iGEM 2025 — Team njtech-china

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![Notebook](https://static.igem.wiki/teams/5881/pages/pages/header/notebook.webp)
![Bottle](https://static.igem.wiki/teams/5881/pages/pages/header/bottle.webp)

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* [Wet Lab](#wet-lab)
* [Dry Lab](#dry-lab)
* [HP](#hp)

Wet Lab

Week 1-2 (6.17-6.30)

Module1: Switch: Controllable Expression of CadA

1.Through literature review and with expert assistance, two dynamic regulation systems were identified: Thermosensitive switch system & Protein encapsulation system.

2.Commissioned a company to synthesize the J23100-RBS-TlpA39-PtlpA1 sequence, and the 0.5A-IDP and 1.0A-IDP gene fragments.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Discussed the selection of mutation sites with dry lab team members.

2.Designed primers required for mutagenesis.

Module3: Cofactor: Improvement of Succinate

1.Screened four NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) enzymes from different organisms: from *Corynebacterium glutamicum* ATCC 13032 (CggapC), *Bacillus subtilis* (BsrocG), *Clostridium saccharobutylicum* DSM 13864 (CsgapC), and *Streptococcus mutans* (SmgapN).

2.Obtained the *BsrocG* gene fragment via genomic extraction; the other three were synthesized by a company.

Week 3 (6.30-7.7)

Module1: Switch: Controllable Expression of CadA

1.Constructed genetic circuits for the Thermosensitive switch system: PJ23100-RBS-TlpA39-PtlpA1-RBS-mWasabi and PJ23100-RBS-TlpA39-PtlpA1-RBS-CadA, using the vector pET28a(ΔlacI).

2.Constructed genetic circuits for the Protein encapsulation system: mWasabi-TEV-site-0.5A-IDP, mWasabi-TEV-site-1.0A-IDP, CadA-TEV-site-0.5A-IDP, CadA-TEV-site-1.0A-IDP, CadA-TVMV-site-0.5A-IDP, CadA-TVMV-site-1.0A-IDP, using the vector pCDFDuet under the Ptrc promoter.

3.Transformed the recombinant plasmids into *E. coli* DH5α competent cells. Selected single colonies for colony PCR verification. Colonies with correct amplification band sizes were chosen, their plasmids were extracted and sent for sequencing verification.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Constructed combinatorial mutant plasmids. Used overlap extension PCR (overlap-PCR) to amplify gene fragments covering all mutation sites. These fragments were connected to the plasmid backbone DNA fragment via homologous recombination to obtain the mutant plasmids: pCDFDuet-Ptrc-CadAH245Q,W333E,E526N,K527S,G655F, pCDFDuet-Ptrc-CadAH245Q,W333H,E526N,K527S,G655F, pCDFDuet-Ptrc-CadAH245Q,W333H,E526N,K527S,G655K, pCDFDuet-Ptrc-CadAH245Q,W333E,E526N,K527S,G655K, pCDFDuet-Ptrc-CadAH245F,W333E,E526N,K527S,G655F.

2.Transformed these into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

Module3: Cofactor: Improvement of Succinate

1.Cloned the four GAP gene fragments into the linearized pCDFDuet-Ptrc vector using In-fusion cloning, resulting in plasmids pCDFDuet-Ptrc-CggapC, pCDFDuet-Ptrc-BsrocG, pCDFDuet-Ptrc-CsgapC, pCDFDuet-Ptrc-SmgapN.

2.Transformed these into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

Week 4 (7.7-7.14)

Module1: Switch: Controllable Expression of CadA

1.All plasmids were successfully constructed.

2.Transformed the sequence-verified plasmids into *E. coli* NT1003.

3.Picked colonies from the plate, inoculated them into 5 mL of seed medium, and cultured for 10 hours. Then transferred to shake flasks containing fermentation medium.

4.After starting fermentation, periodically measured fluorescence intensity, glucose, and L-lysine levels. When glucose was depleted, switched to anaerobic fermentation.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Transformed the sequence-verified plasmids into *E. coli* NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium and cultured for 10h, then transferred to 10 mL LB medium (supplemented with sodium pyruvate, methionine, and threonine). When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce CadA expression, and continued induction culture overnight at 37°C.

3.Collected cells by centrifugation and performed whole-cell catalysis.

4.All combinatorial mutants lost enzyme activity. Decided to proceed with single-point mutations based on analysis.

Module3: Cofactor: Improvement of Succinate

1.Transformed the sequence-verified plasmids into *E. coli* NT1003.

2.Inoculated colonies from the plate into 5 mL of seed medium. At OD₆₀₀ ≈ 0.6, transferred to 30 mL fermentation medium, added IPTG (final conc. 0.5 mmol/L) to induce CadA expression, and cultured at 37°C for over 20 hours.

3.Periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

Week 5 (7.14-7.21)

Module1: Switch: Controllable Expression of CadA

1.Analyzed and organized experimental data. Found that the Protein encapsulation system failed to effectively encapsulate the CadA enzyme, allowing it to leak and catalyze the conversion of L-lysine, preventing L-lysine accumulation. However, the fluorescence intensity of the 1.0 A-IDP group was significantly lower than that of the 0.5 A-IDP group, indicating that 1.0 A-IDP has better encapsulation efficiency for the fluorescent protein compared to 0.5 A-IDP.

2.Based on expert advice to enlarge the A-IDP size, attempted to commission synthesis from a company, but failed due to excessive repetitive sequences. Decided to construct it ourselves.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Constructed alanine mutant plasmids for eight sites: I182, H245, K246, W333, E526, K527, Y652, G655. We designed point mutation primers and cloned the whole plasmid using PCR. The linearized plasmid was isolated and purified by agarose gel electrophoresis.

2.Transformed these into *E. coli* DH5α competent cells, extracted plasmids, and sent them for sequencing verification.

Module3: Cofactor: Improvement of Succinate

1.Measured the activity of the NADP⁺-dependent GAP enzymes by detecting the succinate produced from last week's fermentation samples using HPLC.

2.Analyzed and organized experimental data. All four dehydrogenases showed dehydrogenase activity. Among them, Cggap from *Clostridium saccharobutylicum* showed the highest dehydrogenase efficiency. However, the succinate concentration was much lower than expected.

Week 6 (7.21-7.28)

Module1: Switch: Controllable Expression of CadA

1.Attempted to self-construct the 2.0A-IDP plasmid: Using 1.0A-IDP as a template to synthesize the larger 2.0 A-IDP. Digested the pCDFDuet-Ptrc-cadA-TEV/TVMV-site-1.0A-IDP vector plasmid with KpnI and SalI. Then PCR-amplified the 1.0AIDP fragment with primers containing KpnI and SalI sites. Ligated using T4 DNA Ligase.

2.Transformed the recombinant product into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

Module2: Engineering CadA: Synthesis of Cadaverine

The previously failed G655A construct was re-attempted, but the result was still unsuccessful.

Module3: Cofactor: Improvement of Succinate

1.Decided to switch to the low-copy-number plasmid pACYCDuet.

2.Using plasmids pCDFDuet-Ptrc-CggapC, pCDFDuet-Ptrc-BsrocG, pCDFDuet-Ptrc-CsgapC, pCDFDuet-Ptrc-SmgapN as templates, PCR-amplified the CggapC, BsrocG, CsgapC, and SmgapN fragments. Used In-fusion cloning to perform homologous recombination of CggapC, BsrocG, CsgapC, and SmgapN with the linearized pACYCDuet vector, respectively.

3.Transformed the recombinant products into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

Week 7 (7.28-8.4)

Module1: Switch: Controllable Expression of CadA

1.Transformed the plasmid pCDFDuet-Ptrc-CadA-TEV/TVMV-site-2.0A-IDP into *E. coli* NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium, and cultured for 10 hours. Then transferred to shake flasks containing fermentation medium.

3.After starting fermentation, periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

4.Results showed no L-lysine accumulation, suggesting construction failure. Decided to abandon the Protein encapsulation system strategy. Also observed that cadaverine accumulation in the Thermosensitive switch system gradually decreased as temperature increased, decided to optimize and improve this later.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Transformed the sequence-verified plasmids pCDFDuet-Ptrc-CadAI182A, pCDFDuet-Ptrc-CadAH245A, pCDFDuet-Ptrc-CadAK246A, pCDFDuet-Ptrc-CadAW333A, pCDFDuet-Ptrc-CadAE526A, pCDFDuet-Ptrc-CadAK527A, pCDFDuet-Ptrc-CadAY652A, and the unmutated original plasmid into *E. coli* NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium and cultured for 10h, then transferred to 10 mL LB medium (supplemented with sodium pyruvate, methionine, and threonine). When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce CadA expression, and continued induction culture overnight at 37°C.

3.Collected cells by centrifugation and performed whole-cell catalysis.

Module3: Cofactor: Improvement of Succinate

1.Transformed the sequence-verified plasmids into *E. coli* NT1003.

2.Inoculated colonies from the plate into 5 mL of seed medium. At OD₆₀₀ ≈ 0.6, transferred to 30 mL fermentation medium, added IPTG (final conc. 0.5 mmol/L) to induce CadA expression, and cultured at 37°C for over 20 hours.

3.Periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion

Week 8 (8.4-8.11)

Module1: Switch: Controllable Expression of CadA

1.Received expert advice to control the regulation of TlpA39 from the promoter aspect. Selected promoters BBa\_J23106 and BBa\_J23119 from the part library to replace and compare with the original promoter BBa\_J23100.

2.Replaced the promoter PJ23100 in pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-cadA with PJ23106 and PJ23119.

3.Transformed the recombinant products into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Selected I182, H245, K246, which did not completely lose activity, for subsequent mutation to E, L, S, F, V. Constructed the corresponding mutant plasmids.

2.Transformed the recombinant products into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

3.Sequencing showed the K246V construct failed. Repeated construction was still unsuccessful.

Module3: Cofactor: Improvement of Succinate

1.Measured performance by detecting succinate produced by recombinant *E. coli* using HPLC.

2.Results showed that after recombinant expression in the low-copy-number plasmid pACYCDuet, CggapC from *Clostridium saccharobutylicum* again showed increased succinate production, while L-lysine yield remained stable. Therefore, the CggapC gene was selected for constructing the fermentation strain going forward.

Week 9 (8.11-8.18)

Module1: Switch: Controllable Expression of CadA

1.Transformed plasmids pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-cadA, pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-cadA, pET28a(ΔI7)-PJ23119-RBS-TlpA39-PtlpA-cadA into *E. coli* NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium, and cultured for 10 hours. Then transferred to shake flasks containing fermentation medium.

3.After starting fermentation, periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Transformed the sequence-verified plasmids pCDFDuet-Ptrc-CadAI182E, pCDFDuet-Ptrc-CadAI182L, pCDFDuet-Ptrc-CadAI182S, pCDFDuet-Ptrc-CadAI182F, pCDFDuet-Ptrc-CadAI182V, pCDFDuet-Ptrc-CadAH245E, pCDFDuet-Ptrc-CadAH245L, pCDFDuet-Ptrc-CadAH245S, pCDFDuet-Ptrc-CadAH245F, pCDFDuet-Ptrc-CadAH245V, pCDFDuet-Ptrc-CadAK246E, pCDFDuet-Ptrc-CadAK246L, pCDFDuet-Ptrc-CadAK246S, pCDFDuet-Ptrc-CadAK246F, and the unmutated original plasmid into *E. coli* NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium and cultured for 10h, then transferred to 10 mL LB medium (supplemented with sodium pyruvate, methionine, and threonine). When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce CadA expression, and continued induction culture overnight at 37°C

3.Collected cells by centrifugation and performed whole-cell catalysis.

Module3: Cofactor: Improvement of Succinate

1.Following Dr. Xiao-Jie Guo's suggestion, we chose to overexpress the soluble pyridine nucleotide transhydrogenase (*sthA*) from *E. coli* MG1655 to promote rational allocation and cycling of cofactors.

2.Subcloned the sthA gene fragment together with CggapC into the linearized pACYCDuet vector, generating the pACYCDuet-sthA-CggapC plasmid.

3.Transformed the recombinant product into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, extracted plasmids, and sent them for sequencing verification.

Week 10 (8.18-8.25)

Module1: Switch: Controllable Expression of CadA

1.Analyzed and organized experimental data. Identified PJ23106 as the optimal promoter. The issue of decreasing cadaverine accumulation with increasing temperature was alleviated to some extent but still needed improvement.

2.Learned from experts that RNA thermometers can refine the temperature response gradient. After literature review, selected U7 and U8 for insertion into our plasmids.

3.Commissioned a company to synthesize the U7 and U8 gene sequences.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Analyzed and organized experimental data; no mutations resulted in increased enzyme activity.

2.Conducted literature review and in-depth discussions with experts and the dry lab team. Began attempting to re-predict potential beneficial mutations from the perspective of decamer stability.

Module3: Cofactor: Improvement of Succinate

1.Transformed the sequence-verified plasmids into *E. coli* NT1003.

2.Inoculated colonies from the plate into 5 mL of seed medium. At OD₆₀₀ ≈ 0.6, transferred to 30 mL fermentation medium, added IPTG (final conc. 0.5 mmol/L) to induce CadA expression, and cultured at 37°C for over 20 hours.

3.Periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

Week 11 (8.25-9.1)

Module1: Switch: Controllable Expression of CadA

1.Constructed plasmids pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA1-U7-cadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA1-U8-cadA.

2.Transformed into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Obtained F14C/K44C, V12C/K41C, F14C/L45C, L93C/E445C, Y13C/P36C, E104K via computational simulation analysis, and included V12C/K44C, F14C/D41C, L89C/E445C from literature for mutational.

2.Transformed into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

3.Transformed the sequence-verified plasmids pCDFDuet-Ptrc-CadAF14C/K44C, pCDFDuet-Ptrc-CadAV12C/K41C, pCDFDuet-Ptrc-CadAF14C/L45C, pCDFDuet-Ptrc-CadAL93C/E445C, pCDFDuet-Ptrc-CadAY13C/P36C, pCDFDuet-Ptrc-CadAE104K, pCDFDuet-Ptrc-CadAV12C/K44C, pCDFDuet-Ptrc-CadAF14C/D41C, pCDFDuet-Ptrc-CadAL89C/E445C, and the unmutated original plasmid into *E. coli* BL21.

Module3: Cofactor: Improvement of Succinate

1.Measured performance by detecting L-lysine and succinate produced by *E. coli* using HPLC.

2.Results showed that the strain with pACYCDuet-sthA-CggapC increased succinate production by 26.3% compared to the empty vector, and by 70% compared to the pACYCDuet-CggapC strain, while L-lysine production remained stable. We decided to use this strain in subsequent experiments.

Week 12 (9.1-9.8)

Module1: Switch: Controllable Expression of CadA

1.Transformed plasmids pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-U7-cadA, pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U8-cadA, pET28a(ΔI7)-PJ23119-RBS-TlpA39-PtlpA-cadA (control) into *E. coli* NT1003.

2.Picked colonies from the plate, inoculated into 5 mL of seed medium, and cultured for 10 hours. Then transferred to shake flasks containing fermentation medium.

3.After starting fermentation, periodically measured glucose and L-lysine levels. Switched to anaerobic fermentation after glucose depletion.

Module2: Engineering CadA: Synthesis of Cadaverine

1.Picked single colonies from the plate, inoculated into 5 mL LB seed medium, and cultured for 8h. Then transferred at 1% inoculation volume into 50 mL LB medium for expansion. When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce target protein expression, and continued induction culture overnight at 37°C.

2.Collected cells by centrifugation and performed whole-cell catalysis.

3.Identified Y13C/P36C as the optimal mutant. Performed iterative mutations, constructing mutants Y13C/P36C/E445Q, Y13C/P36C/K477R, Y13C/P36C/L93C/E445C.

4.Transformed into *E. coli* DH5α competent cells. Screened for positive clones via colony PCR, and finally confirmed successful construction by sequencing.

Week 12 (9.8-9.15)

Module1: Switch: Controllable Expression of CadA

1.Analyzed and organized experimental data. Found that leaky expression significantly decreased after adding U7/U8.

2.Replaced the unmutated CadA in the selected optimal mutant Y13C/P36C within the plasmids pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-U7-cadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U8-cadA.

1.Transformed the sequence-verified plasmids pCDFDuet-Ptrc-CadAY13C/P36C/E445Q, pCDFDuet-Ptrc-CadAY13C/P36C/K477R, pCDFDuet-Ptrc-CadAY13C/P36C/L93C/E445C, and the unmutated original plasmid into *E. coli* BL21.

2.Picked single colonies from the plate, inoculated into 5 mL LB seed medium, and cultured for 8h. Then transferred at 1% inoculation volume into 50 mL LB medium for expansion. When OD₆₀₀ reached 0.6-0.8, added IPTG to a final concentration of 0.5 mmol/L to induce target protein expression, and continued induction culture overnight at 37°C.

3.Collected cells by centrifugation and performed whole-cell catalysis.

4.Analyzed and organized experimental data.

Week 13 (9.16-9.23)

Module4: An intelligent cell: Production of cadaverine-succinate

1.Based on previous experiments, we finalized the optimized Thermosensitive switch system and the Cofactor conversion system. Subsequently, we introduced the plasmids for these two modules into the L-lysine-producing *E. coli* NT1003 to construct a cell factory capable of co-producing cadaverine and succinate.

2.Co-transformed plasmids pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA1-U7-cadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA1-U8-cadA respectively with plasmid pACYCDuet-sthA-CggapC into the L-lysine producing strain *E. coli* to construct cell factories capable of co-producing cadaverine and succinate.

3.Inoculated colonies from the plate into 5 mL of seed medium. At OD₆₀₀ ≈ 0.6, transferred to 30 mL fermentation medium, added IPTG (final conc. 0.5 mmol/L) to induce CadA expression, and cultured at 33°C for over 20 hours.

4.Periodically measured glucose and L-lysine levels. After glucose depletion, cultured at 42°C for 4 hours to activate the thermosensitive switch, then switched to anaerobic fermentation.

Week 14 (9.23-9.30)

Module4: An intelligent cell: Production of cadaverine-succinate

1.Measured performance by detecting cadaverine and succinate produced by *E. coli* using HPLC.

2.Fermentation results showed that the recombinant strains successfully achieved synergistic synthesis of cadaverine and succinate, with yields reaching 5.7 g/L and 5.4 g/L, respectively. Through precise metabolic flux regulation, the molar ratio of the two products approached the ideal 1:1. This created excellent thermodynamic conditions for the subsequent acid-base neutral reaction to generate PA54 salt, eliminating the need for additional acid or base, simplifying steps and reducing costs. Furthermore, from the perspective of carbon metabolic flux, the matched yields of the two products indicated a good balance between CO2 fixation and release in their biosynthetic pathways, improving overall carbon economy and laying a solid foundation for the efficient and green preparation of the polymer monomer PA54.

Dry Lab

April

Week 1（4.1-4.6）

**Environment-responsive enzyme-controlled release system:** Established an ODE model from signal sensing to product formation and compiled key parameters for CadC signal perception and cadBA transcriptional regulation.

**Cadaverine-producing enzyme model:** Screened key active sites of CadA and performed molecular docking, preliminarily identifying potential engineering targets.

**Metabolic flux study:** Reviewed project background and challenges to lay the foundation for subsequent modeling.

Weeks 2–3（4.7-4.20）

**Environment-responsive system:** Refined transcriptional regulation and protein release modules, integrated metabolic conversion kinetics, and ensured mass balance.

**Cadaverine enzyme model:** Designed the deep learning model architecture and constructed a data pipeline to extract features from protein sequences and compound graph structures.

**Metabolic flux study:** Studied CadA directed evolution literature to inform the inclusion of enzyme constraints and regulatory logic in models.

Week 4（4.21-4.30）

**Environment-responsive system:** Integrated the four modules into a unified ODE system; initial simulation showed two-stage dynamics: aerobic accumulation and anaerobic production.

**Cadaverine enzyme model:** Implemented feature extraction and custom DataLoader to prepare for training.

**Metabolic flux study:** Installed and tested metabolic modeling software, clarifying dry-lab tasks.

May

Week 1（5.1-5.8）

**Environment-responsive system:** Validated and calibrated the model, designed parameter sensitivity analysis, and developed visualization tools.

**Cadaverine enzyme model:** Implemented feature fusion and activity prediction modules with early stopping to prevent overfitting. Lysine decarboxylase docking simulation to identify key amino acid residues.

**Metabolic flux study:** Set up the modeling environment, including Anaconda, Gurobi, and essential libraries.

Week 2（5.9-5.16）

**Environment-responsive system:** Conducted sensitivity analysis, quantified performance metrics, and predicted cadaverine yield under optimal conditions.

**Cadaverine enzyme model:** Prepared kcat dataset and configured the training environment.

**Metabolic flux study:** Learned FBA and FVA, and tested the iML1515 model.

Week 3（5.17-5.24）

**Environment-responsive system:** Simulated the effects of different A-IDP encapsulation sizes.

**Cadaverine enzyme model:** Trained the generalized model and performed preliminary performance evaluation.

**Metabolic flux study:** Built a project-specific aerobic model including cadaverine and related reactions.

Week 4（5.25-5.31

**Metabolic flux study:** Constructed anaerobic model, added succinate-related reactions, and performed baseline flux analysis.

**Cadaverine enzyme model:** Fine-tuned the model using a specific dataset to improve prediction accuracy.

June

**Metabolic flux study:** Performed FVA and FSEOF analyses to identify key target gene gapA.

**Cadaverine enzyme model:** Designed genetic algorithms to screen high-activity mutants and discussed candidate results with the wet-lab team.

September

**Metabolic flux study:** Compiled analysis reports, submitted prioritized targets, and compared predictions with wet-lab validation data.

**Cadaverine enzyme model:** Organized code, model weights, and predictions; completed iGEM Wiki documentation; summarized methodological innovations and compared computational predictions with wet-lab enzyme activity.

HP

Week 1-2 (May 15 - June 10)

1. Conducted team brainstorming sessions to define our project focus on bio-based nylon development.

2. Interviewed expert Cao Min from Kingfa Science & Technology to understand the market landscape for bio-based polyamides and identify the technological gap for fully bio-based short-chain polyamides.

3. Consulted Professor Chen Kequan to learn about bio-based material synthesis technologies available in the university lab, leading to the final decision to pursue the "Single-cell Synchronous Synthesis of PA54" project.

4. Held discussions with representatives from China National Petroleum Corporation (CNPC) to clarify the environmental, economic, and resource-related challenges within the conventional fossil-based nylon industry.

5. Initiated public awareness research on bio-based materials by designing online and offline survey distribution plans.

Week 3 (June 10 - June 17)

1. Prepared training materials on iGEM competition rules and essential synthetic biology skills for the ZQT-Nanjing high school team.

2. Established initial contact with the LU-NBBMS team from Jilin University to begin planning the collaborative illustration of the "Chassis Organism White Paper".

3. Attended the 3rd Synthetic Biology Conference & Exhibition (SBC), presenting our project and exploring potential collaborations with teams including Nanjing Agricultural University and Xi'an Jiaotong-Liverpool University.

Week 4 (June 17 - June 24)

1. Delivered iGEM rules and synthetic biology skills training to the ZQT-Nanjing high school team.

2. Held an online meeting with the iGEM team from Xi'an Jiaotong-Liverpool University (XJTLU) to discuss potential collaborations in sustainability and public education.

Week 5 (June 24 - June 30)

1. Consulted Professor Wang Xin, leading to the selection of the specialized high-lysine producing chassis strain E. coli NT1003.

2. Organized a "Synthetic Biology Carnival" on campus to introduce PA54 and green production concepts to students and faculty.

3. Participated in the Jiangsu-Zhejiang-Shanghai (JZS) iGEM Exchange, connecting with teams like Shanghai Jiao Tong University and Fudan University to share our project and gather feedback.

Week 6 (June 30 - July 7)

1. Sought advice on dynamic regulation switches from Professor Chen Kequan (recommended thermosensitive system) and Professor Liu Liming (recommended A-IDP encapsulation system).

2. Analyzed results from the public survey on bio-based materials to identify key focus areas for subsequent science communication.

3. Contributed to the writing and proofreading of specific sections for the collaborative "Chassis Organism White Paper" with Jilin University and 32 other teams.

Week 7 (July 7 - July 14)

1. Consulted Professor Xue Chuang to assess project feasibility and gather suggestions for metabolic pathway regulation.

2. Began designing community outreach lecture materials based on the analyzed survey data.

3. Continued collaborative writing and review process for the "Chassis Organism White Paper".

Week 8 (July 14 - July 21)

1. Consulted Professor Liu Guannan regarding leakage expression in the first-generation thermal switch; received advice to test promoters of different strengths.

2. Consulted Manager Guo Xiaojie from Tianmu Biotech regarding metabolic burden caused by high GAP enzyme expression; adopted the suggestion to switch to a low-copy-number plasmid.

3. Team members attended the 12th World Congress on Climate and Energy (WCCE), integrating insights from discussions with field experts into our project.

Week 9 (July 21 - July 28)

1. Visited the "Love Summer Tutoring Program" in Pukou District to deliver a synthetic biology lecture and conduct fun science demonstrations for children.

2. Attended the 11th Microbial Breeding Engineering and Application Evaluation Seminar, discussing potential collaborations and gathering valuable experimental suggestions from experts.

Week 10 (July 28 - August 4)

1. Consulted Professor Zhang Lei to address persistent leakage in the thermal switch system; implemented his suggestion to integrate RNA thermometers (U7/U8) for finer gene regulation.

Week 11 (August 4 - August 11)

1. Consulted Professor Liu Lixia on strategies for optimizing the key enzyme CadA, initiating a machine learning-assisted rational design approach.

2. Attended the 12th Conference of China iGEMer Community (CCiC), presented our project to experts, and received valuable optimization suggestions.

3. Published project updates and synthetic biology educational content on our social media platforms (WeChat Official Account and Xiaohongshu) to broaden our reach.

Week 12 (August 11 - August 18)

1. Communicated with expert Zhang Yutong from Nest.Bio to understand project cost control and explore commercial potential.

2. Collaborated with CJUH-JLU-China and 32 other teams to jointly release the "Synthetic Biology Rumors Debunking White Paper".

Week 13-15 (August 18 - September 8)

1. Interviewed leadership from China Textile Academy to understand challenges in PA54 industrialization and key considerations for optimizing lab protocols.

2. Discussed with Professor Wu Zhansheng from Xi'an Polytechnic University how to align synthetic biology research outcomes with industry regulations.

3. Interviewed experts from Xuan Kai Biotechnology to learn about critical factors for industrial scaling, including site selection and regulatory compliance.

4. After facing obstacles in modifying the CadA active site, consulted Dr. Chen Cai and pivoted our strategy to focus on predicting beneficial mutations that enhance decamer stability.

Week 16 (September 8 - September 15)

1. Consulted Professor Wei Guoguang for final project optimization advice, focusing on overall narrative, presentation logic, and visual improvements.

2. Finalized and officially released the first illustrated "Chassis Organism White Paper" as a core contribution to the iGEM community.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

![Engineering](https://static.igem.wiki/teams/5881/pages/pages/header/engineering.webp)
![Bottle](https://static.igem.wiki/teams/5881/pages/pages/header/bottle.webp)

Switch
Engineering CadA
Cofactor
An intelligent cell

* [Overview](#overview-intro)
* [Cycle 1](#cycle1)
* [Cycle 2](#cycle2)
* [Cycle 3](#cycle3)
* [Cycle 4](#cycle4)
* [Cycle 5](#cycle5)

Design

Build

Test

Learn

Click here!
![](https://static.igem.wiki/teams/5881/svg/9.webp)

Overview

We designed a green strategy for producing PA54 monomers (cadaverine and succinate) using a single cell. The cell grows and accumulates lysine under aerobic conditions; when switching to anaerobic conditions, it activates the expression of lysine decarboxylase to catalyze the synthesis of cadaverine from lysine, while simultaneously synthesizing succinate. This enables synchronous acid-base neutralization during the synthesis of cadaverine and succinate, and the CO2 released from decarboxylation is fixed for succinate synthesis. We selected the lysine-producing strain *Escherichia coli* NT1003 as the chassis cell. Firstly, we constructed and screened the switch circuit to achieve regulatable expression of lysine decarboxylase (CadA) for catalyzing lysine. The switch circuit was further optimized to enhance its accuracy by optimizing the promoters and integrating the RNA thermometers. Subsequently, based on AI-based design and rational design, we performed rational engineering on the key enzyme CadA to improve its catalytic efficiency. Additionally, we adopted cofactor regulation strategy combined the modification site identified by metabolic network model to enhance succinate synthesis. Ultimately, we successfully achieved simultaneous production of cadaverine and succinate from glucose using a single cell.

Module 1: Switch---Controllable expression of CadA

Cycle 1 Constructing a switch genetic circuit to dynamically regulate CadA for controllable synthesis of cadaverine

Design

In *E.coli* NT1003, lysine is a product of aerobic fermentation, while succinate only accumulates during anaerobic processes. To realize the concept of simultaneous production of cadaverine and succinate(Figure 1-1A), we first designed a switch genetic circuit that allowed lysine accumulation without the activity of lysine decarboxylase CadA during the aerobic growth phase, and activated CadA to exert its catalytic activity for converting lysine into cadaverine during anaerobic phase.

After literature research and expert interviews, we ultimately selected two switch systems for regulatory testing: the temperature-responsive system and the protein encapsulation system.

Temperature-responsive system:

This system was constructed using a temperature-sensitive switch. The temperature-responsive repressor protein TlpA39 is expressed under the control of the constitutive promoter J23100. When the temperature is below 39℃, TlpA39 forms a dimer that can bind to the promoter PtlpA, thereby repressing the expression of the downstream *cadA* gene. At temperatures exceeding 39°C, TlpA39 monomerizes and dissociates from PtlpA, switching the circuit from an OFF to an ON state and enabling *cadA* expression. This design ensures that CadA is suppressed during low-temperature aerobic phase to permite lysine accumulation. and *CadA* expression is activated by a temperature upshift during the anaerobic phase to drive the conversion of lysine to cadaverine (Figure 1-1B).

Protein encapsulation system:

This system uses an artificial intrinsically disordered protein (A-IDP), which encapsulates the CadA under aerobic conditions to inhibit its activity, thereby preventing lysine from being catalytically consumed. Under anaerobic conditions, specific proteases (TEV or TVMV protease) cleave the corresponding sites (TEV-site or TVMV-site), separating A-IDP from CadA and releasing CadA from encapsulation. This proteolytic cleavage releases CadA from encapsulation to enable CadA to catalyze lysine into cadaverine, representing the switch from OFF to ON ( Figure 1-1C).

![Figure 1-1 (A) Metabolic diagram of E. coli NT1003 for PA54 production. (B) Mechanism diagram of the temperature-responsive system. (C) Mechanism diagram of the protein encapsulation system.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure1-1.webp)

Figure 1-1 (A) Metabolic diagram of *E. coli* NT1003 for PA54 production. (B) Mechanism diagram of the temperature-responsive system. (C) Mechanism diagram of the protein encapsulation system.

Build

To construct the temperature-responsive system, we first designed a switch genetic circuit composed of the parts, including J23100, RBS, TlpA39, and PtlpA. The J23100-RBS-TlpA39-PtlpA sequence was commercially synthesized and cloned into the pET28a(ΔlacI7) vector. Subsequently, the genes encoding green fluorescent protein (mWasabi) and lysine decarboxylase (CadA) were individually cloned in downstream of the PtlpA promoter to assemble the complete genetic circuits: PJ23100-RBS-TlpA39-PtlpA-RBS-*mWasabi* and PJ23100-RBS-TlpA39-PtlpA-RBS-CadA.

![Figure 1-2 The PCR validation diagram. M: Marker. Lane 1,2:cadA fragment. Lane 3, 4:mWasabi fragment.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure1-2.webp)

Figure 1-2 The PCR validation diagram. M: Marker. Lane 1,2:*cadA* fragment. Lane 3, 4:*mWasabi* fragment.

To construct the protein encapsulation system, we first conducted literature research and then commissioned the synthesis of the 0.5 and 1.0 size A-IDP gene fragments along with the corresponding TEV and TVMV protease cleavage sites. Subsequently, the genes encoding green fluorescent protein (mWasabi) and lysine decarboxylase (CadA) were individually cloned in upstream of A-IDP to construct the genetic circuits, including *mWasabi*-TEV-site-0.5A-IDP, *mWasabi*-TEV-site-1.0A-IDP, CadA-TEV-site-0.5A-IDP, CadA-TEV-site-1.0A-IDP, CadA-TVMV-site-0.5A-IDP, and CadA-TVMV-site-1.0A-IDP. They were inserted into the pCDFDuet-Trc vector to obtain recombinant plasmids.

![Figure1-3 (A) Genetic circuit diagram.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure1-3.webp)

Figure1-3 (A) Genetic circuit diagram.(B) The colony PCR validation diagram. M: Marker. line l~9:*mWasabi*-TEV-0.5A-IDP. line 10~20: *mWasabi*-TEV-1.0A-IDP.

All recombinant plasmids were first transformed into *E. coli* DH5α. Positive clones identified by colony PCR (based on the correct band size) were subjected to commercial sequencing. Sequencing results confirmed the successful construction of the plasmids. These plasmids were then transformed into the *E. coli* NT1003 strain for functional characterization.

Test

For the functional assessment of the temperature-responsive system, we selected low temperature (33℃) and high temperature (39℃) as test conditions. The green fluorescent protein group measured fluorescence intensity at 6, 10, 24, and 34 h, as illustrated in Figure 1-4 A and B. Fluorescence intensity was consistently low at 33°C but exhibited a marked increase at 39°C. After 24 hours, the signal at 39°C was 2.56-fold higher than that at 33°C.

To further investigate the function on CadA expression, lysine and glucose of the CadA-expressing *E. coli* were analyzed using a biosensor analyzer at 0, 6, 10, and 24 h under 33℃ and 39℃. At the inducing temperature (39°C), lysine was rapidly consumed, maintaining low levels, whereas at 33°C, it steadily accumulated, confirming the thermal control of CadA (Figure 1-4 C and D).

![Figure1 Improvements across engineering cycles](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure1-4.webp)

Figure 1-4 Fluorescence intensity of the engineered strain *E. coli* NT1003 .(A) *mWasabi* (33℃). (B) *mWasabi* (39℃) Glucose consumption and lysine accumulation of the engineered strain *E. coli* NT1003. (C) CadA (33℃). (D) CadA (39℃).

To evaluate the protein encapsulation system, we first analyzed the fluorescence signal of recombinant strains expressing the green fluorescent protein mWasabi at 0, 8, and 21 h. Both the 0.5A-IDP and 1.0A-IDP groups produced detectable signals, with the 1.0A-IDP group showing significantly lower intensity than the 0.5A-IDP group. In CadA-expressing strains, both A-IDP groups completely suppressed lysine accumulation despite normal glucose consumption, unlike the empty plasmid control where lysine steadily increased.

![Figure 1-5 (A) Glucose consumption and lysine accumulation of the engineered E. coli (CadA-0.5A-IDP) and (CadA-1.0A-IDP). (B)Fluorescence intensity of the engineered E. coli (mWasabi-0.5A-IDP) and (mWasabi-1.0A-IDP).](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure1-5.webp)

Figure 1-5 (A) Glucose consumption and lysine accumulation of the engineered *E. coli* (CadA-0.5A-IDP) and (CadA-1.0A-IDP). (B)Fluorescence intensity of the engineered *E. coli* (*mWasabi*-0.5A-IDP) and (*mWasabi*-1.0A-IDP).

Learn

The simultaneous synthesis of cadaverine and succinate in a single cell constitutes a green and low-carbon process. To achieve the process, we learned that different switchs could be employed to construct regulatory systems for regulating the key enzyme CadA. In this cycle, we successfully constructed the Temperature-responsive system and the Protein encapsulation system.

In temperature-responsive system, our temperature-sensitive switch successfully regulated the timed expression of CadA, as evidenced by contrasting phenotypes of gene repression (low fluorescence, lysine accumulation) at 33°C and gene activation (high fluorescence, lysine consumption) at 39°C. This functional shift is mediated by the temperature-dependent binding of TlpA39 to the PtlpA promoter, which blocks transcription at low temperatures and releases it upon thermal induction.

The current protein encapsulation system failed to prevent lysine consumption by CadA in both 0.5A-IDP and 1.0A-IDP, suggesting insufficient enzyme encapsulation. The fluorescent result showed 1.0A-IDP conferred significantly greater repression than 0.5A-IDP.

Following Professor Liming Liu's suggestion, CadA may require larger A-IDP constructs for effective encapsulation and shielding, Future work will focus on engineering larger A-IDP variants to achieve complete enzyme encapsulation.

Cycle 2 Optimization of protein encapsulation system

Design

According to Prof. Liu's suggestion, 0.5 A-IDP and 1.0 A-IDP are relatively small with insufficient encapsulation efficiency. Therefore, we constructed the larger 2.0 A-IDP using 1.0 A-IDP as a template.

![Figure 2-1 Construction process of pCDFDuet-Ptrc-CadA-TEV/TVMV-site-2.0A-IDP.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure2-1.webp)

Figure 2-1 Construction process of pCDFDuet-Ptrc-CadA-TEV/TVMV-site-2.0A-IDP.

Build

We used the pCDFDuet-Trc-CadA-TEV-site-1.0A-IDP plasmid as a template to amplify the 1.0A-IDP gene fragment via PCR. The amplified product was inserted into pCDFDuet-Trc-CadA-TEV/TVMV-site-1.0A-IDP between *Kpn I* and *Sal I*, constructing the recombinant plasmid pCDFDuet-Trc-CadA-TEV-site-2.0A-IDP (Figure 2-2A). After transforming into *E. coli* DH5α, positive clones were screened by colony PCR, and then sent for sequencing verification.

![Figure 2-2 (A) Genetic circuit diagram containing 2.0A-IDP. (B) colony PCR.M:Marker, Lane 1-24: products containing 2.0A-IDP.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure2-2.webp)

Figure 2-2 (A) Genetic circuit diagram containing 2.0A-IDP. (B) colony PCR.M:Marker, Lane 1-24: products containing 2.0A-IDP.

Test

![Figure2-3 Glucose consumption and lysine accumulation in the 2.0A-IDP group.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure2-3.webp)

Figure2-3 Glucose consumption and lysine accumulation in the 2.0A-IDP group.

The recombinant plasmid was then transformed into the *E. coli* NT1003 strain for functional characterization. A SBA biosensor was used to determine glucose and lysine at 0 and 26 h. The results showed that glucose was consumed normally, but lysine was still not detected.

Learn

In this cycle, we learned that larger encapsulating proteins titer better encapsulation efficiency. However, a larger-sized encapsulating protein 2.0A-IDP failed to perfectly encapsulate CadA and inhibit its activity. Compared with the temperature-responsive system in lysine accumulation, we decided to adopt the temperature-sensitive switch for subsequent experiments.

Cycle3 The temperature-responsive characterization of temperature-responsive system

Design

By comparision, the Temperature-responsive system was adopted to control the expression of CadA. To quantify its regulatory precision, we then profiled the system's induction response across a spectrum of temperatures from 33 to 42°C.

Build

pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-*mWasabi* and pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-CadA were respectively transformed into *E. coli* NT1003 and cultured aerobically at 33, 35, 37, 39, and 42℃.

![Figure 3-1 Strategy diagram](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure3-1.webp)

Figure 3-1 Strategy diagram

Test

The two engineered strains were aerobically cultured at different temperatures. Samples collected at 0, 6, 10, and 23 h were analyzed using a Thermo fluorescence analyzer and a biosensor to determine fluorescence intensity(reporter for mWasabi expression), lysine, and glucose, respectively. The mWasabi reporter strain showed low fluorescence at ≤37°C but strong induction at ≥39°C (Figure 3-2A). For the CadA group, lysine accumulated to 4.27 g/L, 2.6 g/L, and 2.4 g/L at 33°C, 35°C, and 37°C, respectively (Figure 3-2 B, C, D), but was not detected at 39°C and 42°C (Figure 3-2 E, F).

![Figure 3-2 (A) Fluorescence detection diagram; Glucose consumption and lysine accumulation of the engineered E. coli strain. (B) CadA (33℃). (C) CadA (35℃). (D) CadA (37℃). (E) CadA (39℃). (F) CadA (42℃).](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure3-2.webp)

Figure 3-2 (A) Fluorescence detection diagram; Glucose consumption and lysine accumulation of the engineered *E. coli* strain. (B) CadA (33℃). (C) CadA (35℃). (D) CadA (37℃). (E) CadA (39℃). (F) CadA (42℃).

Learn

In this cycle, we learned that the temperature-responsive system was a clear temperature-dependent switch. Low fluorescence with lysine accumulation at ≤37°C confirmed the "OFF" state, while high fluorescence with no lysine accumulation at ≥39°C confirmed the "ON" state. It defined a clear activation threshold near 39°C, successfully enabling the timed induction of downstream gene through a simple temperature shift.

Cycle 4  Screening different intensity promoters to reduce leaky expression of CadA

Design

From Cycle 3, we found that lysine accumulation gradually decreased from 33°C to 37°C. Professor Guannan Liu hypothesized that insufficient repression stringency resulted in leaky expression of *cadA* and pointed out that the promoter strength affected the stringency and sensitivity. To further enhance the effect of TlpA39, we selected promoters of different strengths ([BBa\_J23106](https://registry.igem.org/parts/bba-j23106) and [BBa\_J23119](https://registry.igem.org/parts/bba-j23119)) from the Part Registry and compared them with the original promoter ([BBa\_J23100](https://registry.igem.org/parts/bba-j23100)). By varying TlpA39 expression via these promoters, we intend to adjust its repression capacity at PtlpA and thus improve control of CadA expression..

![Figure 4-1 Promoter replacement mechanism diagram](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure4-1.webp)

Figure 4-1 Promoter replacement mechanism diagram

Build

Based on the plasmid of pET28a (ΔI7)-PJ23100-RBS-TlpA39-PtlpA-CadA, we designed promoter mutation primers and cloned the whole plasmid using PCR. The linearized plasmid was isolated and purified by agarose gel electrophoresis, and then transferred into *E. coli* DH5α to obtain the targeted plasmids of pET28a (ΔI7)-PJ23106-RBS-TlpA39-PtlpA-CadA and pET28a (ΔI7)-PJ23119-RBS-TlpA39-PtlpA-CadA. Positive colonies were verified by sequencing. Then the plasmids were transferred into *E. coli* NT1003 respectively to evaluate the effect of promoters.

![Figure 4-2 The colony PCR validation diagram.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure4-2.webp)

Figure 4-2 The colony PCR validation diagram.

Test

Three engineered strains with different promotes were cultured aerobically at 33℃ for 22 h, then shifted them to anaerobic culture at 42℃. A biosensor was used to measure the lysine and glucose during the aerobic phase (0 - 23h) and anaerobic phase (after 23h). PJ23106 and PJ23119 led to more lysine accumulation compared to PJ23100. PJ23106 was best, resulting in accumulating 5.1 g/L at 22h of the aerobic phase. Subsequently, during the high-temperature anaerobic phase, lysine was gradually consumed completely.

![Figure 4-3 (A) Schematic Diagram; Glucose consumption and lysine accumulation of the engineered E. coli. (B) PJ23100-CadA (33℃), (C) PJ23106-CadA (33℃), (D) PJ23119-CadA(33℃).](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure4-3.webp)

Figure 4-3 (A) Schematic Diagram; Glucose consumption and lysine accumulation of the engineered *E. coli*. (B) PJ23100-CadA (33℃), (C) PJ23106-CadA (33℃), (D) PJ23119-CadA(33℃).

Learn

In this cycle, we learned that promoters of different strengths determine the expression intensity of downstream genes. The strain with the PJ23106 promoter accumulated the highest lysine accumulation during the aerobic phase, which proved that PJ23106 effectively reduced CadA leaky expression. Additionally, preliminary experiments combining aerobic-anaerobic culture with high-low temperature switching showed that the Temperature-responsive system effectively supported lysine accumulation by repressing CadA during low-temperature aerobic growth of *E. coli* NT1003; while triggering its rapid consumption by inducing CadA expression upon switching to anaerobic conditions with temperature upshift.

Cycle 5 Combining with RNA thermometers for precise temperature-controlled CadA expression

Design

After reviewing the literature, we identified high-efficiency RNA thermometers. At low temperatures, they facilitates the formation of a stem-loop structure at the ribosome-binding site (RBS), blocking ribosome binding and inhibiting translation. Upon temperature increase, the structure unfolds, restoring normal translation. To refine the system's temperature sensitivity and accuracy, we incorporated RNA thermometers U7 and U8 into the plasmid pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-CadA, adding translational control to the existing transcriptional regulatory circuit.

Build

We constructed the plasmids pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U7-CadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U8-CadA using methods consistent with cycle4. Subsequently, the plasmids were transformed into *E. coli* NT1003 to evaluate the effect of RNA thermometers.

![Figure 5-1 RNA thermometer mechanism diagram.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure5-1.webp)

Figure 5-1 RNA thermometer mechanism diagram.

Test

Two engineered strains were cultured aerobically at 33℃ for 23 h, then shifted them to anaerobic culture at 42℃. A biosensor was used to measure the lysine and glucose during the aerobic phase (0 - 23 h) and anaerobic phase (after 23h). The results are shown in Figure 5-2.

The engineered *E. coli* with the U7 and U8 RNA thermometers exhibited higher lysine accumulation (8.7 g/L and 10.1 g/L) at 37℃, while at 33℃, the levels increased by 2-fold and 2.3-fold, respectively, compared to the previous round(Figure 5-2).

![Figure 5-2 Glucose consumption and lysine accumulation of the engineered E. coli. (A) PJ23106-U7-CadA (33℃). (B) PJ23106 -U7-CadA (35℃). (C) PJ23106 -U7-CadA (37℃). (D) PJ23106 -U8-CadA (33℃). (E) PJ23106 -U8-CadA (35℃). (F) PJ23106-U8-CadA (37℃).](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure5-2.webp)

Figure 5-2 Glucose consumption and lysine accumulation of the engineered *E. coli*. (A) PJ23106-U7-CadA (33℃). (B) PJ23106 -U7-CadA (35℃). (C) PJ23106 -U7-CadA (37℃). (D) PJ23106 -U8-CadA (33℃). (E) PJ23106 -U8-CadA (35℃). (F) PJ23106-U8-CadA (37℃).

Learn

In this cycle, we learned that incorporating RNA thermometers into the original temperature-responsive system enables dual-level regulation of CadA expression—at both transcriptional and translational levels via temperature. The optimized system could minimize CadA leakage at low temperatures—maximizing lysine accumulation—and permit accurate induction upon temperature upshift, enabling staged and precise control.

Module 2: Engineering CadA---Synthesis of cadaverine

Cycle 6 AI-guided design of CadA via multi-site combinatorial mutagenesis

Design

CadA is the key enzyme for the synthesis of cadaverine. To improve its catalytic efficiency, we adopted a computationally guided strategy. Molecular docking simulations was performed using the structure of CadA (PDB No. 6YN6) Hydrogen bonds, salt bridges, and hydrophobic interactions between the enzyme and its substrate were systematically analyzed. Based on the interaction analysis, we identified seven residues located near the active pocket that might make key contributions to substrate binding and catalysis: I182, H245, K246, W333, E526, K527 and Y652. These candidate sites were subsequently evaluated using a site-directed mutagenesis prediction model for virtual amino acid substitutions, and their catalytic efficiencies (Kcat) were predicted. The model recommended several pentuple mutants with predicted high enzymatic activity (as shown in Table 6-1).

Table 6-1 Prediction of enzymatic activity for combined mutations

| Mutation type | Enzymatic activity Kcat（s⁻¹） |
| --- | --- |
| Wild type | 3.484 |
| H245Q, W333E, E526N, K527S, G655F | 13.6055 |
| H245Q, W333H, E526N, K527S, G655F | 13.6008 |
| H245Q, W333H, E526N, K527S, G655K | 13.4973 |
| H245Q, W333E, E526N, K527S, G655K | 13.4772 |
| H245F, W333E, E526N, K527S, G655F | 13.2887 |

Build

According to the prediction results, DNA fragment containing five target site-directed mutation sites was amplified by overlap extension PCR (overlap-PCR) using the original CadA plasmid pCDFDuet-Ptrc-CadA as the template and site-directed mutagenesis primers. Subsequently, the homologous recombination method was used to ligate the gene fragment with the plasmid backbone, obtaining the mutant plasmids, including pCDFDuet-Ptrc-CadAH245Q,W333E,E526N,K527S,G655F, pCDFDuet-Ptrc-CadAH245Q,W333H,E526N,K527S,G655F, pCDFDuet-Ptrc-CadAH245Q,W333H,E526N,K527S,G655K, pCDFDuet-Ptrc-CadAH245Q,W333E,E526N,K527S,G655K, pCDFDuet-Ptrc-CadAH245F,W333E,E526N,K527S,G655F, These plasmids were then transformed into competent *E. coli* DH5α. Positive clones were screened by colony PCR, and the successful construction was finally verified by sequencing.

![Figure 6-1 Construction of combined mutations. (A) strategy diagram of combined mutations. (B) PCR validation diagram of fragment. (D) PCR of Vector. (E) The colony PCR validation diagram.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure6-1.webp)

Figure 6-1 Construction of combined mutations. (A) strategy diagram of combined mutations. (B) PCR validation diagram of fragment. (D) PCR of Vector. (E) The colony PCR validation diagram.

Test

We separately transformed the plasmids verified to be correct by sequencing—including pCDFDuet-Ptrc-CadAH245Q,W333E,E526N,K527S,G655F, pCDFDuet-Ptrc-CadAH245Q,W333H,E526N,K527S,G655F, pCDFDuet-Ptrc-CadAH245Q,W333H,E526N,K527S,G655K, pCDFDuet-Ptrc-CadAH245Q,W333E,E526N,K527S,G655K, pCDFDuet-Ptrc-CadAH245F,W333E,E526N,K527S,G655F, as well as the original control plasmid pCDFDuet-Ptrc-CadA into the *E. coli* NT1003. Following protein expression, a whole-cell catalytic assay was performed to detect lysine conversion. Regrettably, we found that all CadA mutants carrying 5 mutations had completely lost their enzymatic activity and were unable to consume the substrate lysine.

Learn

Through this round of cycles, we successfully constructed CadA mutants with 5 mutation sites; however, their enzymatic activity was completely lost. It is speculated that these mutation sites included key amino acid residues in the enzyme's active center or residues related to the binding of the cofactor PLP. It is likely due to structural alterations that disrupt the active site and/or cofactor binding. Therefore, in the next cycle, we chose to perform single-point mutations based on the sites provided by the in silico experiment group to identify the key amino acid residues .

Cycle 7 Engineering key enzyme CadA through single point mutation

Design

Since multi-site mutations resulted in a complete loss of enzymatic activity, we conducted a systematic single-site mutation analysis. Based on autodock result anlysis, the key sites (I182, H245, K246, W333, E526, K527 and Y652) were identified to assess their individual functional importance (Figure7-1).

![Figure 7-1 Interactions of key amino-acid residues](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure7-1.webp)

Figure 7-1 Interactions of key amino-acid residues

First, we performed alanine scanning mutagenesis on all predicted sites. Alanine (A) has a side chain consisting of only a simple methyl group, which can effectively eliminate the specific interactions (e.g., hydrogen bonds, hydrophobic interactions, charge interactions) of the original side chain while minimizing disturbances to the protein backbone conformation and overall structure. Through this step, we quickly screened out the key amino acid residues. Subsequently, the sites with modification potential were mutated into five amino acids of diverse physicochemical properties—glutamic acid (E), leucine (L), serine (S), phenylalanine (F), and valine (V) to explore their functional roles. Specifically: E (negatively charged, hydrophilic, large side chain) is used to probe charge interactions; L and V (hydrophobic, neutral, branched side chains with different sizes) are used to probe hydrophobic packing and spatial volume tolerance; F (hydrophobic, neutral, large-volume rigid aromatic ring) is used to probe π-π stacking interactions and sensitivity to large side chains; S (polar, hydrophilic, small side chain) is used to probe the ability to form hydrogen bonds and tolerance to small side chains.

Build

Based on the plasmid of pCDFDuet-Ptrc-CadA, we designed point mutation primers and cloned the whole plasmid using PCR. The linearized plasmid was isolated and purified by agarose gel electrophoresis, and then transferred into *E. coli* DH5α to obtain the targeted plasmids of pCDFDuet-Ptrc-CadAI182A, pCDFDuet-Ptrc-CadAH245A, pCDFDuet-Ptrc-CadAK246A, pCDFDuet-Ptrc-CadAW333A, pCDFDuet-Ptrc-CadAE526A, pCDFDuet-Ptrc-CadAK527A, pCDFDuet-Ptrc-CadAY652A.

Verified by sequencing, all the plasmids were successfully constructed.

For the potential modification sites identified after alanine scanning—I182, H245, and K246—we mutated each of these three sites into glutamic acid (E), leucine (L), serine (S), phenylalanine (F), and valine (V) respectively using the same method. Verified by sequencing, the construction of pCDFDuet-Ptrc-CadAK246V failed, whereas the remaining fourteen mutant plasmids were successfully constructed.

![Figure 7-2 Construction of single-point mutations. (A) Strategy diagram of single-point mutations. (B) The colony PCR validation diagram of alanine mutations. (C) The colony PCR validation diagram of mutations.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure7-2.webp)

Figure 7-2 Construction of single-point mutations. (A) Strategy diagram of single-point mutations. (B) The colony PCR validation diagram of alanine mutations. (C) The colony PCR validation diagram of mutations.

Test

The mutant plasmids and the unmutated original plasmid were separately transformed into the *E. coli* NT1003. After culturing for expression, a whole-cell catalytic assay was performed to determine the catalytic activity towards the substrate lysine. Under the condition of pH=7, the activity of the unmutated wild type (WT) was set as 100%, and the relative activities of other mutants are shown in Figure 7-3.

![Figure 7-3 Enzymatic activities of single-point mutations. (A) Enzymatic activities of alanine mutations. (B) Mutation results of I182, H245, and K246 Sites (WT activity set as 100%).](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure7-3.webp)

Figure 7-3 Enzymatic activities of single-point mutations. (A) Enzymatic activities of alanine mutations. (B) Mutation results of I182, H245, and K246 Sites (WT activity set as 100%).

Learn

In this cycle, we constructed alanine scanning at positions I182, H245, K246, W333, E526, K527, and Y652. It was found that the activity was almost completely lost after mutation at the W333, E526, K527, and Y652, indicating that these amino acids might be functionally critical residues directly related to substrate binding or the catalytic active site. It is conducive to further understanding the catalytic mechanism of the enzyme in subsequent studies.

We selected I182, H245, and K246 that exhibited less severe activity loss, for subsequent mutations to E, L, S, F, and V. Although their activity was not improved, this information will provide guidance for follow-up research. This avoids the massive screening and sequencing workload caused by full saturation mutagenesis, greatly improving research efficiency.

Faced with the challenges of these results, we did not become discouraged. Instead, we actively reflected, extensively reviewed literature, and took the initiative to conduct in-depth exchanges with experts. Through sufficient discussions and cross-fertilization of ideas, we gradually formed a new hypothesis: rather than directly modifying catalytic residues that are highly sensitive to mutations, could we indirectly improve catalytic performance by enhancing the overall structural stability of the enzyme? Consequently, the in silico experiment group began to attempt re-predicting potential beneficial mutations from the perspective of decamer stability.

Cycle 8 Engineering key enzyme CadA through stabilizing the structure for improvement of enzyme activity

Design

Apart from the aforementioned key amino acid residues, we shifted our modification strategy to enhancing the stability of CadA's decameric structure, as its decameric form is essential for efficiently exerting decarboxylation function. Through computer simulation analysis combined with literature reports, we predicted several pairs of double-site mutations (F14C/K44C, V12C/K41C, F14C/L45C, L93C/E445C, Y13C/P36C, E104K) or single-site mutations (E104K) that may stabilize the decameric structure by forming disulfide bonds or other interactions. We also referenced mutation pairs from the literature (V12C/K44C, F14C/D41C, L89C/E445C).

![Figure 8-1 The structure of CadA.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure8-1.webp)

Figure 8-1 The structure of CadA.

Build

Based on the prediction results, using the original CadA plasmid pCDFDuet-Ptrc-CadA as the template, site-directed mutagenesis primers were designed for the mutation sites. The gene fragments containing the mutation sites were amplified by PCR, and then ligated with the plasmid backbone DNA fragment via homologous recombination method, resulting in the mutant plasmids: pCDFDuet-Ptrc-CadAF14C/K44C, pCDFDuet-Ptrc-CadAV12C/K41C, pCDFDuet-Ptrc-CadAF14C/L45C, pCDFDuet-Ptrc-CadAL93C/E445C, pCDFDuet-Ptrc-CadAY13C/P36C, pCDFDuet-Ptrc-CadAE104K, pCDFDuet-Ptrc-CadAV12C/K44C, pCDFDuet-Ptrc-CadAF14C/D41C, and pCDFDuet-Ptrc-CadAL89C/E445C. These plasmids were transformed into competent *E. coli* DH5α. Positive clones were screened by colony PCR, and the successful construction was finally verified by sequencing.

![Figure 8-2 The colony PCR validation diagram.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure8-2.webp)

Figure 8-2 The colony PCR validation diagram.

Test

Following cultivation and induced expression, the mutated recombinant cells were harvested and subjected to whole-cell catalysis. Under the condition of pH=7 and 20 g/L lysine as the substrate, the consumption of lysine was detected. The activity of the wild type (WT) was set as 100%, and the relative activities of other mutants are shown in Figure 8-3. The Y13C/P36C mutant exhibited a maximum increase of 113.26% in enzymatic activity. Additionally, the enzymatic activities of V12C/D41C, F14C/D41C, and L93C/E445C mutants were also higher than that of the wild type (WT).

![Figure 8-3 Enzymatic activities of mutants modified for disulfide bond formation (WT activity set as 100%).](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure8-3.webp)

Figure 8-3 Enzymatic activities of mutants modified for disulfide bond formation (WT activity set as 100%).

Learn

Through this cycle, we learned that modifications such as Y13C/P36C, V12C/D41C, F14C/D41C, and L93C/E445C promote the formation of disulfide bonds, thereby stabilizing the structure of CadA. We also recognized that the higher-order structure of an enzyme is the material basis for its activity, and maintaining the stability of the structure is one of the effective strategies to improve enzymatic activity.

Module3: Cofactor--- Improvement of succinate

Cycle 9 Overexpression of GAP to promote the synthesis of succinate

Design

For our other co-produced product, succinate, based on the metabolic flux analysis, we chose to overexpress glyceraldehyde-3-phosphate dehydrogenase (GAP) to enhance the supply of reducing power. Succinate is a reductive product that requires the consumption of excess reducing power during its synthesis. To boost succinate synthesis, we screened four NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenases (GAP) originated from *Corynebacterium glutamicum* ATCC 13032 (CggapC), *Bacillus subtilis* (BsrocG), *Clostridium saccharobutylicum* DSM 13864 (CsgapC), and *Streptococcus mutans* (SmgapN). These enzymes catalyze the conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate and simultaneously generate NADPH, thereby increasing intracellular reducing power.

![Figure 9-1 The metabolic pathway catalyzed by glyceraldehyde-3-phosphate dehydrogenase.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure9-1.webp)

Figure 9-1 The metabolic pathway catalyzed by glyceraldehyde-3-phosphate dehydrogenase.

Build

The gene sequences of glyceraldehyde-3-phosphate dehydrogenases (CggapC from [BBa\_25RYKWX3](https://registry.igem.org/parts/bba-25rykwx3), CsgapC from [BBa\_25099LJK](https://registry.igem.org/parts/bba-25099ljk), and SmgapN from [BBa\_259YBJBJ](https://registry.igem.org/parts/bba-259ybjbj)) were synthesized by a company after codon optimization. The gene of BsrocG (from [BBa\_250WWUYW](https://registry.igem.org/parts/bba-250wwuyw)) was obtained by PCR amplification using the *Bacillus subtilis* genome as the template. The different gene fragments were cloned into the plasmid pCDFDuet under the control of the trc promoter via the In-Fusion cloning method, resulting in the plasmids pCDFDuet-Ptrc-CggapC, pCDFDuet-Ptrc-BsrocG, pCDFDuet-Ptrc-CsgapC, and pCDFDuet-Ptrc-SmgapN. These plasmids were transformed into *E. coli* NT1003 for expression.

![Figure 9-2 (A) The schematic of plasmid pCDFDuet-Ptrc-BsrocG. (B) The schematic of plasmid pCDFDuet-Ptrc-CggapC. (C) The schematic of plasmid pCDFDuet-Ptrc-CsgapC. (D) The schematic of plasmid pCDFDuet-Ptrc-SmgapN.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure9-2.webp)

Figure 9-2 (A) The schematic of plasmid pCDFDuet-Ptrc-BsrocG. (B) The schematic of plasmid pCDFDuet-Ptrc-CggapC. (C) The schematic of plasmid pCDFDuet-Ptrc-CsgapC. (D) The schematic of plasmid pCDFDuet-Ptrc-SmgapN.

![Figure 9-3 The colony PCR validation diagram. (A) BsrocG. (B) CggapC. (C) CsgapC. (D) SmgapN.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure9-3.webp)

Figure 9-3 The colony PCR validation diagram. (A) BsrocG. (B) CggapC. (C) CsgapC. (D) SmgapN.

Test

The function of NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase was investigated based on the succinate titer produced by *E. coli*, as quantified by HPLC. The results showed that the expression of CggapC could effectively increase the titer of succinate by 24.5%. However, we also found that it exerted an negative effect on lysine during the aerobic phase, with a 40% decrease compared with the control.

![Figure 9-4 (A) The effect of different glyceraldehyde-3-phosphate dehydrogenases on lysine. (B) The effect of different glyceraldehyde-3-phosphate dehydrogenases on succinate.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure9-4.webp)

Figure 9-4 (A) The effect of different glyceraldehyde-3-phosphate dehydrogenases on lysine. (B) The effect of different glyceraldehyde-3-phosphate dehydrogenases on succinate.

Learn

In this cycle, we learned that enzymes from different sources exhibit varying activities, which affect the titer of target products. We screened four NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenases from different organisms and found that the expression of CggapC achieved the optimal increase in succinate, but it simultaneously reduced lysine synthesis.

We reviewed literatures and discussed with other professors, who provided us with several suggestions. Dr. Xiaojie Guo recommended using a low-copy-number plasmid to mitigate the impact of excessive reducing power on cells and reduce cellular metabolic burden. He also showed strong interest in our modular design concept and suggested adjusting metabolic pathways to increase product titer.

Cycle 10 The coordination of lysine and succinate production by changing plasmid

Design

After consulting experts, we realized that using the pCDFDuet plasmid for expressing glyceraldehyde-3-phosphate dehydrogenase might not be the optimal choice. After in-depth internal team discussions, we decided to adopt the low-copy-number pACYCDuet plasmid to reduce the metabolic burden of the strain.

Build

Using pCDFDuet-Ptrc-CggapC, pCDFDuet-Ptrc-BsrocG, pCDFDuet-Ptrc-CsgapC, and pCDFDuet-Ptrc-SmgapN as templates, the CggapC, BsrocG, CsgapC, and SmgapN fragments were amplified by PCR. Each gene of CggapC, BsrocG, CsgapC, and SmgapN was separately inserted into the linearized pACYCDuet vector using In-Fusion cloning, titering four recombinant plasmids, pACYCDuet-CggapC, pACYCDuet-BsrocG, pACYCDuet-CsgapC, and pACYCDuet-SmgapN.

These plasmids were transformed into *E. coli* DH5α, and positive colonies were verified via PCR and sequencing. Subsequently, the plasmids with correct sequencing results were transformed into the lysine-producing strain *E. coli* NT1003, and direct fermentation was used to evaluate their impact on lysine and succinate production.

![Figure 10-1 (A) The schematic of plasmid pACYCDuet-BsrocG. (B) The schematic of plasmid pACYCDuet-CggapC. (C) The schematic of plasmid pACYCDuet-CsgapC. (D) The schematic of plasmid pACYCDuet-SmgapN.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure10-1.webp)

Figure 10-1 (A) The schematic of plasmid pACYCDuet-BsrocG. (B) The schematic of plasmid pACYCDuet-CggapC. (C) The schematic of plasmid pACYCDuet-CsgapC. (D) The schematic of plasmid pACYCDuet-SmgapN.

![Figure 10-2 The colony PCR validation diagram. (A) BsrocG. (B) CggapC. (C) CsgapC. (D) SmgapN.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure10-2.webp)

Figure 10-2 The colony PCR validation diagram. (A) BsrocG. (B) CggapC. (C) CsgapC. (D) SmgapN.

Test

The performance was evaluated by detecting lysine and succinate produced by recombinant *E. coli* via HPLC. The results showed that the expression of four dehydrogenase using a low-copy-number plasmid pACYCDuet,led to an increase in succinate. Among them, CggapC still exhibited the highest succinate titer, and no decrease in lysine titer (Figure 10-3). Therefore, the CggapC was selected in subsequent work.

![Figure 10-3 (A) The effect of different glyceraldehyde-3-phosphate dehydrogenases on lysine using a low-copy-number plasmid. (B) The effect of different glyceraldehyde-3-phosphate dehydrogenases on succinate using a low-copy-number plasmid.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure10-3.webp)

Figure 10-3 (A) The effect of different glyceraldehyde-3-phosphate dehydrogenases on lysine using a low-copy-number plasmid. (B) The effect of different glyceraldehyde-3-phosphate dehydrogenases on succinate using a low-copy-number plasmid.

Learn

In this cycle, we have learned how to optimize gene expression for the increased product titer. Due to the difference in the replicon, different plasmid has different copy number in the strain, which would affect the gene expression level. Therefore, the selection of appropriate plasmid is also an effective strategy for improving strain producing ability and maintaining activity.

Cycle 11 Constructing cofactor conversion to promote the synthesis of succinate and lysine

Design

Following Dr. Xiaojie Guo’s suggestion, we learned that certain transhydrogenases can also promote the rational distribution and flow of cofactors. Additionally, based on literature research, we ultimately selected the soluble pyridine nucleotide transhydrogenase (sthA) derived from *E. coli* MG1655.

![Figure 11-1 The metabolic pathway catalyzed by soluble pyridine nucleotide transhydrogenase (sthA).](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure11-1.webp)

Figure 11-1 The metabolic pathway catalyzed by soluble pyridine nucleotide transhydrogenase (sthA).

Build

The gene fragment of sthA was subcloned together with CggapC into linearized pACYCDuet, generating the plasmid pACYCDuet-CggapC-sthA. The plasmid was transformed into *E. coli* DH5α, and positive colonies were verified via PCR and sequencing analysis. Finally, the plasmid was transformed into the lysine-producing strain *E. coli*NT1003, and direct fermentation was used to evaluate its impact on lysine and succinate production.

![Figure 11-2 (A) The schematic of plasmid pACYCDuet-CggapC-sthA. (B) The colony PCR validation diagram.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure11-2.webp)

Figure 11-2 (A) The schematic of plasmid pACYCDuet-CggapC-sthA. (B) The colony PCR validation diagram.

Test

Its performance was evaluated by detecting lysine and succinate produced by *E. coli* via HPLC. The results showed that the succinate titer of the strain harboring pACYCDuet-CggapC-sthA increased by 28% relative to control; meanwhile, the lysine titer reached 14.9 g/L. We decided to use this strain in subsequent experiments.

![Figure 11-3 (A) Lysine production of the strain harboring pACYCDuet-CggapC-sthA. (B) Succinate production of the strain harboring pACYCDuet-CggapC-sthA.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure11-3.webp)

Figure 11-3 (A) Lysine production of the strain harboring pACYCDuet-CggapC-sthA. (B) Succinate production of the strain harboring pACYCDuet-CggapC-sthA.

Learn

In this cycle, we recognized the importance of constructing cofactor conversion. The demand for succinate synthesis is NADH. We previously focused mainly on enhancing the overall reducing power by GAP exression. After following Dr. Xiaojie Guo’s suggestion, we introduced soluble pyridine nucleotide transhydrogenase (sthA) to promote the rational distribution and flow of cofactors. As a result, the lysine titer increased to 14.9 g/L, and the succinate titer rose from 5.0 g/L to 6.4 g/L, achieving a further improvement in production. This also provides reference value for the work of other teams.

Module 4: An intelligent cell---Production of cadaverine-succinate

Cycle 12 Construction of a cell factory for synchronized synthesis of cadaverine-succinate

Design

Through preliminary experiments, we optimized the thermo-responsive switch to control the expression of *cadA* and the cofactor system. Subsequently, we introduced two plasmids into *E. coli* NT1003 to construct a cell factory capable of efficiently co-producing cadaverine and succinate..

Build

The plasmids pET28a(I7)-PJ23106-RBS-TlpA39-PtlpA-U7-CadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U8-CadA were co-transformed with the plasmid pACYCDuet-CggapC-sthA into the lysine-producing strain *E. coli* NT1003 to construct a cell factory capable of co-producing cadaverine and succinate.

![Figure 12-1 Construction of a cell factory for synchronized synthesis of cadaverine-succinate.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure12-1.webp)

Figure 12-1 Construction of a cell factory for synchronized synthesis of cadaverine-succinate.

Test

The performance was assessed by detecting cadaverine and succinate via HPLC. According to the fermentation results, the recombinant strain successfully achieved the co-synthesis of cadaverine and succinate, with the highest titers reaching 5.7 g/L and 5.4 g/L, respectively. This indicates that the designed synthetic pathway is feasible in experiments, and a cell factory for the synchronized synthesis of cadaverine and succinate has been successfully developed.

![Figure 12-2 Co-production of cadaverine and succinate in the cell factory.](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure12-2.webp)

Figure 12-2 Co-production of cadaverine and succinate in the cell factory.

Learn

In this cycle, we constructed a cell factory for the synchronized synthesis of cadaverine and succinate. Through precise metabolic flux regulation, the molar ratio of the two products is close to the ideal 1:1. It creates conditions for the direct generation of PA54 salt in the subsequent acid-base neutralization, reducing the demand for acids or bases, and minimizing costs. Secondly, from the perspective of carbon metabolism, the matching of the titers of the two products indicates a good balance between CO2 release and fixation, improving carbon atom economy and laying a robust foundation for the efficient and green production of the PA54.

Summary

The overall goal of the project was to develop an intelligent cell factory for the simultaneous biosynthesis of cadaverine and succinate—key monomers for bio-based PA54—using synthetic biology strategies. We designed and constructed a temperature-sensitive switch to enable lysine accumulation during the aerobic phase at low temperature and its efficient conversion to cadaverine under anaerobic conditions at high temperature, while simultaneously producing succinate.

To enhance the precision of the temperature switch, we optimized both transcriptional and translational controls, increasing lysine accumulation by **2.3-fold**. We also engineered the key enzyme CadA through rational design, obtaining a mutant with **113% higher** catalytic activity than WT. Furthermore, by introducing NADP⁺-dependent GAP dehydrogenase and balancing cofactors via soluble transhydrogenase expression, the results showed that the succinate titer increased by **28%** relative to control; meanwhile, the lysine titer reached **14.9 g/L**.

By integrating the temperature switch, optimized CadA, and cofactor modules, we successfully constructed an intelligent *E. coli* cell factory capable of co-producing **5.7 g/L cadaverine** and **5.4 g/L succinate** from glucose, laying a solid foundation for the green and low-carbon production of PA54.

![Figure13](https://static.igem.wiki/teams/5881/pages/pages/engineering/figure13.webp)

References

[1] Tong T, Chen X, Tang K, et al. A new-to-nature photosynthesis system enhances utilization of one-carbon substrates in *Escherichia coli*. Nat Commun, 2025, 16, 145.

[2] Tang NC, Su JC, Shmidov Y, et al. Synthetic intrinsically disordered protein fusion tags that enhance protein solubility. Nat Commun, 2024, 15, 3727.

[3] Acosta S, Rodríguez-Alonso P, Chaskovska V, et al. Spontaneous self-organized order emerging from intrinsically disordered protein polymers. WIREs Nanomed Nanobi, 2025, 17, e70003.

[4] Gao T, Niu L, Wu X, et al. Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models. Cell Rep Med, 2024, 5(5), 101513.

[5] Chen F, Fang H, Zhao J, et al. Multivariate modular metabolic engineering and medium optimization for vitamin B12 production by *Escherichia coli*. Synth Syst Biotechnol, 2024, 9(3), 453-461.

[6] Juliane N, Daniel K, Ralph B. Design of simple synthetic RNA thermometers for temperature-controlled gene expression in *Escherichia coli*. Nucleic Acids Res, 2008, 36(19), e124.

[7] Johanna R, Franz N. Exploring the modular nature of riboswitches and RNA thermometers. Nucleic Acids Res, 2016, 44(11), 5410-5423.

[8] Gao S, Zhang A, Ma D, et al. Enhancing pH stability of lysine decarboxylase via rational engineering and its application in cadaverine industrial production. Biochem Eng J, 2022, 186, 108548.

[9] Hong EY, Lee SG, Park BJ, et al. Simultaneously enhancing the stability and catalytic activity of multimeric lysine decarboxylase CadA by engineering interface regions for enzymatic production of cadaverine at high concentration of lysine. Biotechnol J, 2017, 12, 1700278.

[10] Kou F, Zhao J, Liu J, et al. Enhancement of the thermal and alkaline pH stability of *Escherichia coli* lysine decarboxylase for efficient cadaverine production. Biotechnol Lett, 2018, 40(4), 719-727.

[11] Xi Y, Ye LD, Yu HW, et al. Enhanced thermal and alkaline stability of L-lysine decarboxylase CadA by combining directed evolution and computation-guided virtual screening. Bioresour Bioprocess, 2022, 9, 24.

[12] Dong J, Kan B, Liu H, et al. CRISPR-Cpf1-assisted engineering of *Corynebacterium glutamicum* SNK118 for enhanced L-ornithine production by NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and NADH-dependent glutamate dehydrogenase. Appl Biochem Biotechnol, 2020, 191, 955–967.

[13] Ben-David J, Chipman DA. Mutual regulatory interactions of the trunk gap genes during blastoderm patterning in the hemipteran *Oncopeltus* fasciatus. Dev Biol, 2010, 346(1), 140-149.

[14] Gu ZY, Tang YS, Chen XL. Research progress on metabolic engineering modification of yeast for succinic acid production. Acta Microbiol Sin, 2025, 65(2), 467-488.

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

![Results](https://static.igem.wiki/teams/5881/pages/pages/header/results.webp)
![Bottle](https://static.igem.wiki/teams/5881/pages/pages/header/bottle.webp)

#####

* [1.Constructing a switch genetic circuit to dynamically regulate CadA for controllable synthesis of cadaverine](#switch-circuit)
* [2.The optimization of the temperature-sensitive switch using different promoters](#temperature-optimization)
* [3.Building a dual switch composed of temperature-sensitive switch and RNA thermometer to precise control cadaverine synthesis](#dual-switch)
* [4.The engineering CadA based on AI-guided design and rational design](#cada-engineering)
* [5.Enhancing the synthesis of succinate by cofactor regulation](#cofactor-regulation)
* [6.A cell factory for synchronized synthesis of cadaverine-succinate](#cell-factory)

1.Constructing a switch genetic circuit to dynamically regulate CadA for controllable synthesis of cadaverine

1.1 Initial Temperature-sensitive Switch

To achieve dynamic regulation of the cadaverine synthesis pathway, we constructed a Temperature-responsive system. The temperature-responsive repressor protein TlpA39 is expressed under the control of the constitutive promoter J23100. At temperatures below 39°C, TlpA39 forms a dimer that can bind to the promoter PtlpA, thereby repressing the expression of downstream genes; at temperatures above 39°C, TlpA39 exists as a monomer, loses the ability to bind to PtlpA, and thus activates the expression of downstream genes.

We placed the reporter gene of mWasabi (a green fluorescent protein) and the lysine decarboxylase gene *cadA* downstream of the PtlpA promoter, respectively. The recombinant plasmids pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-*mWasabi* and pET28a(ΔI7)-PJ23100-RBS-TlpA39-PtlpA-CadA were respectively transformed into *Escherichia coli* NT1003. The two engineered strains were cultured aerobically at 33, 35, 37, 39, and 42°C, respectively. By determining fluorescence intensity, the mWasabi reporter strain showed low fluorescence at ≤37°C but strong fluorescence at ≥39°C (Figure 1-1A). For the CadA group, lysine accumulated to 4.27 g/L, 2.6 g/L, and 2.4 g/L at 33°C, 35°C, and 37°C, respectively (Figure 1-1 B, C, D), but was not detected at 39°C and 42°C (Figure 1-1 E, F). The results indicated the temperature-sensitive switch operated effectively. Low fluorescence with lysine accumulation at ≤37°C confirmed the "OFF" state, while high fluorescence with no lysine accumulation at ≥39°C confirmed the "ON" state. It defined a clear activation threshold near 39°C, successfully enabling the induction of downstream gene through a simple temperature shift.

![Figure 1-1(A) Fluorescence detection diagram; Glucose consumption and lysine accumulation of the engineered E. coli strain. (B) CadA (33°C). (C) CadA (35°C). (D) CadA (37°C). (E) CadA (39°C). (F) CadA (42°C).](https://static.igem.wiki/teams/5881/pages/pages/results/figure1-1-1.webp)

Figure 1-1(A) Fluorescence detection diagram; Glucose consumption and lysine accumulation of the engineered *E. coli* strain. (B) CadA (33°C). (C) CadA (35°C). (D) CadA (37°C). (E) CadA (39°C). (F) CadA (42°C).

1.2 Protein Encapsulation

To achieve dynamic regulation of lysine decarboxylase (CadA), we attempted to construct a protein encapsulation system based on artificial intrinsically disordered proteins (A-IDPs). We designed A-IDPs of different sizes according to literature, including 0.5A-IDP, 1.0A-IDP, and 2.0A-IDP, and inserted TEV/TVMV cleavage sites between CadA and A-IDPs to achieve controllable protein release. To investigated the encapsulation function in *E. coli* NT1003, we applied this encapsulation system to mWasabi (a green fluorescent protein) and lysine decarboxylase, respectively.

![Figure 1-2 (A) Glucose consumption and lysine accumulation of the engineered E. coli (CadA-0.5A-IDP) and (CadA-1.0A-IDP). (B)Fluorescence intensity of the engineered E. coli (mWasabi-0.5A-IDP) and (mWasabi-1.0A-IDP).](https://static.igem.wiki/teams/5881/pages/pages/results/figure1-2-1.webp)

Figure 1-2 (A) Glucose consumption and lysine accumulation of the engineered *E. coli* (CadA-0.5A-IDP) and (CadA-1.0A-IDP). (B)Fluorescence intensity of the engineered *E. coli* (*mWasabi*-0.5A-IDP) and (*mWasabi*-1.0A-IDP).

![Figure 1-3 Glucose consumption and lysine accumulation in the 2.0A-IDP group.](https://static.igem.wiki/teams/5881/pages/pages/results/figure1-3.webp)

Figure 1-3 Glucose consumption and lysine accumulation in the 2.0A-IDP group.

Although the fluorescence results indicated that the 1.0A-IDP group was lower intensity than the 0.5A-IDP group, both A-IDP groups did not accumulate lysine (Figure 1-2). The current protein encapsulation system failed to prevent lysine consumption by CadA in both 0.5A-IDP and 1.0A-IDP, suggesting insufficient enzyme encapsulation. Further increasing the size to 2.0A-IDP, lysine still did not accumulate (Figure 1-3).

Thus, it suggested that 2.0A-IDP failed to perfectly encapsulate the CadA protein to block its activity during the aerobic phase. Therefore, we decided to adopt the temperature-sensitive switch in subsequent experiments.

2.The optimization of the temperature-sensitive switch using different promoters

To optimize the stringency and sensitivity of the temperature-sensitive switch for more accurate control of CadA expression, promoters with different strengths (PJ23100, PJ23106, and PJ23119) were used to regulate the expression of TlpA39. The three engineered strains harboring different promoters were subjected to aerobic culture at 33°C for 23 h, then shifted to anaerobic culture at 42°C. The titers of lysine and glucose during the aerobic and anaerobic phases were measured (Figure 2-1). Both PJ23106 and PJ23119 led to more lysine accumulation compared with PJ23100.Among them, PJ23106 showed the most significant effect, accumulating 5.1 g/L during the aerobic phase. Subsequently, during the high-temperature anaerobic phase, lysine was gradually completely consumed..

This demonstrated that replacing the promoter with PJ23106 could effectively reduce CadA leaky expression, and that anaerobic high temperature allows normal expression of CadA to catalyze lysine consumption.

![Figure 2-1 (A) Schematic Diagram; Glucose consumption and lysine accumulation of the engineered strain E. coli. (B) PJ23100-CadA (33°C). (C) PJ23106-CadA. (33°C) (D) PJ23119-CadA (33°C).](https://static.igem.wiki/teams/5881/pages/pages/results/figure2-1.webp)

Figure 2-1 (A) Schematic Diagram; Glucose consumption and lysine accumulation of the engineered strain *E. coli*. (B) PJ23100-CadA (33°C). (C) PJ23106-CadA. (33°C) (D) PJ23119-CadA (33°C).

3.Building a dual switch composed of temperature-sensitive switch and RNA thermometer to precise control cadaverine synthesis

RNA thermometers U7 and U8 can cause RBS (ribosome binding site) and a segment of sequence to form a stem-loop structure under low-temperature conditions, preventing ribosomes from binding normally to RBS and thus inhibiting the translation of downstream proteins. At high temperatures, they unlock the stem-loop structure, allowing normal translation of downstream proteins. Therefore, we designed to link them into the PJ23106-RBS-TlpA39-PtlpA-CadA construct as a translational-level regulation. The original temperature-sensitive switch, combined with RNA thermometer, formed a dual switch that is controlled at transcriptional and translational levels via temperature.

The different recombinant plasmids pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U7-CadA and pET28a(ΔI7)-PJ23106-RBS-TlpA39-PtlpA-U8-CadA were separately transformed into *E. coli* NT1003 to evaluate the effect of RNA thermometers. The strains were aerobically cultured at 33°C, 35°C, and 37°C for 23 hours, then transferred to an anaerobic environment and cultured at an elevated temperature of 42°C. As shown in Figures 3-1, the engineered *E. coli* with the dual-switch exhibited higher lysine accumulation at temperatures of 33°C, 35°C, and 37°C. It indicated that lysine was hardly consumed during the aerobic stage. Moreover, after increasing the temperature, lysine began to be gradually consumed, producing cadaverine simultaneously conducting anaerobic fermentation to synthesize succinate. These demonstrated that the dual switchs inserting RNA thermometers can achieve more precise temperature-responsive regulation. In addition, it has been initially proven that we can achieve the co-production of cadaverine and succinate through its regulation.

![Figure 3-1 Glucose consumption and lysine accumulation of the engineered strain E. coli. (A) PJ23106-U7-CadA (33°C). (B) PJ23106 -U7-CadA (35°C). (C) PJ23106 -U7-CadA (37°C). (D) PJ23106 -U8-CadA (33°C). (E) PJ23106 -U8-CadA (35°C). (F) PJ23106-U8-CadA (37°C).](https://static.igem.wiki/teams/5881/pages/pages/results/figure3-1-1.webp)

Figure 3-1 Glucose consumption and lysine accumulation of the engineered strain *E. coli*. (A) PJ23106-U7-CadA (33°C). (B) PJ23106 -U7-CadA (35°C). (C) PJ23106 -U7-CadA (37°C). (D) PJ23106 -U8-CadA (33°C). (E) PJ23106 -U8-CadA (35°C). (F) PJ23106-U8-CadA (37°C).

![Figure 3-2 Products synthesis and glucose consumption of aerobic and anaerobic stages. (A) PJ23106-U7-CadA. (B) PJ23106-U8-CadA.](https://static.igem.wiki/teams/5881/pages/pages/results/figure3-2.webp)

Figure 3-2 Products synthesis and glucose consumption of aerobic and anaerobic stages. (A) PJ23106-U7-CadA. (B) PJ23106-U8-CadA.

4.The engineering CadA based on AI-guided design and rational design

Lysine decarboxylase (CadA) is a key enzyme for cadaverine synthesis. To improve its catalytic efficiency, we performed multiple rounds of engineering modification on CadA.

4.1 AI-guided design of CadA via multi-site combinatorial mutagenesis

We first employed a computationally guided strategy to perform molecular docking simulations using the structure of CadA (PDB No. 6YN6). Hydrogen bonds, salt bridges, and hydrophobic interactions between the enzyme and its substrate were systematically analyzed. Based on the interaction analysis, we identified some residues located near the active pocket that might make key contributions to substrate binding and catalysis: I182, H245, K246, W333, E526, K527 and Y652. These candidate sites were subsequently evaluated using a site-directed mutagenesis prediction model for virtual amino acid substitutions, and their catalytic efficiencies (Kcat) were predicted. The model recommended several pentuple mutants with predicted high enzymatic activity (Table1).

We successfully constructed plasmids encoding the five predicted quintuple mutants. However, whole-cell catalytic experiments showed that all of these mutants completely lost enzymatic activity, with no detectable consumption of the substrate lysine. It was likely due to structural alterations that disrupt the active site and/or cofactor binding.

Table 1 Prediction of enzymatic activity for combined mutations

| Mutation type | Enzymatic activity Kcat(s⁻¹) |
| --- | --- |
| Wild type | 3.484 |
| H245Q, W333E, E526N, K527S, G655F | 13.6055 |
| H245Q, W333H, E526N, K527S, G655F | 13.6008 |
| H245Q, W333H, E526N, K527S, G655K | 13.4973 |
| H245Q, W333E, E526N, K527S, G655K | 13.4772 |
| H245F, W333E, E526N, K527S, G655F | 13.2887 |

4.2 Analysis of key residues in CadA based on single-site mutations

Since multi-site mutations resulted in a complete loss of enzymatic activity, we conducted a systematic single-site mutation analysis. Based on autodock result anlysis, the key sites (I182, H245, K246, W333, E526, K527 and Y652) were identified to assess their individual functional importance(Figure4-1). We first performed alanine-scanning, followed by further substitutions at positions that exhibited less severe activity loss (I182, H245, K246) with amino acids of diverse physicochemical properties (E, L, S, F, V) to explore their functional roles.

![Figure 4-1 Interactions of key amino-acid residues](https://static.igem.wiki/teams/5881/pages/pages/results/figure4-1.webp)

Figure 4-1 Interactions of key amino-acid residues

We successfully constructed plasmids for most of the single-site mutants. Enzymatic activity assay results showed that mutations W333A, E526A, K527A, and Y652A led to an almost complete loss of enzymatic activity, indicating that these residues are directly involved in substrate binding or the catalytic active site, and are functionally essential (Figure 4-2(A)). In contrast, alanine substitutions and other mutations at positions I182, H245, and K246 did not significantly enhance activity, but several mutants retained partial activity. These findings provide important clues for the catalytic mechanism of CadA and the functions of its residues (Figure 4-2(B)).

![Figure 4-2 Enzymatic activities of single-point mutations. (A) enzymatic activities of alanine mutations. (B) mutation results of I182, H245, and K246 sites (WT activity set as 100%).](https://static.igem.wiki/teams/5881/pages/pages/results/figure4-2.webp)

Figure 4-2 Enzymatic activities of single-point mutations. (A) enzymatic activities of alanine mutations. (B) mutation results of I182, H245, and K246 sites (WT activity set as 100%).

4.3 Enhancing CadA enzymatic activity by stabilizing the decameric structure

Apart from the aforementioned key amino acid residues, we shifted our modification strategy to enhancing the stability of CadA's decameric structure, as its decameric form is essential for efficiently exerting decarboxylation function. Through computer simulation analysis combined with literature reports, we predicted several pairs of double-site mutations (F14C/K44C, V12C/K41C, F14C/L45C, L93C/E445C, Y13C/P36C, E104K) or single-site mutations (E104K) that may stabilize the decameric structure by forming disulfide bonds or other interactions. We also referenced mutation pairs from the literature (V12C/K44C, F14C/D41C, L89C/E445C).

Whole-cell catalytic experiments showed that multiple mutants exhibited higher enzymatic activity than WT. Among them, the mutant Y13C/P36C showed the most significant effect, with its relative enzymatic activity increasing by approximately 113.26% compared with the wild-type. In addition, the enzymatic activities of mutants V12C/D41C, F14C/D41C, and L93C/E445C also increased to varying degrees compared with the wild-type. These results indicate that stabilizing the decameric structure of CadA through strategies such as introducing disulfide bonds is an effective approach to improve its catalytic efficiency (Figure4-3).

![Figure 4-3 Enzymatic activities of mutants modified for disulfide bond formation (WT activity set as 100%).](https://static.igem.wiki/teams/5881/pages/pages/results/figure4-3.webp)

Figure 4-3 Enzymatic activities of mutants modified for disulfide bond formation (WT activity set as 100%).

4.4 Iterative mutations based on the optimal mutant Y13C/P36C

To further enhance enzymatic activity, we aimed to perform iterative mutations. First, we selected the two best-performing mutants from the previous round, Y13C/P36C and L93C/E445C, for iteration, constructing the mutant Y13C/P36C/L93C/E445C. Considering that multiple disulfide bonds might lead to excessively poor structural flexibility, we speculated a combination of one set of disulfide bonds and a single-site mutation more favorable. Since our current experiments did not screen out single-site mutations with improved enzymatic activity, we selected literature-reported mutations E445Q and K477R, constructing the mutants Y13C/P36C/E445Q and Y13C/P36C/K477R.

Whole-cell catalytic experiments showed that the effect of iterative mutations was not ideal. The mutant Y13C/P36C/E445Q exhibited a significant decrease in enzymatic activity, and the enzymatic activities of the other mutants were also significantly lower than that of the best mutant Y13C/P36C (Figure 4-4).

![Figure 4-4 Enzymatic activity of iterative mutations (WT activity set as 100%).](https://static.igem.wiki/teams/5881/pages/pages/results/figure4-4.webp)

Figure 4-4 Enzymatic activity of iterative mutations (WT activity set as 100%).

4.5 Characterization of the optimal mutant Y13C/P36C

To comprehensively evaluate the catalytic performance of the Y13C/P36C mutant, we compared its catalytic efficiency with that of the wild-type under different substrate concentrations (20 g/L, 50 g/L, 100 g/L, 200 g/L) and different pH conditions (6, 7, 8, 9).

The results showed that CadA(Y13C/P36C) exhibited superior catalytic performance to the wild-type under all tested conditions, with the advantage being more pronounced especially at higher substrate concentrations. In contrast to the wild-type, Y13C/P36C mutant demonstrated superior lysine consumption kinetics across a substrate concentration of 20-200 g/L. More significantly, Y13C/P36C mutant maintained persistent catalytic activity at high substrate. This indicates that the mutant has better industrial application potential. These results indicate that CadA(Y13C/P36C) efficiently improved the substrate tolerance.

![Figure 4-5 Catalytic performance of Y13C/P36C and WT under different substrate concentrations. (A) 20 g/L. (B) 50 g/L.(C) 100 g/L. (D) 200 g/L.](https://static.igem.wiki/teams/5881/pages/pages/results/figure4-5.webp)

Figure 4-5 Catalytic performance of Y13C/P36C and WT under different substrate concentrations. (A) 20 g/L. (B) 50 g/L.(C) 100 g/L. (D) 200 g/L.

Under pH conditions from 6-9, Y13C/P36C mutant exhibited superior catalytic performance. Its relative activities were respectively 1.59, 2.01and 2.07-fold higher than that of WT at pH 6, 7 and 8. Furthermore, the relative activity of WT at pH 9 was 37.07 %, while the relative activities of Y13C/P36C mutant was 74.14% . This indicated that CadA(Y13C/P36C) efficiently improved the pH tolerance and has better application potential.

![Figure 4-6 Enzymatic activity of Y13C/P36C and WT under different pH conditions (WT activity at pH=6 set as 100%).](https://static.igem.wiki/teams/5881/pages/pages/results/figure4-6.webp)

Figure 4-6 Enzymatic activity of Y13C/P36C and WT under different pH conditions (WT activity at pH=6 set as 100%).

5.Enhancing the synthesis of succinate by cofactor regulation

5.1 Overexpression of GAP

For our other co-produced product, succinate, based on the metabolic flux analysis, we chose to overexpress glyceraldehyde-3-phosphate dehydrogenase (GAP) to enhance the supply of reducing power. Succinate is a reductive product that requires the consumption of excess reducing power during its synthesis. To boost succinate synthesis, we screened four NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenases (GAP) originated from *Corynebacterium glutamicum* ATCC 13032 (CggapC), *Bacillus subtilis* (BsrocG), *Clostridium saccharobutylicum* DSM 13864 (CsgapC), and *Streptococcus mutans* (SmgapN). These enzymes catalyze the conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate and simultaneously generate NADPH, thereby increasing intracellular reducing power.

![Figure 5-1 The metabolic pathway catalyzed by glyceraldehyde-3-phosphate dehydrogenase.](https://static.igem.wiki/teams/5881/pages/pages/results/figure5-1.webp)

Figure 5-1 The metabolic pathway catalyzed by glyceraldehyde-3-phosphate dehydrogenase.

The aforementioned strains were cultured in a fermentation medium supplemented with 20 g/L glucose, and the titers of lysine and succinate were measured. As shown in Figure 5-2 (B),the results showed that the expression of CggapC could effectively increase the titer of succinate by 24.5%. However, we also found that it exerted an negative effect on lysine during the aerobic phase, with a 40% decrease compared with the control. It might be due to excessive reducing power led to an imbalance in intracellular redox levels and imposed the metabolic burden on the cells.

![Figure 5-2 (A) The effect of different glyceraldehyde-3-phosphate dehydrogenases on lysine. (B) The effect of different glyceraldehyde-3-phosphate dehydrogenases on succinate.](https://static.igem.wiki/teams/5881/pages/pages/results/figure5-2.webp)

Figure 5-2 (A) The effect of different glyceraldehyde-3-phosphate dehydrogenases on lysine. (B) The effect of different glyceraldehyde-3-phosphate dehydrogenases on succinate.

5.2 The coordination of lysine and succinate production by changing plasmid

To mitigate the effects of excess reducing power on cellular metabolism and lysine accumulation during the aerobic growth phase, the expression of glyceraldehyde-3-phosphate dehydrogenase was further optimized using a low-copy-number plasmid pACYCDuet.

The results showed that the expression of four dehydrogenase using a low-copy-number plasmid led to an increase in succinate. Among them, CggapC still exhibited the highest succinate titer, and no decrease in lysine titer (Figure 5-3). Therefore, the CggapC was selected in subsequent work.

![Figure 5-3. (A) The effect of different glyceraldehyde-3-phosphate dehydrogenases on lysine using a low-copy-number plasmid. (B) The effect of different glyceraldehyde-3-phosphate dehydrogenases on succinate using a low-copy-number plasmid.](https://static.igem.wiki/teams/5881/pages/pages/results/figure5-3.webp)

Figure 5-3. (A) The effect of different glyceraldehyde-3-phosphate dehydrogenases on lysine using a low-copy-number plasmid. (B) The effect of different glyceraldehyde-3-phosphate dehydrogenases on succinate using a low-copy-number plasmid.

5.3 Constructing cofactor conversion

To further enhance succinate production and maintain cell vitality, cofactor distribution and flow were rationally engineered. For the NADH required in the succinate synthesis, the soluble pyridine nucleotide transhydrogenase (sthA) from *E. coli* MG1655 catalyzes the conversion of NADPH to NADH, which was selected to balance different types of cofactor. Co-expression of sthA with CggapC enabled optimization of the intracellular cofactor pool, thereby improving reducing power allocation. CggapC facilitated additional NADPH synthesis, thereby providing the necessary reducing power and then SthA catalyzed the conversion of NADPH to NADH for succinate production. The results showed that the succinate titer of the strain harboring pACYCDuet-CggapC-sthA increased by 28% relative to control; meanwhile, the lysine titer reached 14.9 g/L. These results indicated that proper regulation of cofactors could effectively enhance succinate synthesis.

![Figure 5-4 (A) Lysine production of the strain harboring pACYCDuet-CggapC-sthA. (B) Succinate production of the strain harboring pACYCDuet-CggapC-sthA.s](https://static.igem.wiki/teams/5881/pages/pages/results/figure5-4.webp)

Figure 5-4 (A) Lysine production of the strain harboring pACYCDuet-CggapC-sthA. (B) Succinate production of the strain harboring pACYCDuet-CggapC-sthA.

6.A cell factory for synchronized synthesis of cadaverine-succinate

Through preliminary experiments, we integrated the thermo-responsive switch to control the expression of CadA and the cofactor regulation system. A single-cell factory was constructed to simultaneously produce cadaverine and succinate. According to the fermentation results, the recombinant strain successfully achieved the co-synthesis of cadaverine and succinate, with the highest titer reaching 5.7 g/L and 5.4 g/L from glucose. It laid a robust foundation for the efficient and green production of the PA54.

![Figure 6-1 Co-production of cadaverine and succinate in the cell factory.](https://static.igem.wiki/teams/5881/pages/pages/results/figure6-1.webp)

Figure 6-1 Co-production of cadaverine and succinate in the cell factory.

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