integrated into the host genome, thereby achieving high expression of cystathionine γ-lyase and cystathionine β-synthase in the cell; at the same time, cystathionine β-lyase is inactivated, thereby increasing the cysteine ​​level in the metabolic pool and promoting glutathione synthesis. However, in industrial applications, its cost-effectiveness is not as good as that of optimized Escherichia coli.

[0005] Escherichia coli (E. coli) is an ideal engineering host due to its rapid growth and ease of genetic manipulation. However, wild-type E. coli has weak GSH synthesis capacity and poses a risk of endotoxin contamination, making it difficult to meet the needs of industrial production. Therefore, modifying E. coli for GSH production through biotechnology has become a popular research direction. Chinese patent CN113025592A, filed on April 28, 2021, discloses a high-performance polyphosphate kinase mutant and its application. It constructs a polyphosphate kinase (or its mutant) gene into a pET system plasmid, while a glutathione bifunctional enzyme (glutathione synthase) gene is constructed into an expression system containing a p15A replicon. The two plasmids can be co-expressed in microorganisms, achieving co-expression of the two enzymes. This integrates the ATP regeneration system and the glutathione biosynthesis catalytic system, creating a "hybrid reactor." Only one type of microorganism is needed for fermentation, allowing the bacterial cells to be used as a biocatalyst in the reaction system to synthesize glutathione. This patent uses a dual plasmid system to express the PPK2 mutant and GSH synthase separately. This system faces the risk of genetic instability during long-term fermentation.

[0006] Early research focused on overexpressing key enzymes in GSH synthesis, namely γ-glutamylcysteine ​​synthase (GshA) and glutathione synthase (GshB). However, GshA is strongly inhibited by the end product GSH, severely limiting further increases in synthesis throughput. To overcome this limitation, subsequent research shifted to expressing bifunctional glutathione synthases (GshF / GshAB), which catalyze two consecutive reactions on a single polypeptide chain and exhibit low sensitivity to the end product. However, the enzymatic activity of native GshF is often limited by its catalytic efficiency and the energy supply during the reaction process.

[0007] Besides the synthetic pathway itself, the host's central metabolism and product turnover also profoundly affect the final yield of GSH. The inherent glucose phosphotransferase system (PTS) in *E. coli* exhibits a carbon metabolite repression effect during glucose transport, consuming large amounts of phosphoenolpyruvate (PEP). PEP is also a crucial metabolite for the synthesis of oxaloacetate, a precursor to GSH, leading to uneconomical carbon source utilization. Furthermore, the cell's own γ-glutamyltransferase degrades GSH, and the complex glutathione transport system results in inefficient intracellular and extracellular circulation of synthesized GSH, hindering its efficient accumulation in the fermentation broth.

[0008] In summary, although some progress has been made in modifying Escherichia coli to produce GSH through biotechnology, existing strains and technologies still have many shortcomings. Therefore, there is an urgent need for a systematic technical solution to construct the next generation of engineered bacteria that can efficiently synthesize and secrete glutathione through multi-faceted synergistic modification. Summary of the Invention

[0009] To address the problems existing in the prior art, this invention provides a high-glutathione-producing engineered Escherichia coli strain and its application. The engineered Escherichia coli strain can efficiently synthesize and stably secrete glutathione, and has significant prospects for industrial application.

[0010] The technical solution of the present invention is as follows:

[0011] One of the objectives of this invention is to provide a high-glutathione-producing engineered Escherichia coli strain, with accession number CCTCC M 20252236 and named ZMGSH01F.

[0012] Furthermore, the preservation information of the engineered Escherichia coli is as follows:

[0013] Bacterial strain name: Escherichia coli

[0014] Latin name: Escherichia coli

[0015] Strain number: ZMGSH01F

[0016] Preservation Institution: China Center for Type Culture Collection

[0017] Abbreviation for depository institution: CCTCC

[0018] Address: Wuhan University, Wuhan, China

[0019] Preservation date: October 17, 2025.

[0020] Furthermore, the engineered E. coli strain ZMGSH01F uses E. coli BL21(DE3) as the starting strain and knocks out the gene clusters... ptsH-ptsI-crr And integrate at this site glf-glk Fusion genes, knockout gene clusters gsiA-gsiB-gsiC Integrate the endogenous glutathione transporter gene at this site. cycDC Knockout of γ-glutamyltransferase gene ggt Simultaneously expressing the recombinant plasmid pRSFDuet- gshAB K177E-E275K-V513A - ppk2 .

[0021] Furthermore, the aforementioned glf-glk The nucleotide sequence of the fusion gene is shown in SEQ ID NO. 5, where positions 1-1419 are... glf The gene, positions 1420-1464 are (Gly4Ser)3 flexible linker peptides, and positions 1465-2451 are... glk Gene, glf Genes and glk Genes are linked via a (Gly4Ser)3 flexible linker peptide.

[0022] Furthermore, glucose permease gene glf and glucokinase gene glk Derived from *Mammotrophic motility* Zymomonas mobilis .

[0023] Furthermore, the endogenous glutathione transporter gene cycDC The nucleotide sequence is shown in SEQ ID NO.6.

[0024] Furthermore, the recombinant plasmid pRSFDuet- gshAB K177E-E275K-V513A - ppk2 middle, gshAB K177E -E275K-V513A The mutated gene originates from Actinobacillus pleuropneumoniae in porcines. Actinobacillus pleuropneumoniae The nucleotide sequence is shown in SEQ ID NO. 4.

[0025] Furthermore, gshAB K177E-E275K-V513A Based on the glutathione bifunctional synthase gshAB with the amino acid sequence shown in SEQ ID NO. 2, the amino acid at position 177 is mutated from lysine to glutamic acid; the amino acid at position 275 is mutated from glutamic acid to lysine; and the amino acid at position 513 is mutated from valine to alanine.

[0026] Furthermore, the bifunctional glutathione synthase gene gshAB The nucleotide sequence is shown in SEQ ID NO.1.

[0027] Furthermore, the recombinant plasmid pRSFDuet- gshAB K177E-E275K-V513A - ppk2 middle, ppk2 The gene originates from Pseudomonas aeruginosa. Pseudomonas aeruginosa The nucleotide sequence is shown in SEQ ID NO. 3.

[0028] The second objective of this invention is to provide an engineered Escherichia coli strain that produces high levels of glutathione for the fermentation and culture of glucose, monosodium glutamate, cysteine, and glycine as substrates for the production of glutathione.

[0029] The third objective of this invention is to provide a method for producing glutathione.

[0030] Furthermore, a seed culture of recombinant engineered Escherichia coli ZMGSH01F was obtained, and the seed culture was inoculated into a fermentation medium. The pH was adjusted to 6.9-7.0 during the fermentation process, and the OD... 600At temperatures of 39-41°C, isopropyl-β-D-thiogalactoside IPTG was added and the mixture was induced at 28°C for 10 h. Then, the temperature was raised to 30°C and precursors sodium glutamate, cysteine, and glycine were added. The fermentation broth was collected and purified to obtain glutathione.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] 1. This invention, through multi-faceted synergistic design, for the first time constructs and discloses a high-glutathione-producing engineered Escherichia coli strain, ZMGSH01F. Firstly, a bifunctional glutathione synthase gene is co-expressed using the plasmid pRSFDuet. gshAB and polyphosphate kinase gene ppk2 , gshAB The mutant significantly improved the catalytic efficiency of the synthase. ppk2 Enhancing its ATP regeneration capacity provides sufficient energy for the continuous synthesis of GSH, breaking through the bottleneck of the mismatch between energy supply and enzymatic reaction efficiency in the glutathione synthesis process. Secondly, based on this, by knocking out... ptsH-ptsI-crr Gene clusters and integration of heterogeneous genes glf-glk An expression cassette was constructed to create a glucose transport system independent of the PTS system, eliminating carbon metabolite repression and simultaneously knocking out the γ-glutamyl transferase gene. ggt To reduce GSH degradation, knockout gsiA-gsiB-gsiC The gene cluster inhibits intracellular reuptake of GSH, while simultaneously integrating and overexpressing endogenous transporter genes at this site. cycDC This strongly promotes the extracellular secretion of GSH.

[0033] 2. The *E. coli* engineered strain ZMGSH01F designed in this invention is an engineered strain with a clear genetic background that does not contain exogenous helper plasmids. Its genetic stability has been verified by subculturing experiments, demonstrating its ability to stably inherit high-yield traits. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20252236. From its fermentation curve (… Figure 2 It can be seen that this strain grows well during fermentation, can efficiently utilize glucose, and rapidly synthesizes and accumulates glutathione after induction, demonstrating its excellent metabolic characteristics as a cell factory.

[0034] 3. The innovative engineered *E. coli* strain ZMGSH01F of this invention exhibits ideal yield in the industrial fermentation production of glutathione. Under optimized fermentation technology, strain ZMGSH01F achieved a glutathione yield of up to 35.6 g / L in a small-scale 10 L fermenter experiment, far exceeding the level of shake-flask fermentation, demonstrating its potential in a controlled fermenter environment. The mutant strain in this invention... gshAB and ppk2 Co-expression ensures the synthesis dynamics; glf-glkThe integration makes glucose consumption more efficient, providing ample carbon skeletons and energy for synthesis; and ggt Knockout and cycDC Overexpression minimizes product loss and promotes extracellular secretion, reducing the difficulty and cost of extraction and purification. The construction of this engineered *E. coli* strain not only achieves synergistic optimization in enzyme activity, energy, substrate utilization, and product secretion, but also demonstrates considerable glutathione yield in practical applications, possessing ideal prospects for industrial application and market competitiveness. Attached Figure Description

[0035] Figure 1 The recombinant plasmid pRSFDuet- constructed in this invention gshAB - ppk2 Structural diagram;

[0036] Figure 2 The fermentation curve of the high-glutathione-producing Escherichia coli engineered strain ZMGSH01F described in this invention in a 10 L fermenter. Detailed Implementation

[0037] The present invention will be further described below with reference to preferred embodiments. The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values. These ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed herein.

[0038] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] In the following examples, "codon optimization" refers to gene redesign that utilizes preferred codons and avoids codons with low utilization or rarity. Every organism exhibits some degree of codon utilization difference or preference, with the most frequently used codons being preferred codons.

[0041] Molecular biology experiments not specifically described in the following examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, which were mainly performed in accordance with *Molecular Cloning: A Laboratory Manual* (3rd edition). PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier.

[0042] The whole-genome synthesis, primer synthesis, and sequencing in the following examples were performed by Shanghai Sangon Biotech Co., Ltd. Host strain Escherichia coli BL21 (DE3) competent cells were purchased from Shanghai Sangon Biotech Co., Ltd., and plasmid pRSFDuet-1 was purchased from Changsha Aibiwei Biotechnology Co., Ltd.

[0043] Table 1 Primer sequences in the following examples

[0044]

[0045] The shake-flask fermentation method is described in the following examples:

[0046] Seed culture medium (LB medium): tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0047] Shake-flask fermentation: Pick a single colony from the plate and inoculate it into 5 mL of LB medium containing Kans, incubate overnight at 37°C with shaking at 220 rpm. Inoculate the seed culture at a 1% inoculation rate into 50 mL of LB medium and incubate at 37°C until OD reaches 50%. 600 =0.6, add 0.5 mMIPTG, induce expression at 28℃, add monosodium glutamate, cysteine, glycine and sodium hexametaphosphate.

[0048] The following examples illustrate the glutathione detection method:

[0049] After centrifugation of the fermentation broth, the supernatant was filtered through a 0.22 μm filter membrane and then used for HPLC analysis.

[0050] HPLC conditions: Column: ACE Excel 5 C18-AR; Mobile phase: Sodium heptanesulfonate-phosphate buffer (pH 3.0): methanol = 90:10 (volume ratio); Detection wavelength: 210 nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Column temperature: 30℃.

[0051] The plasmid elimination method in the following examples:

[0052] 1) Elimination of pTarget F series plasmids: Select single clones and inoculate them into LB medium containing kanamycin (Kana) and 0.5 mMIPTG antibiotic. Incubate overnight at 30°C. Dilute the plasmids by a certain factor and spread them on Kana plates. Incubate at 30°C. Select single clones and streak them on Kana plates and spectinomycin Spec plates. If the single clone grows on the Kana plate but not on the Spec plate, it indicates that the pTarget F plasmid has been successfully eliminated.

[0053] 2) Elimination of pCas9 series plasmids: Pick single clones in LB medium and incubate overnight at 37°C. Dilute to a certain factor and spread on LB plates. Incubate at 37°C. Pick single clones and streak them on Kana plates and LB plates. If the single clone grows on the LB plate but not on the Kana plate, it means that the pCas9 plasmid has been successfully eliminated.

[0054] Example 1

[0055] This embodiment provides a method for constructing glutathione-producing recombinant Escherichia coli, and the specific operation steps are as follows:

[0056] I. pRSFDuet- gshAB - ppk2 plasmid construction

[0057] 1. Select strains derived from Actinobacillus pleuropneumoniae (IVPP) Actinobacillus pleuropneumoniae The bifunctional glutathione synthase gene gshAB According to NCBI ID WP_005598599.1 After codon optimization, the nucleotide sequence SEQ ID NO.1 was synthesized, and its amino acid sequence is shown in SEQ ID NO.2;

[0058] 2. Selected from Pseudomonas aeruginosa ( Pseudomonas aeruginosa polyphosphate kinase 2 gene ppk2 According to NCBI ID AY168003.1 After codon optimization, the nucleotide sequence SEQ ID NO.3 was synthesized.

[0059] 3. gshAB Insert pRSFDuet-1 vector at the MCS1 site, ppk2 The specific steps for inserting the pRSFDuet-1 vector into the MCS2 site are as follows:

[0060] (1) PCR recovery using primers gshAB-vF / gshAB-vR gshAB Excerpt ， The reaction conditions were: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 15 s, 57℃ annealing for 30 s, 72℃ extension for 3 min, for 30 cycles, using pRSFDuet-1 support. Sal I and Not After I enzyme digestion, the product is recovered and... gshAB The recovered fragment was ligated and transformed into *E. coli* DH5α via heat shock. The culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 12 h. Positive clones were picked for colony PCR verification. Sequencing confirmed that the vector pRSFDuet- gshAB Successfully built;

[0061] (2) PCR recovery using primers ppk2-vF / ppk2-vR ppk2 Gene, reaction conditions: 95℃ pre-denaturation for 30s, 95℃ denaturation for 15s, 57℃ annealing for 30s, 72℃ extension for 3min, 30 cycles, vector pRSFDuet- gshAB use Nde I and Xho After I enzyme digestion, the product is recovered and... ppk2 The recovered fragment was ligated and transformed into *E. coli* DH5α via heat shock. The culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 12 h. Positive clones were picked for colony PCR verification. Sequencing confirmed that the vector pRSFDuet- gshAB - ppk2 Successfully constructed, its schematic diagram is as follows Figure 1 As shown;

[0062] (3) Plasmid pRSFDuet- gshAB - ppk2 The glutathione-producing strain GSHP1 was obtained by heat shock transformation of Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and cultured at 37°C for 16 h. Positive clones were picked for colony PCR verification.

[0063] The strain GSHP1 was fermented in shake flasks and its GSH content was found to be 0.35 g / L.

[0064] Example 2

[0065] This embodiment provides a bifunctional glutathione synthase gene. gshAB Mutation methods were used to target the key enzyme gene, the bifunctional glutathione synthase gene. gshAB Constructed using error-prone PCR methods gshAB The mutant library was used to screen for mutant strains with improved glutathione synthesis efficiency. The specific steps are as follows:

[0066] I. Construction of mutant expression library

[0067] 1. Error-prone PCR mutations

[0068] Using QuickMutation purchased from Shanghai Beyotime Biotechnology Co., Ltd. TM Gene random mutation kit, using plasmid pRSFDuet- gshAB - ppk2 Using gshAB-vF / gshAB-vR primers as a template, error-prone PCR was performed on the glutathione bifunctional enzyme gene. gshAB Randomly introduce nucleotide mutations;

[0069] Error-prone PCR system (50 μL): ddH2O 32.5 μL, RandomMut buffer (10X) 5 μL, Mutation enhancer (10X) 5 μL, dNTP (2.5 mM each) 5 μL, template DNA 0.5 μL, primer mixture (10 μM each) 1 μL, RandomMut DNA polymerase 1 μL;

[0070] Error-prone PCR procedure: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles; final extension at 72℃ for 3 min, store product at 4℃.

[0071] To improve the mutation rate, the amplification products of the above error-prone PCR were recovered and used as templates for continuous error-prone PCR, with the procedure being the same as the steps described above.

[0072] 2. Construction of mutant libraries

[0073] Error-prone PCR products were recovered using a DNA recovery kit and ligated to DNA-recovered DNA. Sal I and Not pRSFDuet- treated with double enzymes gshAB - ppk2 The ligation product was transformed into BL21(DE3) competent cells using the vector, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated at 37°C for 16 h to obtain... gshAB M Mutant library.

[0074] II. Screening of high-glutathione-producing strains from mutant libraries

[0075] 1. Cu 2+ Initial screening on plates: To determine the appropriate screening pressure, the expression wild-type... gshAB Strain P1 was spotted in samples containing different concentrations of Cu 2+ The original strain was grown on LB agar plates containing 0.5 mM IPTG. The minimum inhibitory concentration (MIC) of the original strain was determined by observing its growth. 10 mM was ultimately selected as the selection concentration; at this concentration, the original strain could barely grow, while the high-glutathione mutant was able to form visible colonies. The mutants grown on the aforementioned LB agar plates... gshAB M Single clones of mutant libraries are crossed out at a concentration of 10 mMCu. 2+ LB plates containing 0.5 mM IPTG were incubated overnight.

[0076] 2. Secondary screening: A secondary screening was performed using a reduced glutathione (GSH) assay kit (DTNB microplate method) to determine the expression of wild-type GSH. gshAB strain P1 was used as a control, and the detection kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0077] Cu 2+ Single colonies from the initial plate screening were picked and transferred to 96-well plates containing 200 μL of LB medium (containing Kans), and incubated overnight at 37°C with shaking at 800 rpm. 10 μL of the bacterial culture from each well was then transferred to a new 96-well plate containing 200 μL of fermentation medium and incubated at 37°C until OD500 was reached. 600 ≈0.6, add IPTG for induction, and continue culturing at 30℃ for 16 h. After culturing, test the samples according to the instructions and measure the absorbance of each well using a microplate reader at 412 nm;

[0078] OD Screening 600 Clones with significantly higher OD values ​​than the original strain were selected. 600 The 10 strains with the highest values ​​were named GSHP2-GM1, GSHP2-GM2, GSHP2-GM3, GSHP2-GM4, GSHP2-GM5, GSHP2-GM6, GSHP2-GM7, GSHP2-GM8, GSHP2-GM9, and GSHP2-GM10, and were subjected to shake-flask fermentation for secondary screening.

[0079] 3. Shake-flask secondary screening: The 10 strains obtained from the initial screening (GSHP2-GM1, GSHP2-GM2, GSHP2-GM3, GSHP2-GM4, GSHP2-GM5, GSHP2-GM6, GSHP2-GM7, GSHP2-GM8, GSHP2-GM9, GSHP2-GM10) were subjected to shake-flask fermentation for verification. The glutathione content was determined by HPLC, and the data are shown in Table 1. Among them, GSHP2-GM3 had the highest glutathione yield, with a glutathione content of 0.68 g / L.

[0080] Plasmids were extracted from the mutant strain GSHP2-GM3, which had the highest yield, and sequenced to determine its mutation sites. Sequencing results showed that, compared to the wild-type strain, this mutant strain had three amino acid mutations: amino acid position 177 was changed from lysine (K) to glutamic acid (E); amino acid position 275 was changed from glutamic acid (E) to lysine (K); and amino acid position 513 was changed from valine (V) to alanine (A). Based on these mutation characteristics, this mutant gene was named... gshAB K177E-E275K-V513A The nucleotide sequence is shown in SEQ ID NO. 4. The plasmid carried by this mutant strain GSHP2-GM3 is named pRSFDuet- gshABK177E-E275K-V513A - ppk2 .

[0081] Table 2. Citicoline content after 24 h of shake-flask fermentation

[0082]

[0083] Example 3

[0084] This embodiment provides a method for constructing a highly efficient PTS-independent glucose transport system by knocking out the PTS system gene cluster. ptsH-ptsI-crr Integrating glucose permease gene from *Mammotrophic motility* glf and glucokinase gene glk To construct an efficient PTS-independent glucose transport system, increase the carbon flux of the GSH biosynthesis pathway, improve glucose utilization in cells, and enhance the efficiency of GSH synthesis, the specific operational steps are as follows:

[0085] I. Knockout of vector pTarget F-Δ ptsH - ptsI - crr Construction

[0086] First, use the CHOPCHOP online design tool (http: / / chopchop.cbu.uib.no / ) to design for... ptsH- ptsI-crr A 20 bp specific target sequence was designed for the gene cluster. Using pTarget F plasmid as a template, reverse PCR amplification was performed using the ptsH-ptsI-crr-N20-F / -R primer pair. The PCR amplification product was digested with the restriction endonuclease DpnI and then transformed into DH5α *E. coli* competent cells. The transformed bacterial culture was evenly spread on LB selective medium plates supplemented with spectinomycin. After single colonies grew, typical colonies were picked for sequencing verification, and the gene cluster was successfully constructed. ptsH- ptsI-crr Gene knockout vector pTarget F-Δ ptsH - ptsI - crr .

[0087] II. DNA homologous recombination insertion fragments— glf-glk Construction of expression box

[0088] 1. The bacteria derived from *Mammotrophic motility* (Mammotrophic motility) Zymomonas mobilis glucose permease gene glf and glucokinase gene glk Codon optimization was performed to adapt to the host expression system. The optimized... glf and glkGenes are linked by (Gly4Ser)3 flexible linker peptides to form... glf-glk A fusion gene, based on which the nucleotide fragment shown in SEQ ID NO. 5 was artificially synthesized, wherein positions 1-1419 of SEQ ID NO. 5 are... glf The gene, positions 1420-1464 are (Gly4Ser)3 flexible linker peptides, and positions 1465-2451 are... glk Gene.

[0089] 2. Using the artificially synthesized SEQ ID NO.5 fragment as a template, the fragment was cloned using primers glf-vF / glk-vR. glf-glk Fusion genes, and with EcoR I / BamH The pTrc99A vector, digested with enzyme I twice, was ligated and transformed into DH5α competent E. coli cells. The cells were plated on LB selective medium supplemented with ampicillin. After single colonies grew, they were picked and sequenced for verification, thus constructing the pTrc99A- vector. glf - glk Carrier.

[0090] 3. A two-round PCR strategy was used to construct... glf-glk Expression cassette: First round PCR with pTrc99A- glf - glk Using the vector as a template, PCR was performed with primers V-LH-F / V-RH-R to obtain... pTrc-glf-glk-rrnB T1 Fragment; using the BL21(DE3) Escherichia coli genome as a template, amplification was performed using primers ptsH-ptsI-crr-LH-F / R. ptsH-ptsI-crr Upstream homology arm LH Amplification was performed using primers ptsH-ptsI-crr-RH-F / R. ptsH-ptsI-crr Downstream homology arm RH The second round of PCR was conducted using... LH , pTrc-glf- glk-rrnB T1 Fragments and RH Using a common template, a complete homologous recombination insert is spliced ​​together using overlap extension PCR technology, i.e. glf-glk Expression box.

[0091] III. Gene Locus ptsH-ptsI-crr Knockout and glf-glk Gene integration

[0092] 1. Prepare Escherichia coli GSHP2-GM3 competent cells, transform pCas9 plasmid into these competent cells, and obtain positive strains through screening, named GSHP2-GM3 / pCas9;

[0093] 2. Prepare competent cells of GSHP2-GM3 / pCas9 strain, and combine the pTarget F-ΔptsH-ptsI-crr knockout vector constructed above with... glf-glk The expression cassette was introduced into the system via electroporation. After electroporation, 1 mL of LB medium containing 10 mM arabinose was added to the system, and the culture was incubated at 37°C and 200 rpm for 2 h. The incubated bacterial culture was then spread onto LB select plates containing chloramphenicol, ampicillin, and 10 mM arabinose.

[0094] 3. Single colonies grown on the plate were initially verified by colony PCR. Positive transformants were screened and their genomes were further extracted and sequenced for verification. The correctly sequenced strains were inoculated into antibiotic-free LB medium and continuously passaged to eliminate the two plasmids pTarget F-ΔptsH-ptsI-crr and pCas9. The strain with pTarget F-ΔptsH-ptsI-crr eliminated was GSHP3 / pCas9, and the final plasmid-eliminated strain was named GSHP3.

[0095] IV. Shake-flask fermentation of strain P3

[0096] The residual sugar and glutathione content in the fermentation broth of strain GSHP3 were detected by shaking flask fermentation.

[0097] After shake-flask fermentation, strain GSHP3 showed improved glucose-to-glutathione conversion efficiency compared to the control strain GSHP2-GM3. The glucose consumption was 10.8 g / L, and the glutathione yield reached 1.03 g / L, representing a 1.51-fold increase in glutathione yield.

[0098] Example 4

[0099] This embodiment uses metabolic engineering techniques to, on the one hand, disrupt... ggt Genes reduce glutathione degradation and disrupt intracellular glutathione transport-related processes. gsiA-gsiB-gsiC Gene clusters, simultaneously overexpressing extracellular transporter genes cydDC This synergistically promotes the extracellular secretion and accumulation of glutathione. The specific steps are as follows:

[0100] I. GGT gene knockout

[0101] Referring to the method in Example 3, design ggt Gene cluster target sites and corresponding sgRNA primers were used to construct the recombinant plasmid pTarget-Δ. ggt Using BL21 (DE3) genomic DNA as a template, the upstream and downstream homologous arms of the ggt gene were amplified using primers ggt-UF / R and ggt-DF / R, and overlap extension PCR was used to amplify the homologous arms.[[ID=1 Connect the upstream and downstream homologous arms and construct the homologous arms into the plasmid pTarget-Δ ​ In the process, plasmid pTarget-Δ was obtained. ​ - ​ .

[0102] Competent cells were prepared from Escherichia coli GSHP3 / pCas9 in Example 3, and the pTarget-Δ cells constructed above were used. ​ - ​ The bacteria were transformed into competent cells of the GSHP3 / pCas9 strain, and colony PCR and genome sequencing were used to select the colony. ​ Gene knockout strain GSHP3-Δ ​ / pCas9.

[0103] II. Destruction ​ Gene clusters and overexpression ​

[0104] 1. pTarget-Δ ​ Plasmid construction: Following the method described in Example 3, the plasmid was designed... ​ Gene cluster target sites and corresponding sgRNA primers were identified, and the recombinant plasmid pTarget-Δgsi was constructed and validated.

[0105] 2. Regarding ​ Gene clusters ​ Construction of overexpression recombinant fragments

[0106] Referring to Example 3, BL21 (DE3) genomic DNA was used as a template and amplified using primers gsi-UF / R and gsi-DF / R. ​ Upstream homologous arm of gene cluster ​ and downstream homologous arm ​ Amplification was performed using primers gsi-Ptac-cydDC-F / R. ​ The fragment will ​ Fragment insertion into pTrc99A to construct plasmid pTrc99A- ​ Using it as a template, PCR was performed using primers V-LH-F / V-RH-R to obtain... ​ Fragments; PCR splicing via overlap extension ​ , ​ ​ Fragments and ​ To form a complete homologous recombination insertion fragment, i.e. pTrc-cydDC Expression box;

[0107] 3. cydDC Gene integration

[0108] Preparation of Escherichia coli GSHP3-Δ ggt / pCas9 competent cells, with the pTarget- constructed above gsi Knockout vector and insertion fragment expression box pTrc-cydDC GSHP3-Δ is introduced via electroconversion. ggt Competent cells of strain / pCas9 were obtained through colony PCR screening and genome sequencing. cydDC The strain was successfully integrated, and after removing the pCas and pTarget plasmids, a plasmid-free recombinant strain was obtained, which was named GSHP4.

[0109] III. Shake-flask fermentation of strain GSHP4

[0110] Strain GSHP4 and control strain GSHP3 were subjected to shake-flask fermentation, and the glutathione content of the supernatant was measured. The results showed that the GSH content of strain GSHP4 was 1.67 g / L, which was significantly higher than that of the control strain, being 1.62 times higher.

[0111] The selected *Escherichia coli* strain GSHP4 was named *Escherichia coli* ZMGSH01F. The engineered *Escherichia coli* strain ZMGSH01F was passaged to investigate its genetic stability. The genetically stable engineered *Escherichia coli* strain ZMGSH01F, capable of simultaneously and efficiently accumulating glutathione, was deposited on October 17, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC M 20252236, located at Wuhan University, Wuhan, China, 430072, China.

[0112] Example 5

[0113] This embodiment conducts a fermentation experiment of strain ZMGSH01F in a 10 L fermenter. The specific operation steps are as follows:

[0114] I. Culture medium formulation

[0115] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0, sterilized at 121℃ for 20 min, with 50 μg / mL kanamycin added;

[0116] Seed shake flask: tryptone 12g / L, yeast extract 20g / L, glycerol 5g / L, dipotassium hydrogen phosphate trihydrate 16.43g / L, potassium dihydrogen phosphate 2.31g / L;

[0117] Fermentation medium: soybean peptone 5 g / L, yeast extract 10 g / L, dipotassium hydrogen phosphate trihydrate 9.85 g / L, potassium dihydrogen phosphate 16.25 g / L, sodium citrate dihydrate 1.35 g / L, magnesium sulfate heptahydrate 2.2 g / L, ammonium sulfate 2.65 g / L, glucose monohydrate 10 g / L, biotin 0.0125 g / L, trace element mixture 2.5 mL (citric acid monohydrate 58 g / L, FeCl3·6H2O 12 g / L, ZnSO4·7H2O 4 g / L, CoCl2·6H2O 0.8 g / L, CuSO4·5H2O 0.8 g / L, boric acid 0.8 g / L, MnSO4·H2O 4.4 g / L, CaCl2·2H2O 4 g / L), defoamer 0.3 g / L;

[0118] Feeding medium: 800 g / L glucose monohydrate, 114.19 g / L sodium glutamate, 61.31 g / L cysteine, 50.63 g / L glycine, 12.22 g / L sodium hexametaphosphate.

[0119] II. Fermentation Parameter Control

[0120] 1. Seed culture: Activate the culture by streaking the glycerol culture tube onto an LB agar plate using an inoculation loop. Pick a single colony and inoculate it into 5 mL of LB medium (50 mg / L kan). Incubate at 37°C and 220 rpm for 14-16 h. Transfer the culture to a seed culture flask at a 2% inoculation rate and incubate at 37°C and 220 rpm for 4-6 h until OD (Organic Dysplasia) occurs. 600 =4-6. As a secondary seed solution;

[0121] 2. Fermentation Culture: Inoculate the seed culture into the fermenter at a 5% inoculum rate. Maintain the following conditions: pH = 6.9-7.0, 37±0.5℃, 200 rpm, 1 vvm. Adjust the rotation speed, aeration rate, and tank pressure to control DO = 20-40%. Cultivate until both DO and pH rise simultaneously, and residual sugar ≤ 1 g / L. Begin feeding the fermenter, controlling the feeding rate to ensure cell growth. OD 600 When the temperature reaches approximately 40°C, begin a gradient cooling process at 3°C / 20 min until reaching 28°C, then add IPTG for induction. After 10 h of induction, begin a gradient heating process at 1°C / 20 min until reaching 30°C, then begin uniformly feeding the substrate amino acid mixture, closely monitoring changes in dissolved oxygen and residual sugar, and adjusting the carbon source feed accordingly.

[0122] 3. Results of a 10 L fermenter

[0123] Samples were taken from the fermenter every 2 hours for OD analysis. 600 The process curve for glutathione content detection is shown below. Figure 2As shown, after 32 h of fermentation in a 10 L fermenter, strain ZMGSH01F produced a total glutathione yield of 35.6 g / L.

[0124] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. An engineered Escherichia coli bacterium for high glutathione production, characterized in that, The accession number is CCTCC M20252236, and the name is ZMGSH01F.

2. The engineered Escherichia coli strain with high glutathione production as described in claim 1, characterized in that, The E. coli engineering bacteria ZMGSH01F takes E. coli BL21 (DE3) as a starting strain, knocks out gene cluster ptsH-ptsI-crr and integrates glf-glk a fusion gene at the site, knocks out gene cluster gsiA-gsiB-gsiC and integrates an endogenous glutathione transporter gene at the site cycDC , knocks out a gamma-glutamyltransferase gene ggt , and expresses a recombinant plasmid pRSFDuet- gshAB K177E -E275K-V513A - ppk2 ; The glf-glk The nucleotide sequence of the fusion gene is shown in SEQ ID NO. 5, where positions 1-1419 are... glf The gene, positions 1420-1464 are (Gly4Ser)3 flexible linker peptides, and positions 1465-2451 are... glk Gene, glf Genes and glk Genes are linked via a (Gly4Ser)3 flexible linker peptide; The endogenous glutathione transporter gene cycDC The nucleotide sequence is shown in SEQ ID NO. 6; The recombinant plasmid pRSFDuet- gshAB K177E-E275K-V513A - ppk2 middle, gshAB K177E-E275K-V513A The mutated gene originates from Actinobacillus pleuropneumoniae in porcines. Actinobacillus pleuropneumoniae The nucleotide sequence is shown in SEQ ID NO. 4; The recombinant plasmid pRSFDuet- gshAB K177E-E275K-V513A - ppk2 middle, ppk2 The gene originates from Pseudomonas aeruginosa. Pseudomonas aeruginosa The nucleotide sequence is shown in SEQ ID NO.

3.

3. The engineered Escherichia coli strain with high glutathione production according to claim 2, characterized in that, glucose permease gene glf and glucokinase gene glk Derived from *Mammotrophic motility* Zymomonas mobilis .

4. The engineered Escherichia coli strain with high glutathione production according to claim 2, characterized in that, gshAB K177E -E275K-V513A Based on the glutathione bifunctional synthase gshAB with the amino acid sequence shown in SEQ ID NO. 2, the amino acid at position 177 is mutated from lysine to glutamic acid; the amino acid at position 275 is mutated from glutamic acid to lysine; and the amino acid at position 513 is mutated from valine to alanine.

5. The application of the engineered Escherichia coli strain with high glutathione production according to any one of claims 1 to 4 in the fermentation culture for glutathione production using glucose, monosodium glutamate, cysteine ​​and glycine as substrates.

6. A method for producing glutathione, characterized in that, The seed liquid of the engineered E. coli ZMGSH01F as claimed in claim 1 is obtained, the seed liquid is inoculated into a fermentation medium, the pH in the fermentation process is regulated to be 6.9-7.0, the OD 600 After 10 h of induction at 28℃, the temperature is increased to 30℃, precursors sodium glutamate, cysteine and glycine are added, the fermentation liquid is collected and separated and purified to obtain glutathione.

Images