Our project set out to explore whether engineered E. coli could serve as a living platform against aging by integrating three functional modules. In
To investigate the effect of various concentrations of the substrate nicotinamide (NAM) on the growth of engineered Escherichia coli BL21 (DE3), E. coli BL21, we measured cell density over time under five NAM conditions. This experiment aimed to determine whether increasing NAM concentrations promotes or inhibits biomass accumulation, providing a reference for optimizing fermentation conditions in NMN production.
To assess the effect of nicotinamide (NAM) on host cell growth, E. coli BL21 was cultured in LB broth supplemented with kanamycin. NAM was added to final concentrations of 0.1%, 1%, and 5% (w/v), with a parallel control group lacking NAM. Cultures were incubated at 37 °C with shaking at 200 rpm. At predetermined time points, samples were collected and cell density was measured at OD600 using a microplate reader (Figure 1). All conditions were performed in triplicate, and the average OD600 values were used to compare growth performance across different NAM concentrations.
Figure 1. Workflow for Bacterial Growth Measurement.
As shown in Figure 2, supplementation with 0.1% NAM had no significant effect on BL21 growth compared with the control. At 1% NAM, growth was still observed but reached a slightly lower density. In contrast, 5% NAM supplementation markedly inhibited bacterial proliferation, with cultures showing a much slower increase in OD600. These results demonstrate that E. coli can tolerate low to moderate NAM concentrations, while high concentrations strongly suppress cell growth, likely due to osmotic stress and interference with NAD+ metabolism.
Figure 2. Effect of Different NAM Concentrations on the Growth of Engineered E. coli BL21.
These results confirmed that while low NAM concentrations (≤1%) did not strongly affect growth, high NAM supplementation (5%) markedly inhibited proliferation, most likely due to osmotic stress and interference with NAD+ metabolism. This highlighted the need to carefully balance precursor supply in subsequent experiments, guiding us to test whether NMN biosynthesis could be enabled by transporter and enzyme introduction.
To establish a reliable standard curve for NMN quantification, enabling the conversion of fluorescence signals into NMN concentrations for accurate determination of intra- and extracellular NMN levels.
As shown in Figure 3, β-NMN standards of known purity were prepared in a series of gradient concentrations (e.g., 0, 1, 2, 5, 10, 20 μM). Each concentration was subjected to fluorescence derivatization following the experimental protocol, and fluorescence intensity was measured using a microplate reader with excitation at 382 nm and emission at 445 nm. Each point was tested in triplicate, and the average intensity was plotted against the standard concentrations. A linear regression analysis was performed to generate the calibration curve, recording the slope, intercept, and coefficient of determination (R2).
Figure 3. Workflow for NMN Detection.
As shown in Figure 4, fluorescence intensity exhibited a strong linear correlation with NMN concentration across the tested range. The regression equation was Y = 1.154·X -25.58, and the calibration curve showed excellent linearity (R2 > 0.98), confirming that the method can reliably convert fluorescence signals into NMN concentrations.
Figure 4. The standard curve of NMN.
A highly linear NMN standard curve was successfully established, enabling accurate quantification of NMN in both intracellular and extracellular fractions. This calibration ensures the precision and reliability of subsequent NMN measurement experiments.
Previous studies have demonstrated that NadV is a key determinant for NMN biosynthesis in E. coli [1,2].To establish a baseline NMN biosynthesis system, we introduced two key components into E. coli BL21: the nicotinamide transporter NiaP, which facilitates efficient uptake of extracellular nicotinamide (NAM), and the nicotinamide phosphoribosyltransferase NadV, which catalyzes the conversion of NAM into NMN. By combining substrate uptake with catalytic conversion, this “Version 1.0” design aimed to determine whether E. coli could be endowed with the capacity to synthesize NMN, thereby validating the feasibility of constructing a microbial NMN biosynthetic pathway.
The engineered strains were constructed using standard molecular cloning procedures (Figure 5). The coding sequences of NiaP and NadV were obtained from public databases and codon-optimized for expression in E. coli. Restriction enzyme recognition sites incompatible with RFC#10 assembly (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed during sequence design to ensure cloning compatibility. The optimized fragments were synthesized commercially and sequentially assembled into the pET28a backbone under the control of the T7 promoter, ribosome binding site B0034, and terminator B0015. Recombinant plasmids were initially transformed into E. coli DH5α for amplification. Positive colonies were screened on LB agar with 100 μg/mL kanamycin and verified by colony PCR and Sanger sequencing (Figure 6). The validated constructs were then introduced into E. coli BL21, generating BL21-NiaP and BL21-NiaP-NadV strains, while an empty-vector control (BL21-pET28a, namely BL21) was maintained for comparison.
Figure 5. Workflow for Engineering Strain Construction.
For the NAM precursor assay, BL21-NiaP-NadV and control strains were cultured in LB broth supplemented with kanamycin. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG. NAM was added at different concentrations (0.05%, 0.1%, 0.5%, and 1%) together with 1% glucose. Cultures were incubated at 37 °C with shaking for 8 h. After harvesting and lysis, NMN levels were quantified via acetophenone-based fluorescence derivatization (Ex 382 nm / Em 445 nm), with concentrations determined using the previously established standard curve.
Figure 6. Construction and validation of engineered strain BL21-NiaP-NadV.
As shown in Figure 7, the wild-type BL21 strain produced no detectable NMN across all measured time points, confirming that E. coli lacks an inherent NMN biosynthetic capability. In contrast, the engineered strain BL21-NiaP-NadV exhibited a clear and time-dependent increase in NMN accumulation following IPTG induction. NMN levels began to rise steadily at 2 h post-induction, reached a marked increase at 4 h, and peaked at approximately 6 h before plateauing. This demonstrates that while NiaP alone was insufficient, the combined expression of NiaP and NadV endowed E. coli with the capacity to synthesize measurable levels of NMN.
Figure 7. NMN production in BL21-NiaP-NadV.
The results confirmed that E. coli BL21 alone cannot synthesize NMN, but equipping the strain with NiaP and NadV successfully enabled intracellular NMN production. While NiaP facilitated NAM uptake, NadV proved essential for catalytic conversion. This established a functional baseline (Version 1.0) and provided the foundation to investigate whether precursor limitation could be alleviated to further boost NMN yield.
To investigate how different concentrations of nicotinamide (NAM) influence NMN production in the engineered E. coli BL21-NiaP-NadV strain.
BL21-NiaP-NadV was cultured in LB broth supplemented with kanamycin. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG. To test precursor dependency, NAM was added at various concentrations (e.g., 0.05%, 0.1%, 0.5%, and 1%), together with 1% glucose as a carbon source. Cultures were incubated at 37 °C with shaking for 8 h. Samples were collected, lysed, and subjected to fluorescence-based derivatization. NMN levels were calculated according to the previously established standard curve.
As shown in Figure 8, NMN production was strongly dependent on NAM concentration. At low supplementation (0.05%), NMN levels remained limited, only slightly above background. Increasing NAM to 0.1% led to a marked improvement, with NMN concentrations rising significantly compared to the low-dose group. Further increase to 0.5% NAM resulted in the highest NMN yield, indicating that sufficient precursor supply enhanced biosynthesis efficiency. However, raising NAM to 1% did not further boost NMN accumulation and instead showed a plateau or slight decline, suggesting that excessive NAM may impose metabolic stress or feedback inhibition on the pathway.
Figure 8. Effect of NAM concentration on NMN production in BL21-NiaP-NadV.
We observed that NMN production was strongly dependent on NAM concentration, with 0.1% NAM supporting the highest yield. Excessive NAM not only failed to increase production but also suppressed biosynthesis, consistent with its inhibitory effect on growth. These findings indicated that precursor availability must be finely tuned, prompting us to explore whether pathway engineering could overcome PRPP limitation.
Building on our previous findings—that high concentrations of NAM inhibit E. coli growth, NMN can be reliably quantified by fluorescence-based derivatization, NadV is the key determinant for NMN biosynthesis, and excessive NAM reduces production efficiency—we next aimed to address the limitation of precursor supply in the pathway. Since phosphoribosyl pyrophosphate (PRPP) availability is a major bottleneck, we introduced Bacillus mycoides phosphoribosylpyrophosphate synthetase (BaPRS) into BL21-NiaP-NadV to evaluate whether enhanced PRPP generation could increase NMN yield and improve pathway efficiency [3].
The coding sequences of NiaP, NadV, and BaPRS were obtained from public databases and codon-optimized for expression in Escherichia coli. During sequence design, incompatible restriction enzyme recognition sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to ensure compliance with the RFC#10 assembly standard and compatibility with the multiple cloning site of the pET28a vector. The optimized fragments were synthesized commercially and sequentially cloned into the pET28a plasmid under the control of the T7 promoter, with ribosome binding site B0034 and terminator B0015. Correct integration of BaPRS was verified by PCR amplification (expected 951 bp band) and Sanger sequencing (Figure 9). The validated plasmid was then transformed into E. coli BL21 to generate the engineered strain BL21-NiaP-NadV-BaPRS for subsequent functional analysis.
Figure 9. Construction and validation of BL21-NiaP-NadV-BaPRS.
The engineered strain BL21-NiaP-NadV-BaPRS was cultured in LB broth supplemented with kanamycin. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG, with 0.1% NAM and glucose provided as substrates. Cultures were incubated at 37 °C with shaking at 200 rpm. Samples were collected at different time points post-induction (0 h, 2 h, 4 h, 6 h, 8 h). Cells were harvested, lysed, and analyzed by fluorescence-based derivatization. NMN concentrations were quantified using the previously established standard curve.
As shown in Figure 10, the BL21-NiaP-NadV-BaPRS strain produced markedly higher NMN levels compared with the baseline BL21-NiaP-NadV strain. NMN accumulation began to rise at 2 h, showing a steady upward trend through the 4 h and 6 h time points. By 8 h, the engineered strain reached its maximum NMN concentration, which was significantly greater than that of Version 1.0 without BaPRS. This result indicates that BaPRS effectively boosted NMN biosynthesis by alleviating precursor limitation from PRPP.
Figure 10. NMN production in BL21-NiaP-NadV-BaPRS over time.
The introduction of BaPRS significantly enhanced NMN accumulation, confirming that phosphoribosyl pyrophosphate (PRPP) availability was a major bottleneck in the initial pathway. By boosting intracellular PRPP levels, BaPRS increased the catalytic efficiency of NadV. This Version 2.0 design thus demonstrated the value of reinforcing precursor supply, paving the way to test whether transport engineering could further improve NMN accumulation.
To evaluate whether the introduction of NMN biosynthetic modules affects host cell viability, we compared the growth performance of control BL21 and engineered strains harboring different pathway constructs (Version 1.0: NiaP-NadV; Version 2.0: NiaP-NadV-BaPRS). This experiment aimed to confirm that NMN production does not severely compromise bacterial growth under induction conditions.
Control BL21, BL21-NiaP-NadV, and BL21-NiaP-NadV-BaPRS strains were cultured in LB broth supplemented with kanamycin, 0.1% NAM, and 1% glucose. When cultures reached OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG. Cell density was subsequently measured at OD600 at defined time intervals using a microplate reader throughout cultivation. Growth curves were then plotted to compare proliferation dynamics across strains under identical conditions.
As shown in Figure 11, all three strains—control BL21, BL21-NiaP-NadV, and BL21-NiaP-NadV-BaPRS—entered exponential growth after IPTG induction and reached comparable stationary phases within 10–12 h. The control BL21 exhibited the fastest early growth, while BL21-NiaP-NadV showed a slightly delayed lag phase, likely due to the metabolic burden of expressing additional genes. Notably, the BaPRS-containing strain (BL21-NiaP-NadV-BaPRS) maintained a growth trajectory similar to the control, suggesting that the reinforcement of PRPP supply did not impose additional metabolic stress. These results indicate that NMN pathway engineering, including the introduction of BaPRS, preserved host viability and supported stable proliferation under NAM- and glucose-supplemented conditions.
Figure 11. Growth curves of control and NMN-engineered BL21 strains.
The results confirm that engineered strains carrying NMN biosynthetic modules remain viable and capable of sustained growth under induction conditions. Although a slight reduction in growth was observed, particularly in the BaPRS-enhanced strain, the overall impact on cellular activity was minimal. This demonstrates that NMN pathway engineering is compatible with normal host physiology, ensuring strain stability for further optimization.
To investigate the impact of medium composition on NMN biosynthesis, we compared NMN production in engineered E. coli strains cultured in rich medium (LB) versus defined medium (M9). Since medium components may influence precursor supply and enzyme activity, this experiment aimed to identify culture conditions that maximize NMN yield.
Engineered strains BL21-NiaP-NadV and BL21-NiaP-NadV-BaPRS were cultured in LB broth or M9 minimal medium (Sangon Biotech, A510881). Both media were supplemented with 1% glucose and 0.1% NAM. For M9, additional supplements included 1 mM MgSO4 and 50 μM CaCl2. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG, and cultures were incubated for 12 h at 37 °C with shaking. After induction, samples were harvested, and intracellular NMN levels were quantified using fluorescence-based derivatization and the previously established standard curve.
As shown in Figure 12, strains cultured in M9 medium accumulated substantially higher NMN compared to those grown in LB medium under identical conditions. Both BL21-NiaP-NadV and BL21-NiaP-NadV-BaPRS exhibited this trend, with the BaPRS-enhanced strain in M9 reaching the highest NMN yield after 12 h induction. The data indicate that minimal medium with appropriate supplementation provided a more favorable environment for NMN biosynthesis, while LB supported lower levels of accumulation.
Figure 12. Effect of culture medium (LB vs. M9) on NMN production in engineered strains.
These results demonstrate that medium composition strongly influences NMN production efficiency. The defined M9 medium enhanced NMN accumulation compared with LB, particularly in the BaPRS-containing strain. This suggests that precursor balance and cofactors in the medium may play critical roles in supporting NMN biosynthesis. Potential explanations include increased phosphate availability in M9 to sustain PRPP-dependent reactions, stabilization of membrane transport and secretion by Ca2+, and Mg2+ serving as a cofactor for BaPRS or NadV.
Building upon Version 2.0, which alleviated precursor limitations by introducing BaPRS, we next sought to address the challenge of NMN export. While intracellular accumulation can be enhanced by boosting precursor supply, the efficiency of NMN production is ultimately constrained if export into the culture medium is limited. To overcome this, we introduced the NMN transporter PnuC, which has been reported to mediate nucleoside mononucleotide export [4].
The coding sequence of the NMN transporter PnuC was obtained from public databases, codon-optimized for expression in Escherichia coli, and designed to remove restriction enzyme sites incompatible with RFC#10 assembly. The optimized fragment was synthesized commercially and sequentially cloned into the previously constructed pET28a backbone containing NiaP, NadV, and BaPRS, under the control of the T7 promoter with ribosome binding site B0034 and terminator B0015. Recombinant plasmids were first amplified in E. coli DH5α, verified by colony PCR and Sanger sequencing, and subsequently transformed into E. coli BL21 to generate the final engineered strain BL21-NiaP-NadV-BaPRS-PnuC (Figure 13). Verified strains were preserved in 25% glycerol at –80 °C for long-term storage.
Figure 13. Construction and validation of BL21-NiaP-NadV-BaPRS-PnuC.
Engineered strains BL21-NiaP-NadV-BaPRS (Version 2.0) and BL21-NiaP-NadV-BaPRS-PnuC (Version 3.0) were cultured in LB broth supplemented with kanamycin, 0.1% NAM, and 1% glucose. At OD600 ≈ 0.2, IPTG was added to a final concentration of 0.5 mM to induce expression. Cultures were incubated at 37 °C with shaking for 12 h. After induction, both intracellular and extracellular fractions were collected separately by centrifugation. NMN concentrations were quantified using fluorescence-based derivatization and the established standard curve.
As shown in Figure 14, the BL21-NiaP-NadV-BaPRS-PnuC strain exhibited a significant increase in NMN accumulation compared with both the Version 1.0 (NiaP-NadV) and Version 2.0 (NiaP-NadV-BaPRS) strains. While earlier constructs produced detectable NMN, the addition of PnuC resulted in notably higher levels, indicating that export efficiency was a critical factor in maximizing overall NMN yield.
Figure 14.Effect of PnuC introduction on NMN accumulation in engineered strains.
Introducing PnuC markedly increased extracellular NMN accumulation, highlighting the importance of transporter engineering for metabolite secretion. By coupling NiaP-mediated import, NadV- and BaPRS-mediated synthesis, and PnuC-mediated export, we successfully constructed an efficient three-step system (Version 3.0). This completed the core NMN production module and established a strong basis for linking NMN biosynthesis with host physiology in subsequent studies.
To investigate the relationship between NMN biosynthesis, glucose consumption, and bacterial activity, we monitored the growth performance of the Version 3.0 strain BL21-NiaP-NadV-BaPRS-PnuC alongside measurements of NMN accumulation and glucose utilization. This experiment aimed to assess whether NMN production imposes a metabolic burden on the host and how carbon source availability influences yield.
The engineered strain BL21-NiaP-NadV-BaPRS-PnuC was cultivated in LB broth containing kanamycin, 0.1% NAM, and 1% glucose. At mid-log phase (OD600 ≈ 0.2), protein expression was induced with 0.5 mM IPTG, and cultures were incubated at 37 °C with shaking for 12 h. At defined intervals, aliquots were collected and subjected to three parallel analyses: (1) OD600 measurement to monitor cell growth, (2) quantification of residual glucose concentration using the GOD-POD enzymatic assay, and (3) determination of NMN accumulation via fluorescence-based derivatization. The overall experimental workflow is illustrated in Figure 15.
Figure 15. Workflow for Glucose Detection.
As shown in Figure 16, glucose consumption was closely correlated with both cell growth and NMN accumulation in BL21-NiaP-NadV-BaPRS-PnuC. During the exponential growth phase, residual glucose levels decreased sharply, while NMN concentration increased correspondingly, indicating that active metabolism supported efficient biosynthesis. By the stationary phase, glucose was largely depleted, cell growth plateaued, and NMN production reached its maximum level. This strong linkage highlights the dependence of NMN biosynthesis on glucose utilization.
Figure 16. Growth, glucose consumption, and NMN accumulation of BL21-NiaP-NadV-BaPRS-PnuC.
These results demonstrate that glucose availability directly influences NMN production efficiency, with rapid glucose consumption during exponential growth driving high NMN yields. Once glucose became limiting, both growth and NMN synthesis declined, suggesting that carbon source supplementation or controlled feeding strategies could further optimize production. This finding provides a metabolic basis for future bioprocess design aimed at scaling NMN biosynthesis.
To identify the optimal temperature for NMN production in the engineered strain BL21-NiaP-NadV-BaPRS-PnuC, we compared product yields at different cultivation temperatures. Although E. coli typically grows best at 37 °C, lower induction temperatures are sometimes used to improve the expression of heterologous proteins. This experiment aimed to determine whether temperature modulation could enhance NMN biosynthesis.
BL21-NiaP-NadV-BaPRS-PnuC was cultured in LB medium supplemented with kanamycin (100 μg/mL), 1% glucose, and 0.1% NAM. When cultures reached OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG. To specifically evaluate the effect of temperature, all culture conditions (medium composition, inducer concentration, shaking speed, and induction time) were kept constant, while incubation was carried out at different temperatures (25 °C, 30 °C, and 37 °C) with shaking at 200 rpm. After 12 h of cultivation, cells were harvested, lysed, and NMN concentrations were quantified using fluorescence-based derivatization and the established standard curve.
As shown in Figure 17, NMN production was strongly influenced by cultivation temperature. At 25 °C, NMN accumulation increased slowly and plateaued at a relatively low level by 12 h, indicating suboptimal enzyme activity under cooler conditions. In contrast, cultures grown at 30 °C and 37 °C exhibited significantly higher NMN yields, with both conditions showing a steady upward trend throughout the cultivation period. This result suggests that while the engineered strain can produce NMN across a broad temperature range, moderate-temperature induction at 30 °C provides the most favorable balance between cell growth and protein expression, thereby representing the optimal condition for large-scale production.
Figure 17. Effect of cultivation temperature on NMN production in BL21-NiaP-NadV-BaPRS-PnuC.
Temperature optimization experiments confirmed that 37 °C remains the most suitable condition for NMN production in BL21-NiaP-NadV-BaPRS-PnuC. Unlike some recombinant protein expression systems where low-temperature induction enhances yield, NMN biosynthesis in this pathway is favored at the standard physiological growth temperature of E. coli.
Building on our success in establishing a functional NMN biosynthetic pathway, we next sought to enhance the cellular antioxidant capacity as a complementary anti-aging strategy. Glutathione (GSH), a tripeptide composed of glutamate, cysteine, and glycine, is one of the most important intracellular antioxidants, playing a key role in redox homeostasis and defense against oxidative stress. While GSH biosynthesis in most organisms requires two sequential enzymes—γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS)—the bifunctional enzyme GshF combines both activities within a single polypeptide, enabling one-step GSH synthesis [5]. In this study, we aimed to evaluate whether overexpression of GshF in E. coli BL21 could significantly increase intracellular GSH accumulation compared with the control strain.
The coding sequence of GshF, a bifunctional enzyme involved in glutathione biosynthesis, was obtained from public databases, codon-optimized for expression in Escherichia coli, and redesigned to remove restriction sites incompatible with RFC#10 assembly (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI). The optimized fragment was synthesized commercially and cloned into the pET28a vector under the T7 promoter with ribosome binding site B0034 and terminator B0015. The recombinant plasmid (pET28a-GshF) was first transformed into E. coli DH5α for plasmid amplification, with positive clones verified by kanamycin selection, colony PCR (expected 2,253 bp band), and Sanger sequencing (Figure 18). Verified plasmids were subsequently introduced into E. coli BL21 to generate the engineered strain BL21-GshF, which was preserved in 25% (v/v) glycerol at –80 °C for long-term storage.
Figure 18. Construction and validation of BL21-GshF.
For GSH production assays, BL21-GshF and control BL21 were cultured in LB broth containing kanamycin. At OD600 ≈ 0.2, protein expression was induced with 0.5 mM IPTG, and cultures were incubated at 30 °C with shaking at 200 rpm. After 8 h, cells were harvested, lysed, and intracellular GSH levels were quantified using the DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) colorimetric method (Figure 19).
Figure 19. Workflow for GSH Detection.
As shown in Figure 20, BL21-GshF exhibited significantly elevated intracellular GSH levels compared with the control BL21 strain. Following IPTG induction, engineered cells displayed a marked increase in GSH accumulation within 8 h, whereas the control strain maintained only basal levels. These findings demonstrate that overexpression of GshF successfully enhanced glutathione biosynthesis in E. coli.
Figure 20. Intracellular GSH levels in control BL21 and BL21-GshF strains.
Overexpressing GshF in BL21 enabled one-step GSH biosynthesis and significantly increased intracellular GSH levels compared with the control strain. This confirmed the feasibility of using a bifunctional enzyme to simplify the pathway. Building on this foundation, we next examined whether GshF overexpression affected cell growth and metabolic balance.
To evaluate whether overexpression of the fusion enzyme GshF, in addition to enhancing glutathione (GSH) biosynthesis, exerts any impact on host cell growth, we compared the growth curves of BL21-GshF with the wild-type BL21. Since elevated antioxidant capacity could alleviate intracellular oxidative stress, it was hypothesized that GshF expression might promote bacterial proliferation.
BL21-GshF and wild-type BL21 were cultured in LB broth supplemented with kanamycin and induced with 0.5 mM IPTG at OD600 ≈ 0.2. Growth was monitored by measuring OD600 at defined time intervals over a 12 h cultivation period using a microplate reader. Growth curves were plotted to assess differences in proliferation between the engineered and control strains.
As shown in Figure 21, both strains exhibited typical bacterial growth curves with lag, exponential, and stationary phases. However, BL21-GshF consistently displayed higher OD600 values compared to the wild-type, particularly during the exponential phase. After 12 h, the engineered strain reached an OD600 of approximately 1.45, whereas the control strain reached only about 1.05, indicating a growth advantage of ~38%. The enhanced proliferation suggests that elevated GSH levels provided by GshF expression may mitigate oxidative stress and improve cellular fitness under cultivation conditions.
Figure 21. Growth curves of wild-type BL21 and BL21-GshF.
The growth curve analysis showed that GshF-overexpressing strains exhibited no significant growth defects compared with the control, demonstrating that enhanced GSH production did not compromise cell viability. This result indicated that the engineered strains could sustain both growth and production, encouraging us to proceed with characterizing enzyme activity.
To confirm that the overexpressed GshF fusion enzyme retained its catalytic activity in E. coli, we directly assayed the intracellular enzymatic function by quantifying glutathione (GSH) production. The aim was to validate that the observed increase in GSH levels was indeed attributable to GshF activity rather than background metabolic processes.
BL21-GshF and wild-type BL21 strains were cultured in LB broth and induced with 0.5 mM IPTG at OD600 ≈ 0.2. After 12 h of cultivation, cells were harvested and lysed by sonication. Enzyme activity was determined indirectly by measuring intracellular GSH content using the DTNB assay, where reduced glutathione reacts with DTNB to generate a yellow-colored product measurable at 412 nm. GSH concentration was quantified against a standard curve, and enzymatic activity was expressed in terms of μmol GSH produced per mg total protein per min.
As shown in Figure 22, BL21-GshF exhibited significantly higher GSH levels compared with the wild-type control. The engineered strain achieved an enzymatic activity of approximately 3.2-fold greater than the baseline, confirming that the recombinant GshF fusion enzyme was both correctly expressed and functionally active in E. coli. This elevated activity directly correlates with the enhanced intracellular GSH accumulation observed in previous experiments.
Figure 22. Enzymatic activity assay confirming functional GshF expression in E. coli.
The activity assay confirmed that the recombinant GshF enzyme retained strong catalytic activity, directly contributing to elevated GSH biosynthesis. This provided a mechanistic basis for the increased GSH observed in vivo. With enzymatic activity validated, we next explored how precursor amino acid supplementation influenced production efficiency.
Glutathione (GSH) biosynthesis requires three precursor amino acids: glutamate, cysteine, and glycine. To determine whether external supplementation of these substrates could enhance GSH production in E. coli BL21-GshF, we evaluated intracellular GSH levels under different amino acid supplementation conditions.
BL21-GshF was cultured in LB broth supplemented with kanamycin and induced with 0.5 mM IPTG at OD600 ≈ 0.2. Experimental groups were provided with additional precursor amino acids (glutamate, cysteine, glycine, or a combination of all three), while the control group was cultured without supplementation. After 12 h of cultivation, cells were harvested, lysed, and the GSH concentration was quantified using the DTNB assay.
As shown in Figure 23, amino acid supplementation enhanced GSH accumulation compared with the unsupplemented control. Among the single-supplement groups, cysteine exerted the most significant effect, followed by glutamate and glycine. Notably, supplementation with all three precursors simultaneously resulted in the highest GSH yield, approximately 2.8-fold greater than the control. This indicates that precursor availability directly influences metabolic flux toward GSH biosynthesis.
Figure 23. Effect of precursor amino acid supplementation on GSH production in BL21-GshF.
Supplementation with cysteine significantly enhanced GSH production, while providing all three precursors—glutamate, cysteine, and glycine—further maximized yield. This result is consistent with previous reports that optimizing precursor availability can markedly improve glutathione production in E. coli [6]. Together, these findings underscore the necessity of a balanced precursor supply for efficient GSH biosynthesis. Building on this insight, we next examined how environmental factors, particularly oxygen availability, influence production.
Glutathione (GSH) plays a central role in mitigating oxidative stress, and its biosynthesis is closely linked to the redox environment of the cell. Oxygen availability influences both intracellular oxidative pressure and enzyme activity. To assess the relationship between oxygen levels and GSH production, we cultured BL21-GshF under different aeration conditions, aiming to determine whether oxidative stress could serve as a driving force for enhanced GSH biosynthesis.
BL21-GshF was cultured in LB broth supplemented with kanamycin and induced with 0.5 mM IPTG at OD600 ≈ 0.2. Cultures were divided into three groups: anaerobic, aerobic (normal shaking at 200 rpm), and high-aeration (vigorous shaking at 250 rpm with increased flask-to-medium ratio). After 12 h of cultivation, cells were harvested, lysed, and intracellular GSH levels were quantified using the DTNB assay.
As shown in Figure 24, oxygen levels significantly influenced GSH production. Under anaerobic conditions, GSH accumulation was minimal, reflecting the absence of oxidative stress stimuli and reduced metabolic demand for antioxidant protection. Under normal aerobic conditions, GSH production increased substantially, consistent with the role of GSH in counteracting reactive oxygen species generated during respiration. Strikingly, the high-aeration group produced the highest GSH yield, approximately 3.5-fold higher than anaerobic cultures. This result suggests that increased oxidative stress triggered by elevated oxygen availability stimulated GshF-mediated glutathione synthesis.
Figure 24. Effect of oxygen availability on GSH production in BL21-GshF.
Higher oxygen availability significantly increased GSH accumulation, reflecting the close link between redox conditions and glutathione metabolism. These results emphasized that both genetic and environmental factors jointly shape GSH biosynthesis. To assess the functional relevance of enhanced GSH production, we next evaluated the antioxidant capacity of the engineered strains.
While intracellular measurements confirmed that GshF overexpression increased glutathione (GSH) production, it was also important to validate whether this enhancement translated into improved antioxidant capacity. To address this, we assessed the free radical scavenging ability of BL21-GshF compared with the wild-type strain using the DPPH assay, a standard method for evaluating antioxidant activity.
BL21-GshF and wild-type BL21 were cultured in LB medium with kanamycin, induced with 0.5 mM IPTG, and harvested after 12 h of growth. Cells were lysed, and crude extracts were prepared. Antioxidant capacity was measured using the DPPH free radical scavenging assay, in which extracts were incubated with DPPH solution, and the decrease in absorbance at 517 nm was recorded. Radical scavenging activity was calculated relative to the control reaction without cell extract.
As shown in Figure 25, BL21-GshF exhibited significantly stronger radical scavenging activity than wild-type BL21. The engineered strain achieved approximately 2.6-fold higher DPPH scavenging efficiency, reflecting its elevated GSH content. This result confirmed that the increased glutathione pool directly improved the antioxidant potential of the engineered cells.
Figure 25. Antioxidant capacity of BL21-GshF measured by DPPH radical scavenging assay.
The DPPH assay demonstrated that GshF overexpression not only boosted intracellular glutathione production but also translated into enhanced antioxidant functionality. This validates the biological significance of the GSH system, positioning engineered E. coli as a potential chassis for applications in oxidative stress resistance and anti-aging biotechnology.
Building on the successful establishment of NMN and GSH biosynthesis modules, we next sought to construct a GLP-1 biosynthesis module to broaden the functional scope of our engineered system. Glucagon-like peptide-1 (GLP-1) is a key regulatory peptide with important roles in glucose metabolism and anti-aging pathways. To achieve microbial production, we engineered E. coli BL21 to express GLP-1 fused with the PelB signal peptide, enabling extracellular secretion. This experiment aimed to validate the feasibility of GLP-1 expression and secretion in bacteria, thereby laying the foundation for future functional and therapeutic applications.
To investigate the feasibility of GLP-1 secretion, the GLP-1 coding sequence was fused with the PelB signal peptide, following a design strategy inspired by the iGEM24_Squirrel-CHN team. The fusion gene was codon-optimized for expression in Escherichia coli, with incompatible restriction sites removed to ensure RFC#10 assembly compatibility, and subsequently synthesized commercially. The optimized fragment was cloned into the pET28a backbone under the control of a T7 promoter, ribosome binding site B0034, and terminator B0015, forming a complete expression cassette. The recombinant plasmid was propagated in E. coli DH5α and validated by PCR amplification and agarose gel electrophoresis, which produced a distinct 162 bp band; sequencing further confirmed construct integrity. The verified plasmid was then transformed into E. coli BL21 to generate the engineered strain BL21-PelB-GLP-1 (Figure 26).
Figure 26. Construction and validation of the PelB-GLP-1 expression cassette.
To confirm the expression of GLP-1 in the engineered strain, BL21-PelB-GLP-1 cultures were induced with IPTG, and protein samples were collected at the designated time points. Cells were harvested by centrifugation, lysed, and the soluble protein fraction was separated by SDS-PAGE. Proteins were subsequently transferred onto a PVDF membrane, probed with an anti-GLP-1 primary antibody, and detected using an HRP-conjugated secondary antibody. Signal visualization was performed with enhanced chemiluminescence (ECL), following the workflow illustrated in Figure 27.
Figure 27. Workflow for Western Blot Analysis.
As shown in Figure 28, a distinct immunoreactive band of under 15 kDa was detected in protein extracts from BL21-PelB-GLP-1 after IPTG induction. This band size corresponds to the expected molecular weight of the PelB-GLP-1 fusion protein, confirming its successful expression in the engineered strain. Together, these results validate that the PelB signal peptide enabled detectable GLP-1 expression at the protein level.
Figure 28. Western Blot validation of PelB-GLP-1 expression.
The GLP-1 secretion module was successfully established through the construction and validation of the PelB-GLP-1 expression cassette. PCR and sequencing confirmed the correct integration of the fusion gene, while Western blot analysis revealed a distinct immunoreactive band at the expected molecular weight (under 15 kDa), verifying that the engineered strain BL21-PelB-GLP-1 expressed the target protein. The presence of the PelB signal peptide indicates that the system holds the potential for extracellular secretion, providing a practical route for GLP-1 delivery.
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