The coding sequences of niaP, nadV, baPRS, and pnuC were retrieved from public databases and codon-optimized for Escherichia coli. Incompatible restriction sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to ensure compliance with the RFC#10 assembly standard and compatibility with the pET28a(m) vector. The optimized genes were synthesized (Generalbiol, China) and cloned into the pET28a(m) plasmid using NdeI/XhoI restriction sites, under the control of the T7 promoter. The recombinant plasmid was first transformed into E. coli DH5α (D0351, Beyotime) for plasmid amplification and storage, followed by plasmid extraction (Tiangen, China) and transformation into E. coli BL21(DE3) (D1009S, Beyotime) for protein expression. Positive clones were selected on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin, verified by colony PCR, and confirmed by Sanger sequencing (Qingke, Beijing). Verified strains were stored in 25% (v/v) glycerol stocks at –80 °C. Routine cultivation was carried out in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin at 37 °C and 200 rpm. In parallel, an empty-vector control strain (BL21-pET28a) was maintained to assess the contribution of the engineered NMN biosynthetic pathway.
To assess growth activity, frozen glycerol stocks of engineered strains and corresponding control strains were thawed and inoculated (1:100, v/v) into pre-warmed selective medium containing the appropriate antibiotic. Cultures were incubated at 37 °C with shaking at 250 rpm for 3–5 h until reaching an OD600 of approximately 0.4–0.6. The bacterial suspensions were then diluted under sterile conditions to normalize the starting OD600 to 0.05–0.20, and 200 μL aliquots were dispensed into transparent 96-well microplates. Blank controls (medium with antibiotic only) and at least three biological replicates were included for each group. Plates were incubated at the target temperature, and OD600 values were recorded at defined time intervals to generate growth curves.
Frozen glycerol stocks of engineered strains were thawed and inoculated (1:100, v/v) into 5 mL of LB broth supplemented with 100 μg/mL kanamycin in 15 mL culture tubes, and incubated at 37 °C with shaking at 200 rpm for 3–5 h until the culture reached an OD600 of approximately 0.6. Subsequently, 2% (v/v) of the pre-culture was transferred into 30 mL of fresh medium, either LB broth (A507002, Sangon Biotech) or M9 broth (A510881, Sangon Biotech), supplemented with 1% glucose, 1 mM MgSO4 , and 50 μM CaCl2. The cultures were incubated at 37 °C, 200 rpm until reaching an OD600 of 0.2, at which point protein expression was induced with 0.5 mM IPTG. Simultaneously, 0.1% nicotinamide (NAM, A30252, Innochem, China) and 1% glucose (G6500, Innochem, China) were added as precursor and carbon source to promote NMN biosynthesis. Induced cultures were maintained at 37 °C with shaking at 200 rpm and sampled at designated time points for subsequent analysis.
To enable accurate quantification of NMN, a calibration curve was first established using β-NMN standards (A00974, Innochem, China). Serial dilutions of the standard were subjected to the same fluorescence derivatization procedure as experimental samples. Fluorescence intensity was measured using a microplate reader (FlexStation 3, Molecular Devices, USA) with an excitation wavelength of 382 nm and emission wavelength of 445 nm. A concentration–fluorescence response curve was generated, and linear regression was applied to ensure a correlation coefficient of R2 ≥ 0.98 and to define the validated linear detection range.
For sample measurement, 1 mL of fermentation broth was centrifuged at 10,000 g for 5 min at 4 °C to separate the supernatant (extracellular fraction) and cell pellet. The pellet was washed with PBS, resuspended, and disrupted on ice using a sonicator (Shanghai Jingxin, China) at 70 W with 1 s on/3 s off cycles for 20 min. Cell lysates were centrifuged at 10,000 g for 30 min at 4 °C, and the resulting supernatant was collected as the intracellular fraction. For derivatization, 69 μL of sample was sequentially mixed with 27.7 μL of 20% acetophenone in DMSO and 27.7 μL of 2 M KOH, incubated on ice for 2 min, followed by the addition of 125 μL of 88% formic acid and incubation at 37 °C for 10 min. The resulting derivatives were analyzed by fluorescence spectroscopy (excitation 382 nm, emission 445 nm), and NMN concentrations were calculated based on the established standard curve.
Glucose concentration in the culture medium was determined using the GOD-POD method (Yeasen, 60408ES60). At designated time points, 1 mL of fermentation broth was collected and centrifuged at 10,000 g for 5 min to remove cells, and the supernatant was used as the assay sample. Samples were diluted appropriately when necessary and mixed with the colorimetric reagents provided in the kit. Absorbance was measured at 505 nm in a 96-well microplate reader, and glucose concentrations were calculated based on a standard calibration curve. The residual glucose levels were then compared with the initial medium concentration to determine glucose consumption during fermentation.
The coding sequence of gshF (glutathione synthetase fusion) was obtained from GenBank and codon-optimized for Escherichia coli. Incompatible restriction sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to ensure compliance with the RFC#10 standard and compatibility with the multiple cloning site of the pET28a(m) vector. The optimized gene was synthesized commercially (Generalbiol, China) and directionally cloned into the pET28a(m) plasmid via NdeI/XhoI restriction sites under the control of the T7 promoter. The recombinant plasmid was first transformed into E. coli DH5α (D0351, Beyotime) for plasmid amplification and storage. Purified plasmids were subsequently transformed into E. coli BL21(DE3) (D1009S, Beyotime) for protein expression. Positive colonies were screened on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin and incubated at 37 °C. PCR-verified colonies and further confirmed by Sanger sequencing (Qingke, Beijing), yielding the engineered strain E. coli BL21-GshF. Verified recombinant strains were stored in 25% (v/v) glycerol stocks at –80 °C for long-term preservation.
The engineered strain E. coli BL21-GshF was inoculated into LB broth supplemented with 100 μg/mL kanamycin and cultured at 37 °C and 200 rpm until an optical density at 600 nm (OD600) of approximately 0.6 was reached. Protein expression was induced with 0.5 mM IPTG at 30 °C for 4 hours. After induction, the cells were harvested by centrifugation at 8000 g, 4 °C for 10 minutes, and washed twice with pre-chilled PBS (pH 7.4). Precisely 1 g of wet cell pellet was resuspended in PBS at a ratio of 1:5 (w/v), followed by sonication on ice (150 W, 1 s on/3 s off cycles, 20 min) to lyse the cells. The lysate was centrifuged at 12,000 g, 4 °C for 15 min, and the resulting supernatant was collected as the crude enzyme extract.
The enzymatic reaction was performed in a 10 mL PBS system containing 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamic acid, 20 mM L-cysteine, and 20 mM glycine. Reactions were incubated at 37 °C for 1 h, then terminated by the addition of 10% (v/v) trichloroacetic acid (TCA). After centrifugation at 12,000 g for 10 min, the supernatant was collected for analysis. The amount of reduced glutathione (GSH) produced was quantified using a commercial detection kit (Solarbio, China). Samples were incubated with DTNB reagent at 37 °C in the dark for 15 min, and absorbance was measured at 412 nm. GSH concentration was calculated based on a standard calibration curve. One unit of enzyme activity (U) was defined as the amount of enzyme required to catalyze the formation of 1 μmol of GSH per minute under the described conditions.
The engineered strain E. coli BL21-GshF was inoculated into LB broth supplemented with 100 μg/mL kanamycin and cultured at 37 °C and 200 rpm until an optical density at 600 nm (OD600 ) of ~0.6 was reached. The culture was then inoculated at 1% (v/v) into 50 mL LB broth containing 100 μg/mL kanamycin and transferred to an incubator with controlled gas composition. Three oxygen levels were applied: 0% O2 (strict anaerobic conditions), 20% O2 (ambient atmospheric conditions), and 30% O2 (hyperoxic conditions). Cultures were incubated statically at 37 °C for 3 h. At the end of incubation, 1 mL of culture from each group was collected in triplicate, centrifuged at 12,000 g, 4 °C for 10 min, and washed twice with pre-chilled PBS (pH 7.4) to remove residual medium. The washed pellets were resuspended in PBS and lysed by sonication on ice (150 W, 1 s on/3 s off, 20 min). Cell lysates were centrifuged at 12,000 g, 4 °C for 15 min, and the supernatant was collected as the crude enzyme extract. Reduced glutathione (GSH) levels were quantified using a commercial detection kit (DTNB method, Solarbio, China). Extracts were incubated with DTNB at 37 °C in the dark for 15 min, and absorbance was measured at 412 nm. GSH concentrations were calculated from a standard calibration curve, and production levels were compared across different oxygen conditions.
The engineered strain E. coli BL21-GshF was inoculated into LB broth containing 100 μg/mL kanamycin and cultured at 37 °C and 200 rpm until an optical density at 600 nm (OD600) of ~0.6 was reached. The seed culture was inoculated at 1% (v/v) into two groups of 50 mL LB broth with kanamycin, with three biological replicates per group to ensure equal starting cell densities. For the experimental group, the medium was supplemented with 20 mM L-glutamic acid, 20 mM L-cysteine, and 20 mM glycine, sterilized by 0.22 μm filtration. The control group received an equal volume of sterile deionized water. Cultures were incubated at 37 °C, 220 rpm for 3 h. At the end of incubation, 1 mL of culture from each replicate was centrifuged at 12,000 g, 4 °C for 10 min, and cell pellets were washed twice with pre-chilled PBS (pH 7.4). Washed cells were resuspended in PBS and disrupted by sonication on ice. The lysates were centrifuged, and the supernatant was collected as the crude enzyme extract. GSH content was quantified using a reduced glutathione detection kit (DTNB method, Solarbio, China). Extracts were incubated with DTNB at 37 °C in the dark for 15 min, and absorbance was measured at 412 nm. GSH concentrations were determined using a standard calibration curve, and the results were compared between the experimental and control groups.
Frozen glycerol stocks of E. coli BL21-GshF were inoculated (100 μL) into 5 mL LB broth supplemented with 100 μg/mL kanamycin and cultured at 37 °C, 200 rpm for 12 h. The pre-culture was then transferred at a 1:100 inoculation ratio into 100 mL LB broth with kanamycin and grown until reaching an OD600 of ~0.6. Cells were harvested by centrifugation at 8000 g, 4 °C for 10 min, washed twice with pre-chilled PBS (0.1 mol/L, pH 7.4), and resuspended in 10 mL PBS. Cell suspensions were disrupted on ice by sonication (150 W, 3 s on/5 s off cycles, 20 min), followed by centrifugation at 12,000 g, 4 °C for 20 min. The resulting supernatant was filtered through a 0.22 μm membrane to obtain the crude enzyme extract.
For antioxidant activity determination, a 0.1 mmol/L DPPH working solution was prepared in absolute ethanol and stored at 4 °C in the dark. A GSH standard curve was established using serial dilutions (0, 20, 40, 60, 80, 100 μmol/L) in PBS (0.1 mol/L). For the assay, 50 μL of sample supernatant or GSH standard was mixed with 50 μL of DPPH solution in a 96-well microplate and incubated at 25 °C in the dark for 30 min. Absorbance was measured at 517 nm using a microplate reader (FlexStation 3, Molecular Devices, USA). The radical scavenging activity was expressed as DPPH clearance percentage, calculated using the formula:
The coding sequence of the PelB-GLP-1 fusion protein was retrieved from public databases and codon-optimized for Escherichia coli. Incompatible restriction sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to ensure compliance with the RFC#10 standard and compatibility with the pET28a(m) vector. The optimized gene was synthesized commercially (Generalbiol, China) and cloned into the pET28a(m) plasmid using NdeI/XhoI restriction sites, under the control of the T7 promoter for expression. The recombinant plasmid was first transformed into E. coli DH5α (D0351, Beyotime) for plasmid amplification and storage, and subsequently extracted and introduced into E. coli BL21(DE3) (D1009S, Beyotime) for protein expression. Positive clones were selected on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin and incubated at 37 °C. PCR-verified colonies and further confirmed by Sanger sequencing (Qingke, Beijing). Verified recombinant strains were stored in 25% (v/v) glycerol stocks at –80 °C for long-term preservation.
For expression, E. coli BL21(DE3) carrying the PelB-GLP-1 construct was first inoculated into 3 mL LB broth containing 100 μg/mL kanamycin and cultured overnight at 37 °C, 220 rpm. The pre-culture was then expanded into 50 mL LB + kanamycin and incubated at 37 °C until reaching an OD600 of 0.5–0.8, followed by induction with IPTG at a final concentration of 0.2–0.5 mM for 4 h at 37 °C. Cells were harvested by centrifugation at 10,000 g, 4 °C for 10 min, and the supernatant was collected. Protein samples were mixed with 5× reducing sample buffer and denatured at 95–100 °C for 5–10 min, then cooled on ice. Proteins were separated by 15% SDS-PAGE at 120 V and transferred to PVDF membranes under wet transfer conditions (100 V, 75 min, 4 °C). Membranes were blocked with 5% skim milk in TBST at room temperature for 1 h, then incubated overnight at 4 °C with anti-His antibody (mouse, Beyotime AH367, 1:1000). After washing three times with TBST (10 min each), membranes were incubated with HRP-conjugated goat anti-mouse IgG (Beyotime A0216, 1:1000) at room temperature for 1 h, followed by three additional washes. Finally, membranes were incubated with ECL reagents (A: B = 1:1) for 1–2 min and imaged using a chemiluminescence detection system according to the manufacturer’s protocol.
All experiments were carried out with at least three independent repeats to ensure reliability. Data were processed and visualized using GraphPad Prism. Results are shown as mean ± SD, with individual data points displayed whenever possible. Statistical comparisons between groups were performed using one-way ANOVA with Tukey’s test for multiple groups, or Student’s t-test for two-group comparisons. A p-value less than 0.05 was considered statistically significant.
Plasmid Extraction Kit (DP103)
Reduced Glutathione (GSH) Content Detection Kit Instruction Manual
Glucose Content Detection Kit (GOD-POD, Microplate Method)
