Through iterative
Abstract Figure. Overview of DBTL Cycles for NMN, GSH, and GLP-1 Modules.
The NMN module illustrates how iterative engineering converted a minimal pathway into a scalable production system. Successive introduction of precursor-boosting and transporter elements enabled high-yield extracellular secretion, and final optimization confirmed its robustness in E. coli [1–3].
Nicotinamide mononucleotide (NMN) is an essential precursor in NAD+ metabolism, and our first goal was to enable its production in E. coli. To achieve this, we designed a minimal module consisting of
Figure 1. Design of the first-generation NMN module (Cycle 1.1: NiaP + NadV) for intracellular synthesis in E. coli.
The genes
Figure 2. Construction and validation of engineered strain BL21-NiaP-NadV (Cycle 1.1, Version 1.0).
To comprehensively evaluate the performance of the first-generation NMN biosynthesis module, we first examined the effect of nicotinamide (NAM) supplementation on the growth of E. coli BL21, ensuring that substrate addition did not adversely affect cell viability (Figure 3A). We then established a fluorescence-based derivatization assay and generated a robust NMN standard curve with excellent linearity (R2 > 0.98), which enabled accurate quantification (Figure 3B). Using this method, intracellular NMN levels were compared across three groups: wild-type BL21, BL21 expressing only NiaP, and BL21 co-expressing NiaP and NadV. The co-expression strain demonstrated a significant increase in NMN accumulation, confirming successful establishment of a minimal biosynthetic pathway (Figure 3C). Finally, by testing different NAM concentrations, we identified an optimal supplementation level that maximized NMN production while maintaining stable growth, providing a practical guideline for pathway utilization (Figure 3D). Collectively, these results validated the functionality of the NiaP + NadV construct, established reliable measurement tools, and laid a solid baseline for subsequent cycle optimization.
Figure 3. Validation of the first-generation NMN biosynthesis module (Cycle 1.1: NiaP + NadV). (A) NAM effect on cell growth. (B) Standard curve for NMN quantification. (C) Intracellular NMN levels in control vs engineered strains. (D) Optimal NAM concentration for NMN production.
The first-generation NMN module demonstrated that E. coli could produce NMN, giving us a baseline for measurement and confirming pathway feasibility. Yet yields plateaued even with additional NAM, suggesting that the limitation might lie elsewhere. Based on both our results and previous reports, we speculated that insufficient PRPP supply could be restricting NadV activity[4]. This learning inspired our next step: introducing BaPRS as a possible way to boost precursor availability and potentially improve NMN production.
Building on the insights from Cycle 1.1, we sought to address the production plateau caused by limited precursor supply. Literature and our own data suggested that insufficient PRPP was constraining the conversion of NAM to NMN by NadV. To overcome this, we designed the second-generation module by introducing
Figure 4. Design of the second-generation NMN biosynthesis module (Cycle 1.2: +BaPRS).
To construct the second-generation NMN module, we combined NiaP (BBa_25KTH04H) with the composite part NadV-BaPRS (BBa_25SCJT4H), which encodes NadV and BaPRS in tandem to enable both nicotinamide conversion and PRPP regeneration. All sequences were codon-optimized for E. coli and designed to comply with RFC#10 standards. The operon was assembled into the pET28a(m) backbone under the T7 promoter, and correct construction was confirmed by PCR and sequencing. Successful validation of the plasmid carrying the second-generation module is shown in Figure 5.
Figure 5. Verification of the second-generation NMN biosynthesis module (Cycle 1.2: +BaPRS).
The second-generation NMN module was systematically evaluated under multiple conditions. Time-course analysis revealed that Version 2.0 (NiaP + NadV-BaPRS) accumulated NMN much faster and to significantly higher levels than Version 1.0, confirming that BaPRS effectively boosted precursor supply (Figure 6A). Growth monitoring showed comparable OD600 trends among BL21, V1.0, and V2.0 strains, indicating that the additional metabolic burden did not compromise cell viability (Figure 6B). Furthermore, culture optimization revealed that M9 medium supported markedly higher NMN yields than LB, with Version 2.0 in M9 reaching the highest titers observed in this cycle (Figure 6C). Together, these results validated the benefit of BaPRS introduction, highlighted the importance of culture conditions, and provided strong evidence that precursor regeneration substantially improved pathway efficiency.
Figure 6. Validation of the second-generation NMN biosynthesis module (Cycle 1.2: NiaP + NadV-BaPRS). (A) Time-course analysis showing improved NMN accumulation in Version 2.0 compared to Version 1.0. (B) Cell growth profiles of control and engineered strains. (C) Comparison of NMN production in LB and M9 media, showing superior yield in M9 with Version 2.0.
Yield improved, but most NMN still accumulated inside the cells, especially under certain media conditions. These observations suggested that secretion might now be the key barrier. We therefore hypothesized that adding a transporter such as PnuC could facilitate extracellular accumulation [4,5].
Building on the progress of Cycle 1.2, we next aimed to overcome the limitation of intracellular NMN accumulation. Although BaPRS successfully enhanced PRPP availability and boosted production, most NMN remained inside the cells, restricting its accessibility for downstream applications. From both our observations and literature reports, we reasoned that secretion rather than synthesis had become the new bottleneck. To address this, we designed the third-generation module by introducing
Figure 7. Design of the third-generation NMN biosynthesis module (Cycle 1.3: NiaP + NadV-BaPRS + PnuC).
To assemble the third-generation NMN module, we extended the construct from Cycle 1.2 by adding the transporter PnuC (BBa_25P5WGA3) to the existing NiaP + NadV-BaPRS backbone. This design created a four-gene under the T7 promoter in the pET28a(m) plasmid. All sequences were codon-optimized for E. coli and verified to comply with RFC#10 standards, ensuring modular compatibility with iGEM Registry requirements. Correct construction was confirmed through sequencing and PCR amplification of the PnuC insert, which produced the expected 717 bp band. As a result, we successfully obtained the engineered strain BL21-NiaP-NadV-BaPRS-PnuC, which served as the working chassis for Cycle 1.3. The plasmid map and validation results are shown in Figure 8..
Figure 8. Verification of the third-generation NMN biosynthesis module (Cycle 1.3: NiaP + NadV-BaPRS + PnuC).
To evaluate whether transport engineering could resolve the intracellular accumulation bottleneck, we compared extracellular NMN levels between Version 2.0 (NiaP + NadV-BaPRS) and Version 3.0 (NiaP + NadV-BaPRS + PnuC). The PnuC strain displayed significantly higher extracellular titers, demonstrating that secretion capacity was the limiting step in Cycle 1.2 and that the addition of PnuC effectively enhanced NMN export (Figure 9).
Figure 9. Comparison of extracellular NMN production between Version 2.0 and Version 3.0 strains.
Having confirmed the benefit of PnuC, we next investigated how NMN secretion was linked to central metabolism. Monitoring glucose utilization during cultivation showed a tight correlation: as glucose was consumed, NMN steadily accumulated in the culture supernatant, while cell growth remained robust. This indicated that extracellular secretion did not impose a major metabolic burden and that carbon flux was efficiently directed toward NMN production (Figure 10).
Figure 10. Glucose consumption profile and its correlation with NMN accumulation in Version 3.0 strain.
Finally, we examined how culture temperature affected NMN yield in the Version 3.0 strain. At 25 °C, NMN accumulation was limited, while both 30 °C and 37 °C supported much higher production, revealing a favorable range for efficient secretion. Based on productivity and practicality, 37 °C was chosen as the working condition for subsequent experiments. This optimization concluded the third DBTL cycle, establishing a robust strain for high-yield extracellular NMN biosynthesis, while also providing parameters that will guide future cycles and downstream applications (Figure 11).
Figure 11. Effect of cultivation temperature on extracellular NMN production, showing optimal performance at 30–37 °C.
By the third generation, we had established a stable NMN production system capable of efficient extracellular secretion, marking the completion of our first module. Yet, boosting NAD+ metabolism alone could not address the broader complexity of aging. This prompted us to ask: beyond NMN, what other factors could reinforce cellular resilience? With oxidative stress emerging as a parallel challenge, we next focused on glutathione (GSH), a central antioxidant [6], to explore whether strengthening redox balance could complement NMN biosynthesis.
The GSH module shows how a single DBTL cycle can yield meaningful improvements. Replacing the native two-enzyme pathway with the bifunctional GshF enzyme immediately enhanced glutathione production. Functional assays further confirmed its antioxidant activity, highlighting how targeted enzyme design can efficiently strengthen cellular defense[7,8.
To enhance antioxidant capacity, we designed a construct expressing
Figure 12. Schematic diagram of the metabolic engineering pathway for one-step intracellular synthesis of GSH in E. coli using GshF.
The codon-optimized GshF (BBa_25PR4M2U) was cloned into the expression vector pET28a(m) under the control of the T7 promoter, RBS, and terminator, ensuring RFC#10 compatibility. The construct was verified by sequencing and colony PCR, which showed the expected 2253 bp band. The recombinant plasmid was transformed into E. coli BL21, generating the engineered strain BL21-GshF (Figure 13).
Figure 13. Construction and validation of engineered strain BL21-GshF (Cycle 2, GSH Module).
The engineered strain BL21-GshF accumulated up to ~350 μM GSH within 8 hours, whereas the wild-type strain produced almost none, and its growth remained robust without metabolic burden (Figure 14A–B). Enzyme activity assays further confirmed that GshF was functionally expressed, with BL21-GshF showing ~3 U/g prot compared to negligible activity in the control (Figure 14C). Moreover, precursor supplementation markedly enhanced production: cysteine exerted the strongest effect, and the combination of all three substrates (glutamate, cysteine, glycine) yielded the highest titers, nearly 2.8-fold above the unsupplemented group (Figure 14D). These results demonstrated that the single-enzyme pathway effectively boosted intracellular GSH biosynthesis, while also revealing that precursor availability is a major determinant of yield.
Figure 14. GSH production and enzyme activity of engineered strain BL21-GshF. (A) Time-course of GSH accumulation. (B) Growth curves. (C) Enzyme activity assay. (D) Effect of precursor supplementation.
To assess environmental factors affecting the GshF module, we varied dissolved oxygen and evaluated antioxidant function. Raising oxygen from 0% to 20% or 30% markedly increased GSH accumulation during cultivation, with the 30% condition giving the highest titers, indicating that oxygen availability positively drives flux toward GSH biosynthesis (Figure 15A). Consistent with higher intracellular GSH, the engineered strain exhibited a substantially greater DPPH radical–scavenging rate than the control, confirming that enhanced production translates into improved antioxidant capacity (Figure 15B).
Figure 15. Oxygen availability and antioxidant function of BL21-GshF. (A) Effect of dissolved oxygen level (0%, 20%, 30%) on GSH production over time. (B) DPPH radical scavenging assay comparing BL21-GshF with the control strain.
This cycle showed that GshF alone enabled efficient and functional GSH production, strengthening the cell’s antioxidant defense. However, combining NMN and GSH suggested that further anti-aging potential might be unlocked by introducing new bioactive molecules.
Building on the progress of NMN and GSH modules, we next explored whether our chassis could be extended from metabolic products to regulatory peptides. To this end, we designed a construct where GLP-1 was fused with the PelB signal peptide, enabling its secretion in E. coli. Protein analysis confirmed both expression and extracellular release, showing that even a single DBTL cycle can expand our design space to include bioactive peptides [9].
Building on the success of NMN and GSH modules, we aimed to further enhance the anti-aging capacity of our system by introducing a regulatory peptide with clinically validated benefits. Glucagon-like peptide-1 (GLP-1) is widely known for improving glucose metabolism and has also been reported to provide cellular protective effects. To achieve its secretion in E. coli, we designed a construct where the GLP-1 coding sequence was fused to the PelB signal peptide, ensuring its export across the cell membrane (Figure 15). This design allowed us to directly evaluate the feasibility of heterologous GLP-1 secretion as a functional expansion of our engineered platform.
Figure 16. Schematic diagram of the GLP-1 secretion module with PelB signal peptide.
The codon-optimized PelB-GLP-1 (BBa_K5083001) was cloned into the expression vector pET28a(m) under the control of the T7 promoter, RBS, and terminator, ensuring RFC#10 compatibility. The construct was verified by sequencing and colony PCR, which showed the expected 162 bp band. The recombinant plasmid was transformed into E. coli BL21(DE3), generating the engineered strain BL21-PelB-GLP-1 (Figure 16).
Figure 17. Construction and validation of engineered strain BL21-PelB-GLP-1.
As shown in Figure 28, a distinct immunoreactive band of <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. These results verified that the PelB signal peptide successfully mediated detectable GLP-1 expression at the protein level.
Figure 18. Western blot validation of GLP-1 expression in BL21-PelB-GLP-1.
The validation of PelB-GLP-1 confirmed that E. coli can be engineered to secrete a functional regulatory peptide through a single DBTL cycle. Although expression was successful, the yield appeared relatively low, suggesting that future optimization of secretion efficiency or peptide stability may be beneficial. Importantly, this result demonstrates the feasibility of extending our platform beyond metabolic products like NMN and GSH to include bioactive peptides. Building on this, we integrated the GLP-1 module with the other systems to establish a more comprehensive anti-aging strategy.
Through three interconnected engineering cycles, we established a multi-layered system addressing different aspects of cellular health. The NMN biosynthesis module demonstrated how iterative pathway design can achieve high-yield extracellular production; the GSH module showed that strengthening redox balance is both feasible and effective; and the GLP-1 module validated that bioactive peptides can be secreted in a bacterial chassis. Together, these modules illustrate how stepwise DBTL cycles can turn separate genetic designs into a coherent platform with measurable outcomes.
At this stage, our work remains at the level of laboratory validation, yet the results highlight clear potential for synergy between metabolic enhancement, antioxidant defense, and signaling peptides. Moving forward, further engineering may optimize flux distribution, stabilize performance under varying conditions, and expand the range of protective factors. While not a final product, our system provides a robust proof-of-concept that multiple anti-aging–related functions can be integrated within one chassis, offering a foundation for future research exploring safe and scalable applications of synthetic biology in longevity science.
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