Results

Module 1: Exploration of Pathway Feasibility

1. Molecular Docking Predicted Feasibility

Fig.1 Comparison of binding modes for the natural and non-natural substrates.

The left panel shows the docked pose of the natural substrate, chorismic acid (yellow sticks), in the active site. The right panel displays the docked pose of the non-natural substrate, vanillic acid (yellow sticks). Key interacting amino acid residues are highlighted (blue sticks). Potential hydrogen bonds are indicated by yellow dashed lines, with distances measured in angstroms (Å).

Molecular docking simulations predicted that vanillic acid (VA) could bind effectively to the active site of the PabAB enzyme, in a manner similar to its native substrate, chorismic acid (Figure 1). Therefore, we propose that VA could serve as a substrate to enter and complete the metabolic pathway leading to the synthesis of acetaminophen (AAP) (Figure 2).

Fig 2. Hypothetical Pathway from Vanillic Acid (VA) to Acetaminophen (AAP).

There are two steps of transformation, which are catalyzed by two enzymes, PabAB and PabC, respectively

2. Plasmid Construction

Fig 3. Plasmid pYB1a-pabABC.

pabA, pabB and pabC were constructed on the same plasmid to form a toolbox.

Fig 4. Colony PCR results of pYB1a-pabABC

The coding sequences of pabA, pabB, and pabC amplified by PCR were assembled into the pYB1a vector using Gibson Assembly. Subsequently, restriction digest analysis confirmed the correct assembly of the plasmid (Figure 4).

Fig 5. HPLC detection of fermentation results.

From top to bottom, liquid chromatography comparison of p-ABA standard, 0 h fermentation of pabABC engineered strain, and 12 h fermentation of pabABC engineered strain, respectively.

The plasmid was transformed into BW25113 strain. After 12 hours of fermentation with vanillic acid, HPLC analysis showed a distinct peak corresponding to the p-ABA standard, which was absent at 0 hours (Figure 5). This result demonstrates that the pabABC genes were successfully expressed and the two proteins it expresses are functional, confirming that the metabolic pathway from vanillic acid to p-ABA is feasible.

Fig 6. Line chart of p-ABA production within 48 hours of fermentation.

The yield of p-ABA continued to rise from 0 to 36 hours and decreased from 36 to 48 hours.

After 48 hours of fermentation, the p-ABA titer reached 1.13 mM (Figure 6), with a conversion rate of 22.6% from VA.

To improve the conversion rate, we optimized two key fermentation parameters: L-Glutamine concentration and fermentation medium.

Fig 7. Conditions are optimised: Line chart of p-ABA production over time with different concentrations of L-Gln.

L-glutamine can be used as a nitrogen source in the fermentation broth to support the growth of the bacteria and protein synthesis, and also has the effect of regulating pH to maintain the cell function and ensure the stability of fermentation.

We found that supplementing the medium with 15 mM L-glutamine (L-Gln) maximized production: the p-ABA titer reached 4.786 mM (Figure 7), with a conversion rate of 47.6%.

Fig 8. Optimisation of conditions: comparing p-ABA yield results of different fermentation broths.

The effect of M9 medium within 48 hours is significantly better than that of M9g.

We tried to change the fermentation broth to increase the yield, and the results showed that M9 medium yielded better, which provided guidance for our subsequent fermentation conditions (Figure 8).

Module 2: Enzyme Optimization and Bypass Optimization

Fig 9. Hypothetical Pathway from p-ABA to p-AP.

1. Plasmid Construction

Fig 10. Plasmid pYB1a-pabABC-MNX1

The MNX1 coding sequence was amplified by PCR and inserted into the linearized pYB1a-pabABC vector using the Gibson assembly method. Subsequently, colony PCR was performed to verify the presence of the insert (Figure 11).

Fig 11 .Colony PCR results of pYB1a-pabABC-MNX1

Fig 12. Plasmid pYB1a-pabABC-ABH60

We replaced the MNX1 gene on the original plasmid with ABH60 and speculated that it had a better catalytic effect.

Fig 13. Plasmid R6K-dCas9(sg-nhoA)

Construct was used to achieve the role of interfering with nhoA expression in this step, preventing the conversion of p-ABA to the bypass pathway.

Fig 14. Colony PCR Results of R6K-dCas9(sg-nhoA)

2. Efficiency comparison between ABH60 and MNX1

Fig 15.SDS-PAGE Verification of ABH60 Protein Expression.

The leftmost lane (Lane 1) shows the molecular weight marker. Induced ABH60 expression is shown in Lane 2 (supernatant) and Lane 4 (pellet), while uninduced ABH60 expression is shown in Lane 3 (supernatant) and Lane 5 (pellet). The rightmost lane (Lane 6) shows the molecular weight marker.

Fig 16.SDS-PAGE Verification of ABH60 and MNX1 Protein Expression.

The molecular weight marker is shown in the leftmost lane (Lane 1). Induced ABH60 expression is shown in Lane 2 (supernatant) and Lane 4 (pellet), while uninduced ABH60 is shown in Lane 3 (supernatant) and Lane 5 (pellet). Induced MNX1 expression is shown in Lane 6 (supernatant) and Lane 8 (pellet), and uninduced MNX1 is shown in Lane 7 (supernatant) and Lane 9 (pellet). The rightmost lane (Lane 10) shows the parental strain without plasmid.

In the protein expression analysis, both ABH60 and MNX1 displayed bands consistent with the expected sizes of the markers and were primarily present in the soluble supernatant. No corresponding bands were detected in the BW25113 parental strain (Figure 16). These results indicate that ABH60 and MNX1 proteins were successfully expressed in the BW25113 strain and mainly existed in a soluble form.

Fig 17.HPLC Analysis of p-AP Production after 48-Hour Fermentation in the Strain Harboring pYB1a-pabABC-ABH60.

From top to bottom, they are the p-AP standard, the sample after 48 hours of fermentation, and the sample after 0 hours of fermentation.

The HPLC results (Figure 17) show a characteristic peak matching the p-AP standard. This indicates that, after 48 hours of fermentation and ABH60 expression, the metabolic pathway successfully converted p-ABA into p-AP.

Fig 18.Comparison of p-AP Production after 48-Hour Fermentation between Strains Expressing ABH60 and MNX1 Proteins

The experimental results (Figure 18) show that under the same conditions, the strain expressing ABH60 produced p-AP with higher concentration compared to the strain expressing MNX1. This indicates that ABH60 exhibits higher efficiency in converting p-ABA to p-AP.

3. nhoA Gene Knockout Outperforms Knockdown for Eliminating Byproducts

Despite the improved enzyme, p-AP yield was limited by a competing pathway involving the native E. coli enzyme NhoA, converting p-ABA to the by-product paracetaminobenzoic acid.

Based on this finding, we designed and implemented two strategies: knocking out the nhoA gene and interfering with nhoA expression, in order to evaluate which approach more effectively suppresses the competing pathway and enhances p-AP production.

Fig19. Colony PCR Results of Donor-U500D500-nhoA

The D500U500 sequence was amplified by PCR and inserted into the linear Donor vector using the Gibson assembly method. Colony PCR was then performed to verify the insertion, and plasmids with successful D500U500 integration were selected. This resulted in the construction of the Donor-D500U500-nhoA plasmid containing the targeting fragment.

Fig 20. Colony PCR Results of nhoA Knockout (95% Positive Rate).

Primers were designed at 1000bp upstream and downstream of the knockout gene respectively. If the gene was successfully knocked out, a colony pcr result of 2000bp in size would be obtained.

We employed the CRISPR-Cas9 gene knockout method. The constructed plasmid was introduced into the BW25113 strain via electroporation, and the transformants were plated on selective media containing kanamycin and streptomycin. Finally, colony PCR verification (Figure 20) confirmed that the nhoA gene was successfully knocked out.

As shown by the electrophoresis bands in Figure 20, the nhoA gene was successfully knocked out.

Fig 21. Line Chart of p-AP Production over Time in the ABH60-Expressing nhoA Knockout Strain and the ABH60-Expressing nhoA Knockdown Strain

As shown in Figure 21, the nhoA knockout strategy outperformed the knockdown approach in enhancing the target product yield. The knockout strain reached a maximum p-AP titer of 4.8 mM, significantly higher than the knockdown strain, indicating that completely blocking the nhoA pathway is more effective in improving p-AP production.

Fig 22. Replicate experiments were performed to verify p-AP yield

Here, in order to ensure the credibility of the experimental conclusion, we conducted repeated experiments to detect the p-AP yields at 36 hours and 48 hours in the two engineered strains. This result shows that we were more efficient using the knockout strain than importing the plasmid used to interfere with nhoA expression.

Fig 23. Time-Course of p-AP Production in the nhoA Knockout Strain Expressing ABH60

In the engineered strain with the nhoA gene knocked out and ABH60 protein expressed, the p-AP production was significantly enhanced during fermentation. As fermentation progressed, the concentration of p-AP gradually increased, ultimately reaching a yield of 4.47 mM, with a vanillic acid conversion rate of 86% (Fig 23).

This result was substantially higher than that of the control strain expressing only ABH60 under identical conditions, indicating that the strategy of blocking competing pathways while introducing a highly efficient catalytic enzyme can effectively redirect metabolic flux and promote the accumulation of the target product. Moreover, it was observed that after 36 hours, p-AP production gradually plateaued, entering a stable phase (Fig 23).

Module 3: Design of Temperature-Sensitive Promoter and Enzyme Optimization for Final Product Synthesis

Fig 24. Hypothetical pathway from p-AP to Acetaminophen (AAP)

1. Testing of Temperature-Sensitive Promoter

Fig 25. Fluorescence Results of pSB1c-I38-mCherry Expression in BW25113

The pSB1c-I38-mCherry plasmid was constructed and transformed into E. coli BW25113. The strains were induced at 30°C and 37°C for 18 hours, and fluorescence intensity was measured using a microplate reader (excitation: 552 nm, emission: 600 nm). The results showed that the I38 promoter is highly temperature-sensitive (Fig 25).

2. Plasmid Construction

Fig 26. Plasmid pSB1c-I38-nhoA

Fig 27. Plasmid pSB1c-I38-PANAT

Fig 28. Colony PCR Results of Plasmid pSB1c-I38-PANAT

Fig 29. Our final AAP production is calculated based on this standard curve

Fig 30. 10-Minute HPLC Analysis after 48-Hour Fermentation of the Engineered Strain

Based on the HPLC chromatogram, comparison with the AAP standard shows that the engineered strain produced detectable amounts of AAP after 48 hours of fermentation, confirming our hypothesis.

Fig 31. Temporal profiles of AAP production after 96h when pSB1c-I38-nhoA and pSB1c-I38-PANAT were transfected into BW-ΔnhoA strain for fermentation, respectively

At 30°C, the temperature-sensitive promoter I38 remains inactive, during which the synthesis and accumulation of p-AP constitute the primary metabolic phase. At 37°C, the temperature-sensitive promoter is activated, leading to the conversion of the accumulated p-AP into the final product AAP (Fig 31). Our results demonstrate that induction of the temperature-sensitive promoter at 36 hours resulted in a gradual increase in AAP production, which reached its peak concentration of 0.78 mM at 72 hours (Fig 31).

Fig 32. Repeated experiments using the best engineered bacteria to confirm yields, 36h and 72h AAP yields

To confirm the final yield, we repeated the experiment with 3 sets of the same optimal engineered bacteria (E. coli with knockout nhoA containing pabABC, ABH60, and PANAT genes) fermented at the same time, and samples were taken at 36h as well as at 72h for HPLC.

The results demonstrated that our optimised bacteria were able to synthesise AAP from VA and achieved a yield of 1.17mM AAP at 72h (Fig. 32), with a conversion rate of 23.4%.

New Composite Part

Fig 33. Schematic diagram of the mechanism of action of the pabABC toolbox

Our project's primary innovation is the development of a novel biosynthetic route utilizing vanillic acid, a lignin derivative, while preserving its core aromatic structure. This was enabled by our new composite part combining pabAB gene and pabC gene. This part successfully catalyzes the efficient conversion of VA to p-ABA, achieving a 47.6% conversion rate for this module.

At the same time, we identified and overcame two key bottlenecks by replacing underperforming enzymes. The exogenous hydroxylase MNX1 was substituted with the far more efficient ABH60, while the endogenous E. coli enzyme NhoA was replaced by the acetyltransferase PANAT. In temperature control Under the premise of the system, with the joint action of two key enzymes, we finally obtained 5.0mM vanillanic acid that can produce 1.17mM of AAP, with a conversion rate of 23.4%.

We believe our PabABC part provides a useful new tool for the iGEM community, enabling future teams to easily use vanillic acid as an alternative to chorismic acid for synthesizing valuable aromatic compounds.