iGEM 2025 — Team nefu-china

# Notebook

Part 1 From Vanillic Acid to p-ABA

Part 2 From p-ABA to p-AP

Part 3 From p-AP to AAP

## Part 1 From Vanillic Acid to p-ABA

Week1 | 4.1-4.7

Validation of the hypothesis: vanillic acid and chorismic acid can undergo the same reaction to produce p-ABA. This week, we first constructed the plasmid pYB1a-pabABC.

![](https://static.igem.wiki/teams/5974/notebook/notebook-1.webp)

Week2| 4.8-4.14

Calibration of the standard curve for vanillic acid and p-ABA. The standard curve for AAP was established using standard solutions of vanillic acid and p-ABA at concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, and 1.

![](https://static.igem.wiki/teams/5974/notebook/notebook-3.webp)
![](https://static.igem.wiki/teams/5974/notebook/notebook-4.webp)

The pYB1a-pabABC plasmid was introduced into the BW25113 strain. Fermentation was conducted with the BW25113 strain into which the pYB1a-pabABC plasmid had been induced, and samples were taken at 0h and 12h to detect the production of p-ABA using High-Performance Liquid Chromatography (HPLC).

![](https://static.igem.wiki/teams/5974/notebook/notebook-5.webp)

Week3 | 4.15-4.21

Bypass optimisation: Construct and introduce the R6K-dCas9(sg-nhoA) plasmid to interfere with nhoA expression. Colony PCR results of R6K-dCas9 (sg-nhoA) plasmid.

![](https://static.igem.wiki/teams/5974/notebook/notebook-6.webp)

Week4 | 4.22-4.29

Conducting HPLC detection of the interference and non-interference of the *nhoA* group p-ABA yield. One group involved the single transformation of pYB1a-pabABC into BW25113, while another group consisted of the dual transformation of pYB1a-pabABC and R6K-dCas9(sg-nhoA) into BW25113. Samples were taken at set intervals of 0h, 6h, 12h, 18h, and 24h from 1.5mL centrifuge tubes, 10mL centrifuge tubes, and 100mL conical flasks (OD50) and OD6 to detect p-ABA yield using HPLC.

![](https://static.igem.wiki/teams/5974/notebook/notebook-7.webp)

Week5 | 4.29-5.5

Bypass optimisation: Knocking out the *nhoA* gene. This week, we first constructed the Donor-U500D500-nhoA and pTarget-nhoA plasmids.

![](https://static.igem.wiki/teams/5974/notebook/notebook-8.webp)

Week6| 5.6-5.13

Obtain the target fragment via PCR and electroporate it along with the pTarget-nhoA plasmid into the BW strain. Incubate at 37°C for 12 hours. Perform colony PCR to validate if the knockout was successful.

![](https://static.igem.wiki/teams/5974/notebook/notebook-9.webp)

Positive rate 95%

Week7 | 5.14-5.21

Introduce the pYB1a-pabABC plasmid into the *nhoA* knockout strain BW25113 and measure the concentration of the intermediate product p-ABA at different fermentation times.

![](https://static.igem.wiki/teams/5974/notebook/notebook-10.webp)

Week8 | 5.22-5.29

Optimisation of fermentation conditions:

1. Glutamine concentration gradient

2. Comparison of M9 fermentation broth and M9g fermentation broth

Week9 | 5.30-6.6

1.Glutamine concentration gradient

![](https://static.igem.wiki/teams/5974/notebook/notebook-11.webp)

2.Comparison of M9 fermentation broth and M9g fermentation broth

![](https://static.igem.wiki/teams/5974/notebook/notebook-12.webp)

## Part 2 From p-ABA to p-AP

Week10 | 6.7-6.14

Verification of hypotheses: validating the enzymatic activity of MNX1 and optimizing the enzymatic activity efficiency of ABH60.

This week, we first constructed the plasmids pYB1a-pabABC-MNX1 and pYB1a-pabABC-ABH60.

![](https://static.igem.wiki/teams/5974/notebook/notebook-13.webp)

Week11 | 6.15-6.22

We constructed plasmids pYB1a-pabABC-ABH60 and pYB1a-pabABC-MNX1 and separately introduced them into the engineered strain BW-ΔnhoA. After shaking the cultures for 12 hours, we took 1 ml of the culture and divided it into two groups: one prior to induction and one after induction. For the post-induction group, we added arabinose to induce the expression of *pabABC* and *ABH60* genes. After incubating at 30°C for 16 hours, we took 6 OD of the culture, disrupted it by sonication, and centrifuged it at 10,000 rpm for 5 minutes. We then collected the supernatants from both the pre-induction and post-induction groups, as well as the precipitates from both groups, and added an equal volume of loading buffer for SDS-PAGE experiments.

We constructed plasmids pYB1a-pabABC-ABH60, pYB1a-pabABC-MNX1 and introduced the engineered bacteria BW-ΔnhoA respectively. After 12 hours, 1 ml bacterial suspension was divided into two groups: pre-induction group and post-induction group. After induction, Arabinose was added to induce the expression of *pabABC* and *ABH60* genes. After 16 h at 30 °C, 6 OD bacterial suspensions were taken respectively, and centrifuged for 5 min at 10000 rpm after ultrasonic disruption. The supernatants were taken before induction, and after induction, the supernatants were precipitated before induction.

![](https://static.igem.wiki/teams/5974/notebook/notebook-14.webp)
![](https://static.igem.wiki/teams/5974/notebook/notebook-15.webp)

Week12|6.23-6.30

Transfer the pYB1a-pabABC-MNX1 and pYB1a-pabABC-ABH60 plasmids into BW-ΔnhoA. Sample at 12, 24, 36, and 48 hours during fermentation and use HPLC to detect the p-AP yield.

![](https://static.igem.wiki/teams/5974/notebook/notebook-16.webp)
![](https://static.igem.wiki/teams/5974/notebook/notebook-17.webp)
![](https://static.igem.wiki/teams/5974/notebook/notebook-18.webp)

Week13|6.31-7.6

Introduce pYB1a-pabABC-ABH60 into the BW25113 engineered strains with *nhoA* interference and knockout, and take samples at 12h, 24h, 36h, and 48h during fermentation for HPLC detection of p-AP yield.

pYB1a-pabABC-ABH60 was introduced into BW25113 with *nhoA* interference and knockout, respectively. Samples were taken at 12h, 24 h, 36 h and 48 h of fermentation and p-AP production was detected by HPLC.

![](https://static.igem.wiki/teams/5974/notebook/notebook-19.webp)

## Part 3 From p-AP to AAP

Week14| 7.7-7.14

Verification of the hypothesis: validating the enzyme activity of NhoA controlled by the thermosensitive promoter and optimizing the enzymatic efficiency of enzyme PANAT.

We constructed an amplified pSB1c and connected it with the promoter obtained through PCR amplification, creating a vector carrying the thermosensitive promoter. We linked pSB1c-I38 with the reporter gene *mCherry*. We introduced the plasmid pSB1c-I38-mCherry into BW25113 and conducted three parallel experiments at 30°C and 37°C respectively. After 16 hours, we took 200μl samples and measured OD600 using a microplate reader, exciting with 552nm light and detecting the absorption at 600nm, then normalizing the absorption results using OD600.

![](https://static.igem.wiki/teams/5974/notebook/notebook-20.webp)

Week15|7.15-7.22

This week, we first constructed the plasmids pSB1c-I38-nhoA and pSB1c-I38-PANAT.

We first constructed the plasmids pSB1c-I38-nhoA and pSB1c-I38-PANAT this week

Week16|7.23-7.30

We introduced pSB1c-I38-nhoA and pYB1a-pabABC-MNX1 into the *nhoA* knockout strain BW25113. After shaking the culture for 12 hours, pabABC-MNX1 expression was induced, and 36 hours after inducing MNX1 expression, the culture was maintained in a shaking incubator at 30℃ for a further 96 hours. Samples were taken every 12 hours starting from 12 hours after induction at 12h, 24h, 36h, 48h, and so on.

Use AAP standard products to calibrate the standard curve of AAP with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.

We transferred pSB1c-I38-nhoA and pYB1a-pabABC-MNX1 into the BW-ΔnhoA. The expression of *pabABC* and *MNX1* genes was induced after 12h of shaking, and after 36h of induction of expression of MNX1, the bacteria were placed in a 37 °C shaking table to continue cultivation for 96 h, where 12h, 24h, 36h, 48h from the beginning of induction... Samples were taken every 12 hours.

The AAP standard curve was calibrated with AAP standard at concentrations of 0.2,0.4,0.6,0.8, and 1, respectively.

At 12h, 24h, 36h, 48h after induction. The concentration of AAP at 96h.

Detect the concentration of AAP at 12h, 24h, 36h, 48h...96h after induction using HPLC.

![](https://static.igem.wiki/teams/5974/notebook/notebook-21.webp)

Week17 | 7.31-8.6

![](https://static.igem.wiki/teams/5974/notebook/notebook-22.webp)

After transforming pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT to BW-ΔnhoA strain and inducing expression at 30℃ for 36 hours, it was incubated at 37℃ for 48 hours, with samples taken at the 48-hour mark from the start of induction. The concentration of AAP after 48 hours of induction was measured using HPLC.

![](https://static.igem.wiki/teams/5974/notebook/notebook-23.webp)

Week18 | 8.7-8.14

Compare the conversion efficiencies of NhoA and PANAT enzymes.

The pYB1a-pabABC-ABH60 and pSB1c-I38-nhoA, as well as pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT, plasmids were transformed into the BW-ΔnhoA. After induction at 30°C for 36 hours, the cultures were transferred to a shaker at 37°C and grown for 96 hours. Samples were taken every 12 hours from the start of induction at 12h, 24h, 36h, 48h, etc. The concentration of AAP at 12h, 24h, 36h, 48h... up to 96h post-induction was detected using HPLC.

![](https://static.igem.wiki/teams/5974/notebook/notebook-24.webp)

Optimization of glycerol content in fermentation broth

Week19 | 8.15-8.22

We cotransfected pYB1a-pabABC-ABH60 and pSB1c-I38-nhoA, pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT, respectively, into BW-ΔnhoA, and each was divided into two groups to ferment in fermentation broth containing 1% and 10% glycerol, respectively, all were induced at 30 °C for 36h and then changed to 37 °C for 96h. 12h, 24h, 36h, 48h... Samples were taken every 12 hours. At 12h, 24h, 36h, 48h after induction. The concentration of AAP at 96h.

![](https://static.igem.wiki/teams/5974/notebook/notebook-25.webp)

Week20 | 8.23-8.30

PYB1a-pabABC-ABH60 and pSB1c-I38-PANAT were cotransferred to BW-ΔnhoA, and fermented in broth containing 1% and 10% glycerol, respectively. Samples were taken at 0h, 48h, 60h and 72h, respectively. The yields of p-AP, p-ABA and AAP in 1% and 10% glycerol groups were determined by HPLC.

![](https://static.igem.wiki/teams/5974/notebook/notebook-26.webp)
![](https://static.igem.wiki/teams/5974/notebook/notebook-27.webp)

Week21 | 8.31-9.6

Cotransfection of pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT in BW-ΔnhoA was performed, and both were changed to 37 °C shaker for 96h after induction at 30 °C for 36h. 12h, 24h, 36h, 48h... Samples were taken every 12 hours. At 12h, 24h, 36H, 48H after induction. 96 h, the final product AAP yield was detected.

![](https://static.igem.wiki/teams/5974/notebook/notebook-28.webp)

# Engineering

Module 1：Construction of the Metabolic Pathway From Vanillic Acid to p-aminobenzoic Acid (p-ABA)

Module 2: Construction of the Metabolic Pathway from p-ABA to p-Aminophenol (p-AP)

Module 3: Establishing the Metabolic Pathway from p-AP to AAP

Summary

## Module 1：Construction of the Metabolic Pathway From Vanillic Acid to p-aminobenzoic Acid (p-ABA)

### Cycle 1: Molecular Docking Simulation

Design

As shown in Figure 1, in the natural metabolic pathway, p-aminophenol (AAP) can be transformed from chorismic acid through a series of chemical reactions. Since our ideal substrate, vanillic acid, is structurally very similar to chorismic acid (Figure 2), we conducted molecular docking simulations to predict whether the binding sites of the two compounds with the enzymes PabA and PabB are similar.

![](https://static.igem.wiki/teams/5974/engineering/engineering-1.webp)

Figure 1: The metabolic pathway of AAP for the synthesis of chorismic acid

![](https://static.igem.wiki/teams/5974/design/design123.webp)

Figure 2: Structural comparison of vanillic acid (left) and chorismic acid (right)

Build

We used the Autodock Vina for molecular docking. First, we found the structures of the PabA and PabB enzymes on Autodock Vina. Then, we imported the two compounds, chorismic acid and vanillic acid, and conducted the docking.

Test

![](https://static.igem.wiki/teams/5974/engineering/engineering-3.webp)

Figure 3: Molecular docking simulation results of chorismic acid (left) and vanillic acid (right)

Learn

The molecular docking simulation results show that the two compounds have extremely similar binding sites in the PabAB enzyme and can both interact with the 386th and 62nd amino acids. This suggests that vanillic acid could be a substrate for the PabABC enzyme system.

### Cycle Two: Construction and Expression of Recombinant Plasmids to Convert VA to p-ABA

Design

We hypothesized that the endogenous *E. coli* PabABC enzyme system, which naturally converts chorismic acid to p-aminobenzoic acid (p-ABA), could be engineered to use vanillic acid (VA) as a novel substrate. We directly obtained these three genes from *E. coli* and constructed them on the expression vector pYB1a.

Build

We obtained the three genes *pabA, pabB*, and *pabC* by PCR using the *E. coli* genome as a template. The linearized vector of pYB1a was obtained through restriction enzyme digestion. Then, we connected the expression vector and the target genes through Gibson Assembly to obtain the recombinant plasmid pYB1a-pabABC (Figure 4). The recombinant plasmid was transformed into Escherichia coli BW25113, followed by induction of gene expression. A 48-hour fermentation was subsequently conducted. After fermentation, high-performance liquid chromatography (HPLC) was employed for analysis.

![](https://static.igem.wiki/teams/5974/engineering/engineering-4.webp)

Figure 4: Map of the pYB1a-pabABC plasmid

Test

We took the fermentation broth after 12 hours for HPLC detection and found the generation of p-ABA (Figure 5), which proved that vanillic acid could be converted into p-ABA under the catalysis of PabABC enzymes. Then we continued the fermentation until 48 hours and plotted the time curve of p-ABA production (Figure 6).

![](https://static.igem.wiki/teams/5974/engineering/engineering-5.webp)

Figure 5: HPLC detection peak chart of 12h fermentation liquid (p-ABA)

![](https://static.igem.wiki/teams/5974/engineering/engineering-6.webp)

Figure 6: Time curve of p-ABA production after 48 hours of fermentation

Learn

The test results show that vanillic acid can be converted into p-ABA under the catalysis of enzymes, proving that our idea is feasible. After 48 hours of fermentation, 1.13 mM of p-ABA can be synthesized, and the conversion rate of vanillic acid is 22.6%.

Since the conversion of vanillic acid to p-ABA requires the participation of L-glutamine (Figure 7), we aimed to explore the optimal addition amount of L-glutamine to enhance the conversion rate of vanillic acid in the next cycle.

![](https://static.igem.wiki/teams/5974/engineering/engineering-7.webp)

Fig 7: L-Glutamine can serve as a cofactor to provide a nitrogen source, enabling the conversion of vanillic acid to o-methoxy-p-aminobenzoic acid.

### Cycle Three: Exploring the Optimal Addition Amount of L-Glutamine

Design

In the process of converting vanillic acid to p-ABA, L-glutamine is an important cofactor. Since the addition amount of L-glutamine can be controlled when preparing the fermentation broth, we designed to improve the yield of p-ABA by optimizing the concentration of the co-substrate L-glutamine.

Build

When configuring the fermentation medium, we set up a concentration gradient of L-glutamine at 5 mM, 10 mM, 15 mM, 20 mM, and 25 mM, while keeping the rest of the fermentation conditions unchanged. The fermentation was carried out for 24 hours, and then the samples were analyzed using HPLC.

Test

We measured p-ABA production by HPLC after 24 hours in each condition, and the highest yield of p-ABA was achieved when 15 mM L-glutamine was added (Fig. 8).

![](https://static.igem.wiki/teams/5974/engineering/engineering-8.webp)

Figure 8: Exploring the Optimal Addition Amount of L-Glutamine

Learn

The results indicated that 15 mM L-glutamine was the optimal addition amount, at which the yield of p-ABA was 4.786 mM and the conversion rate of vanillic acid was 47.6%. This condition was adopted for all the next experiments.

## Module 2: Construction of the Metabolic Pathway from p-ABA to p-Aminophenol (p-AP)

### Cycle 1: Comparison of MNX1 and ABH60 Enzymes

Design

To efficiently convert p-aminobenzoic acid (p-ABA) into p-aminophenol (p-AP), we compared the catalytic efficiency of two reported isoenzymes from different sources. The candidates were MNX1, a monooxygenase from *Candida parapsilosis* CBS604, and ABH60, an N-hydroxylase from *Agaricus bisporus*.

![](https://static.igem.wiki/teams/5974/engineering/engineering-9.webp)

Figure 9: Metabolic pathway from vanillic acid to p-aminophenol

Build

The MNX1 and ABH60 enzymes were synthesized and respectively constructed on the pYB1a-pabABC plasmid to obtain the pYB1a-pabABC-MNX1 plasmid and the pYB1a-pabABC-ABH60 plasmid (as shown in Figure 10). After successful construction, the plasmids were transferred into the BW25113 strain, and subsequent detection of protein synthesis was carried out after induction expression.

![](https://static.igem.wiki/teams/5974/engineering/engineering-10.webp)

Figure 10: Maps of the pYB1a-pabABC-MNX1 plasmid and the pYB1a-pabABC-ABH60 plasmid

Test

The whole cell lysate was subjected to 12% SDS-PAGE and stained with Coomassie Brilliant Blue. As shown in Figure 11, obvious overexpression bands appeared at 50.9 kDa (MNX1) and 52 kDa (ABH60), while no corresponding bands were observed in the blank control. This indicates that both exogenous genes can be normally expressed in Escherichia coli. Both engineered strains produced p-AP, which was confirmed by an HPLC peak at a retention time of 2.8 minutes, matching the standard (Fig. 12). This peak was absent in the blank control, demonstrating that both MNX1 and ABH60 are functional. A comparison of product yields, however, showed that the strain expressing ABH60 performed better (Fig. 13).

![](https://static.igem.wiki/teams/5974/engineering/engineering-11.webp)

Figure 11: Protein Gel Electrophoresis Results of ABH60 and MNX1

![](https://static.igem.wiki/teams/5974/engineering/engineering-12.webp)

Figure 12: HPLC detection peak chart of fermentation liquid (p-AP)

![](https://static.igem.wiki/teams/5974/engineering/engineering-13.webp)

Figure 13: Bar Chart of Products of ABH60 and MNX1

Learn

ABH60 exhibits higher catalytic efficiency within the cell, thus it was determined that the ABH60 enzyme would be the core enzyme for optimizing the pathway from p-ABA to p-AP. However, literature review revealed that the endogenous *nhoA* gene in*E. coli* converts p-ABA to p-acetylsalicylic acid instead of p-AP, directly reducing the yield of p-AP, as shown in Figure 14. Therefore, in the next cycle, we plan to use CRISPRi technology to inhibit the expression of the *nhoA* gene or CRISPR-Cas9 technology to knockout *nhoA* to block the side pathway and increase carbon flux.

![](https://static.igem.wiki/teams/5974/engineering/engineering-14.webp)

Figure 14: Metabolic pathway from p-ABA to p-AP, NhoA can convert p-ABA into by-products.

### Cycle 2: Construction of the Inhibitory Plasmid R6K-dCas9(sg-nhoA)

Design

To block the diversion of p-ABA to the by-product p-acetamidobenzoic acid, this study employed a CRISPRi system to repress the chromosomal nhoA gene. To this end, we aimed to construct an R6K-dCas9(sg-nhoA) plasmid for subsequent genetic inhibition. A suitable sgRNA sequence for CRISPRi was identified using the website https://crispy.secondarymetabolites.org, which was designed to be incorporated into the R6K-dCas9 plasmid.

Build

After introducing the sgRNA sequence by PCR, the R6K-dCas9 (sg-nhoA) plasmid was constructed through Gibson Assembly. Once the construction was successful, it was transferred into BW25113 for subsequent detection.

Test

As shown in Figure 15, the sequencing results indicate that the plasmid construction was successful.

![](https://static.igem.wiki/teams/5974/engineering/engineering-15.webp)

Figure 15: Sequencing results of R6K-dCas9-nhoA

Learn

The R6K-dCas9(sg-nhoA) inhibitory plasmid was successfully constructed, and the dCas9 protein can be normally expressed. It will be used to interfere with the *nhoA* gene in the subsequent steps.

### Cycle 3: Knockout of the *nhoA* Gene

Design

We designed the sgRNA sequences suitable for CRISPR-Cas9 on the website https://crispy.secondarymetabolites.org, and constructed them on the pTarget plasmid. We have also prepared to construct the targeting fragment to achieve the complete deletion of the *nhoA* coding region while retaining the original promoter and terminator.

Build

The sgRNA sequence was introduced by PCR method and the pTarget-nhoA plasmid was constructed by Gibson Assembly. After successful construction, it was transformed into BW25113. We obtained the complete sequence of the *nhoA* gene and used PCR to obtain 500 bp upstream and downstream of the *nhoA* gene. The two fragments were connected by Overlap PCR method to obtain the targeting fragment. Subsequently, the pTarget-nhoA plasmid and the targeting fragment were electroporated into BW25113 for gene knockout.

Test

Colony PCR screening was performed. The results are shown in Figure 16, with a knockout efficiency of 95%.

![](https://static.igem.wiki/teams/5974/engineering/engineering-16.webp)

Figure 16: PCR results of *nhoA* gene knockout colonies

Learn

The *nhoA* gene knockout was successfully achieved, and a genetically stable BW-ΔnhoA strain was obtained. Subsequently, parallel fermentation will be conducted with the CRISPRi inhibition strain, and the p-AP production will be quantitatively compared to determine the optimal strategy for blocking the side pathway.

### Cycle 4: Detect and Compare the Effects of Interference and Knockout.

Design

Compare the final concentration and conversion rate of p-AP of the BW25113-sgnhoA interference strain and the BW-ΔnhoA knockout strain under consistent fermentation conditions.

Build

Both the BW25113-sgnhoA interference strain and the BW-ΔnhoA knockout strain were introduced with the same pathway plasmid pYB1a-pabABC-ABH60 under consistent fermentation conditions.

Test

We performed HPLC analysis on the p-AP production of *nhoA* knockout strains and *nhoA* inhibition carrying that the knockout of the *nhoA* gene is more effective than the interference of the *nhoA* gene.

![](https://static.igem.wiki/teams/5974/engineering/engineering-17.webp)

Figure 17: Comparison Chart of Knockout and Interference

Learn

By comparison, it was found that the knockout of the *nhoA* gene had a better effect. The yield and conversion rate of p-AP were significantly higher than those of the CRISPRi interference strategy. Therefore, the BW-ΔnhoA strain was subsequently used as the chassis strain for further experiments.

### Cycle 5: Determine the Maximum Yield of p-AP

Design

In the previous four rounds of cycles, we determined to use ABH60 enzyme as one of the key enzymes for building the pathway, and to use BW-ΔnhoA strain as the chassis strain. Based on this, we conducted fermentation experiments to determine the highest yield of p-AP and the conversion rate of vanillic acid.

Build

The constructed recombinant plasmid pYB1a-pabABC-ABH60 was transformed into the BW-ΔnhoA strain. Following this, protein expression was induced, and the bacterial culture was subjected to a 48-hour fermentation. Subsequently, the production yield was analyzed by high-performance liquid chromatography (HPLC).

Test

During the fermentation process, samples were taken at 6-hour intervals for HPLC analysis to plot the time-course profile of p-AP production.

![](https://static.igem.wiki/teams/5974/engineering/engineering-18.webp)

Figure 18: Time Curve of p-AP Production

Learn

The results showed that the p-AP yield after fermentation was 4.47 mM, and the vanillic acid conversion rate was 86%. Moreover, the p-AP yield gradually stabilized after 36 hours.

## Module 3: Establishing the Metabolic Pathway from p-AP to AAP

### Cycle 1: Testing the Temperature-Sensitive Promoter I38

Design

To complete our pathway, we needed an enzyme to convert p-AP to AAP. While both NhoA and PANAT are known to catalyze this reaction, expressing them constitutively could lead to unwanted byproducts. Moreover, we already incorporated an nhoA CRISPR construct in a previous step. To overcome this issue, we designed a time-delayed expression strategy using the I38 temperature-sensitive promoter. This design functions as a thermal switch, repressing expression at 30°C and activating it only after shifting to 37°C.

![](https://static.igem.wiki/teams/5974/engineering/engineering-19.webp)

Figure 19: Metabolic pathway from vanillic acid to AAP

However, before fermentation, the first thing we need to do is test the effect of the temperature-sensitive promoter I38. We linked I38 with the red fluorescent protein gene *mCherry* and detected the relative fluorescence intensity after induction at 30℃ and 37℃ respectively. By comparing the relative fluorescence intensity, we can evaluate the effect of the I38 promoter.

Build

We obtained the temperature-sensitive promoter I38 and the red fluorescent protein gene *mCherry* through PCR, and obtained the linear expression vector pSB1c by restrict enzymatic digestion. The recombinant plasmid pSB1c-I38-mCherry was obtained through Gibson Assembly (Figure 10). Subsequently, the successfully constructed recombinant plasmid was transformed into Escherichia coli BW-ΔnhoA, and induced expression was carried out at 30℃ and 37℃ respectively.

![](https://static.igem.wiki/teams/5974/engineering/engineering-20.webp)

Figure 20: Maps of the pSB1c-I38-mCherry plasmid

Test

After 16 hours of induction, the fluorescence intensity and the OD600 of the induction liquid were detected using an microplate reader, and the relative fluorescence intensity was calculated. The results are shown in Figure 21.

![](https://static.igem.wiki/teams/5974/engineering/engineering-21.webp)

Figure 21: Relative fluorescence intensity at 30℃ and 37℃

Learn

The test results show that the promoter worked as an effective temperature-controlled switch, showing strong induction at 37°C. This validated its use for controlling our final pathway step.

### Cycle 2: Testing and Comparing nhoA and PANAT

Design

We designed to compare the efficiency of two final-step enzymes, NhoA and PANAT, under the control of the I38 promoter. The fermentation would involve a temperature shift at 36 hours, a time point learned from previous tests where p-AP accumulation plateaus.

Build

We obtained the I38 promoter, the *nhoA* gene and the *PANAT* gene by PCR, and the linear expression vector pSB1c was obtained by PCR. Then, the recombinant plasmids pSB1c-I38-nhoA and pSB1c-I38-PANAT were obtained by Gibson Assembly. Subsequently, the constructed pSB1c-I38-nhoA plasmid and the successfully constructed pYB1a-pabABC-ABH60 in the second part were co-transformed into the BW-ΔnhoA strain, and the pSB1c-I38-PANAT and pYB1a-pabABC-ABH60 were co-transformed into the BW-ΔnhoA strain. Induction expression was carried out. Following fermentation, the bacterial culture was incubated in a shaker flask at 30°C. After 36 hours, the temperature was shifted to 37°C to activate the thermosensitive promoter, thereby inducing the expression of nhoA and PANAT genes. The fermentation was continued for a total of 96 hours, after which the samples were analyzed by HPLC. Replicate experiments were conducted to determine the maximum yield of AAP and the conversion rate of vanillic acid.

![](https://static.igem.wiki/teams/5974/engineering/engineering-22.webp)

Figure 22: Maps of the pSB1c-I38-nhoA and pSB1c-I38-PANAT plasmid

Test

During the fermentation process, samples were taken every 12 hours and analyzed by HPLC. The results are shown in Figure 22.

![](https://static.igem.wiki/teams/5974/engineering/engineering-23.webp)

Figure 23: HPLC detection peak chart of fermentation liquid (AAP)

![](https://static.igem.wiki/teams/5974/engineering/engineering-24.webp)

Figure 24: Time curve of AAP production after 96 hours of fermentation

![](https://static.igem.wiki/teams/5974/engineering/engineering-25.webp)

Figure 25: Repeat the experiment to determine the maximum yield of AAP

Learn

In terms of AAP yield, the PANAT enzyme demonstrated superior performance compared to the NhoA enzyme. The AAP production reached its peak at 72 hours of fermentation, resulting in a final yield of 1.17 mM AAP from 5.0 mM vanillic acid.

## Summary

Following a series of experiments, we successfully established a novel heterologous metabolic pathway from vanillic acid to AAP and performed systematic optimization. Ultimately, 5 mM vanillic acid was converted to 1.17 mM AAP. Our work provides a novel strategy for the efficient utilization of lignin, demonstrating its potential conversion into high-value compounds via synthetic biology.

# Experiments

Plasmid Preparation

Transformation: Electroporation Procedure, Chemical Transformation

PCR

SDS-PAGE

Preparation of Competent Cells

Preparation of Competent Cells for Chemical Transformation

Gel Preparation

Colony PCR

Restriction Digestion

Microplate Reader Detection

Digestion

Induction

Whole-Cell Catalysis

Sample Preparation

## Plasmid Preparation

1. Harvest Cells: Transfer the overnight cultured medium to a 1.5 mL EP tube using a pipette. Centrifuge at 10,000×g for 1 min to collect the cell pellet. Discard the supernatant. Repeat this step to enrich the cell pellet.

2. Resuspend Cells: Add 250 μL of Solution I (containing RNase A and stored at 4°C) to the cell pellet. Use a pipette to resuspend the cells until no clumps are visible.

3. Lysis: Add 250 μL of Solution II and gently invert the tube 4-7 times. Incubate for 2-3 min to lyse the cells.

4. Neutralization: Add 350 μL of Solution III and gently invert the tube 4-7 times. Centrifuge at maximum speed (15,000×g) for 10-15 min.

5. Bind DNA: Carefully transfer the supernatant to a blue adsorption column and centrifuge at maximum speed for 1 min. Discard the waste liquid and return the column to an empty collection tube.

6. Wash: Add 500 μL of HBC Buffer (containing isopropanol) to the adsorption column. Centrifuge at maximum speed for 1 min, discard the waste liquid, and return the column to an empty collection tube.

7. Wash Again: Add 700 μL of DNA Wash Buffer(add absolute ethanol) to the adsorption column. Centrifuge at maximum speed for 1 min, discard the waste liquid, and repeat this step twice.

8. No-Load Operation: Place the adsorption column in an empty collection tube and centrifuge at maximum speed for 2 min to remove any residual wash buffer.

9. Ethanol removal: Transfer the adsorption column to a clean 1.5 mL EP tube. Place the tube in a metal bath at approximately 55°C for 3-5 min to dry.

10. Elution: Add 30-50 μL of distilled water to the center of the adsorption membrane. Let it stand at room temperature for 1 min, then centrifuge at maximum speed for 1 min to elute the DNA.

11. Storage: Store the extracted DNA at -20°C.

## Transformation

### Electroporation Procedure

1. Add the target fragment (The target practice fragment should not exceed 500 ng, the plasmid should not exceed 300 ng, and the total volume added should not exceed 10 μL) to 100 μL of competent cells and place on ice for 10-30 min.

2. Sterilize and dry 2 mm electroporation cuvettes under UV in a laminar flow hood for 20 min, pre-chill on ice, and quickly transfer the competent cells to the cuvette, ensuring cells are at the bottom.

3. Perform the following steps quickly: Wipe the outside of the cuvettes dry. For 2 mm cuvettes, use electroporation program Ec2; for 1 mm cuvettes, use program Ec1. After electroporation, immediately add 900-1000 µL of pre-warmed LB at 37°C, gently pipette, and transfer to a 1.5 mL centrifuge tube. Incubate at 37°C on a shaking incubator at 200 rpm for 45-60 min.

4. Plate an appropriate volume onto Kana+Str double antibiotic plates and incubate at 37°C.

### Chemical Transformation

1. Thaw competent cells (stored in a -80°C freezer) on ice, add the ligation product/plasmid, and incubate on ice for 20 min.

2. Heat shock at 42°C for 45 sec, then let the cells rest on ice for 2 min.

3. Add 600 µL of LB medium without antibiotics and incubate at 37°C with shaking for 45 min.

4. Centrifuge at 4200 rpm for 3 min, discard an appropriate amount of supernatant, and plate the cells (using both diluted and concentrated samples if needed). Label the plates accordingly.

5. Incubate the plates upside down at 37°C for 12 h.

## PCR

1. Prepare the reaction mixture in a PCR tube on ice and mix thoroughly using a vortex mixer.

2. Collect any residual liquid on the tube walls using a mini centrifuge.

3. Transfer the PCR tube to the thermal cycler and set the program to initiate the PCR thermal cycling process.

x=10~50ng (the addition volume depends on the concentration)

![](https://static.igem.wiki/teams/5974/experiment/experiment-1.webp)

## SDS-PAGE

### 1. Gel preparation:

Assemble the module and secure the gel mold. Prepare the lower gel according to the following recipe, pour it into the mold, and add water to level the surface. Let it sit for 40-60 min until the gel solidifies. Once solidified, discard the water on the surface. Prepare the upper gel according to the recipe, pour it into the mold, insert the comb, and allow it to solidify for approximately 1 h. After solidification, dismantle the mold to complete the gel preparation.

### 2. Sample preparation:/h3> Collect the induced and uninduced bacterial cultures, measure and obtain 6 OD units of bacterial suspension. Centrifuge at 10,000 g for 10 min, discard the supernatant, and resuspend the pellet in 600 µL of sterile water. Use an ultrasonic cell disruptor to lyse the cells. Centrifuge the lysate at 10,000 g for 10 min to separate the supernatant and pellet. Resuspend the pellet with 600 μL sterile water. Completing the sample preparation. 3. Sample loading: Prepare the protein buffer solution according to the table below. Place the gel into the electrophoresis apparatus. After checking for leaks with the buffer solution, mix 16 µL of the prepared sample with 4 µL of loading buffer. The mixed sample was put into the PCR instrument and heated at 100 ℃ for 5min to denature it. After cooling, load 16 µL of the mixture into the wells of the gel, followed by 4 µL of the protein marker. Then, sample loading is complete. 4. Electrophoresis: Connect the power supply and run the gel at 80 V for 30 min, followed by 120 V for about 1 h. 5. Staining: Carefully peel the gel off in water, then place the gel into the staining solution. Boil the solution and then use the heated staining solution to stain the gel on a small shaker at room temperature for about 45min. 6. Destaining: Rinse the gel with clean water, wash the staining solution on the surface, add protein decolorizing solution, boil the solution, and decolorize the heated staining solution on a small shaker at room temperature for 30 minutes. 7. Composition: Upper gel: ![](https://static.igem.wiki/teams/5974/experiment/experiment-2.webp) Lower Gel: ![](https://static.igem.wiki/teams/5974/experiment/experiment-3.webp) Protein Destaining Solution (500 mL system): Anhydrous ethanol (75 mL), acetic acid (50 mL), distilled water (375 mL). 5× Protein Buffer Solution (5× Tris-Glycine Buffer; 1 L system): Tris base (15.1 g), glycine (94 g), SDS (5 g); adjust the pH to 8.3 with precise measurements. SDS-PAGE Staining Solution (400 mL system): Coomassie Brilliant Blue R-250 (0.4 g), isopropanol (100 mL), glacial acetic acid (40 mL), distilled water (260 mL); stir thoroughly and filter using filter paper, frequently replacing the paper to remove particulate matter. Prepare two bottles at once, which can be reused.

## Preparation of Electroporation Competent Cells

1. Transfer 200 μL of the glycerol stock into 5 mL of LB liquid medium and culture at 30°C, 200 rpm for approximately 12 h. Subsequently, inoculate into LB medium at a 2% concentration and incubate for 0.5 h before adding arabinose to a final concentration of 0.2%.

![](https://static.igem.wiki/teams/5974/experiment/experiment-4.webp)

2. After culturing at 30°C for approximately 2 to 2.5 h, achieving an OD₆₀₀ of about 0.55-0.6 (values significantly higher or lower can severely affect electroporation efficiency), remove the culture and incubate on ice for 30 min.

3. Pre-cool the centrifuge rotor in a freezer for 10 min. Collect the cells by centrifugation using pre-cooled centrifuge tubes and keep on ice. Centrifuge at 4000 rpm for 10 min, aliquoting 50 mL per tube.

4. Using 20 mL of pre-cooled, sterile 10% glycerol per tube, gently pipette while keeping the tube on ice. Aliquot into two 10 mL centrifuge tubes and centrifuge at 4200 rpm for 10 min in a low-temperature centrifuge.

5. Immediately after centrifugation, discard the supernatant. Add pre-cooled, sterile 10% glycerol and then pipette gently while keeping the tube on ice.

6. Repeat steps 4 and 5 three times. For the first two washes, use 10 mL of pre-cooled, sterile 10% glycerol. For the final wash, resuspend in 0.5 mL of 10% glycerol and combine the two tubes into one.

7. Aliquot 100 μL per tube into pre-cooled centrifuge tubes. Proceed with electroporation or store immediately at -80°C. The competent cells can be stored at -80°C for up to six months.

## Preparation of Competent Cells for Chemical Transformation

1. From -20°C or -80°C glycerol stock, use a sterile loop or white pipette tip to pick up the bacterial culture. Streak it onto an agar plate and incubate for 12 h (to isolate single colonies).

2. Pick single colonies and inoculate into 5 mL of liquid LB medium using a white pipette tip (or by pipetting up and down). Label the tubes and incubate at 37°C with shaking for 12 h.

3. Transfer 1 mL of the LB culture into 100 mL of LB medium and incubate at 37°C with shaking for about 2 h. Measure the OD until it reaches approximately 0.4 (monitor time carefully).

4. Place the culture into a 16°C shaker and incubate for about 1 huntil the OD reaches 0.5.

5. At this stage, do not use the alcohol lamp. Perform all procedures under strict aseptic conditions on ice. Aliquot 100 mL of the culture into four 50 mL centrifuge tubes and let sit on ice for 30 min to stop active growth.

6. Centrifuge at 4°C and 4200 rpm for 10 min. After centrifugation, place the tubes on ice, quickly discard the supernatant, and remove residual liquid using a pre-cooled pipette tip. Add 1 mL of a mixed solution of 80 mM MgCl₂ and 20 mM CaCl₂ to each tube (you may combine centrifuge tubes, ensuring balance).

7. Incubate on ice for 30 min.

8. Centrifuge at 4°C and 4200 rpm for 10 min. After centrifugation, place the tubes on ice, quickly discard the supernatant using aseptic techniques, and remove residual liquid with a pre-cooled pipette tip. Resuspend the pellet in 4 mL of a 15% glycerol and 100 mM CaCl₂ mixture, gently shaking on ice.

9. Aliquot 100 μL into pre-cooled tubes. Record the number of aliquots prepared. Prepare labeling for the storage bags, including preparation date, preparer, competent cell type, and aliquot volume. Place the labeling bags in ice for cooling.

10. Store at -80°C.

## Gel Preparation:

1. Prepare agarose gels of appropriate concentration: 1% for detection gels and 2% for recovery gels. Heat in a microwave until the agarose is fully dissolved.

2. Allow the solution to cool to approximately 60°C, then add 1/10,000 Goldview and mix thoroughly.

3. Place an appropriately sized comb into the gel mold. Pour the warm agarose gel into the mold, ensuring the gel thickness is between 3-5 mm. Allow the gel to solidify for approximately 20 min.

4. Once the gel has completely solidified, gently remove the comb and place the gel tray into the electrophoresis chamber. Add 1×TAE buffer so that the buffer level is about 1 mm above the gel surface, ensuring proper conductivity.

Sample Loading:

1. Add the following to the wells: (PCR products + loading buffer) or DNA marker.

Electrophoresis:

1. Cover the electrophoresis chamber and run the gel at 80-120V.

2. When the dye front has migrated an appropriate distance through the gel (approximately 30 min), turn off the power, remove the gel, and observe the DNA bands under a UV lamp.

Gel Imaging:

1. Place the gel onto the gel imaging system and capture an image under ultraviolet light to visualize the gel.

## Colony PCR

1. Prepare the colony PCR reaction system. For a 10 µL reaction volume, use: 1 µL of bacterial suspension, 0.4 µL of upstream and downstream primers, 5 µL of PCR enzyme Master Mix, and 3.2 µL of sterile water. If directly picking a colony, add 4.2 µL of sterile water to a final volume of 10 µL. For a 50 µL reaction volume, scale up the components proportionally.

2. Set up the PCR program according to the length of the bacterial PCR fragment and run the PCR.

3. Prepare the electrophoresis gel based on the number of bacterial PCR products and the system.

4. Load the bacterial PCR products and an appropriately sized DNA marker into the wells of the gel. Place the gel into the electrophoresis chamber and run the gel at 120-180V.

5. Determine the electrophoresis completion time based on the marker bands. After completion, remove the gel, visualize under UV light, and photograph for analysis.

## Restriction Digestion

1. Select appropriate restriction sites based on the plasmid map.

2. Identify the corresponding restriction enzymes and system instructions, and prepare the reaction mixture according to the instructions.

3. Incubate the prepared mixture at the specified temperature in a metal bath or PCR machine for the recommended time.

## Microplate Reader Detection

1. Inoculate the bacterial strain into 6 tubes of LB medium. Incubate three tubes in a shaking incubator at 30°C and the other three tubes in a shaker at 37°C, with all tubes cultured for 12 hours.

2. Multi-well plate preparation: Sterilize and label the 96-well plate.

3. Add 200 μL of bacterial solution dissolved in LB to each well.

4. Set the corresponding parameters on the microplate reader to measure the growth and fluorescence intensity of each well strain.

## Digestion

1. Add Dpn I enzyme: Add 1 µL of Dpn I enzyme (10 U/µL) to each 50 µL PCR reaction mixture.

2. Gently mix the reaction mixture to avoid vigorous shaking and incubate at 37°C for 1~2 h to ensure complete digestion of the methylated template plasmid by Dpn I.

3. After digestion is complete, proceed with bacterial transformation without the need to deactivate Dpn I.

![](https://static.igem.wiki/teams/5974/experiment/experiment-5.webp)

## Induction

1. Turn on the UV light in the laminar flow hood and sterilize for 10-20 min.

2. Turn off the UV light, then turn on the fan and lighting. Sterilize and place the required glycerol stocks, antibiotics, and LB medium into the laminar flow hood.

3. Add 5 µL of the antibiotic corresponding to the bacterial strain (the volume of antibiotic added should be 1/1000 of the medium volume) to 5 mL of LB medium in a test tube.

4. Transfer 50 µL of bacterial culture from the glycerol stock (the volume of glycerol stock added should be 1/100 of the medium volume) into the LB medium with the added antibiotic.

5. Turn off the fan and lighting, remove the test tube, and incubate at 37°C with shaking for 12 h.

6. After incubation, prepare ZY medium (shaking flask), 50×M, 50×5052, trace elements, MgSO₄, and antibiotics, and sterilize them before placing them into the sterilized laminar flow hood.

7. Add the following to the ZY medium (shaking flask) using a pipette: 2 mL of 50×M, 2 mL of 50×5052, 200 µL of trace elements, 200 µL of MgSO₄, 100 µL of the corresponding antibiotic, and 1 mL of the bacterial culture. Seal the flask with parafilm and secure it with a rubber band.

8. Remove the flask and incubate at 37°C with shaking for 1-2 h until the OD600 reaches 0.5.

9. After incubation, prepare 20% arabinose and IPTG as needed, sterilize them, and place them into the sterilized laminar flow hood.

10. Add 1 mL of 20% arabinose (final concentration 0.2%) and 100 µL of IPTG to the ZY medium (shaking flask) using a pipette. Seal the flask with parafilm and secure it with a rubber band.

11. Remove the flask and incubate at 30°C with shaking for 16 h.

## Whole-Cell Catalysis

1. Remove the induced shaking flask, take the bacterial culture with the corresponding OD value, and transfer it to a 10 mL centrifuge tube. Centrifuge at 4200 rpm and 4°C for 10 min.

2. Discard the supernatant and add 5 mL of fermentation broth. Mix thoroughly.

3. Transfer the mixed solution to a 250 mL shaking flask.

Fig. 2 Whole-cell catalyzed sample vials made

![](https://static.igem.wiki/teams/5974/experiment/experiment-6.webp)

4. Incubate the shaking flask at 30°C and sample at fixed time intervals. (during the fermentation process, the conical flasks need to be transferred from 30°C to 37°C midway)

Composition of fermentation broth (40 mL system): Na₂HPO₄·12H₂O is 23.88 mM, NaH₂PO₄ is 11.02 mM, NaCl is 4.28 mM, NH₄Cl is 9.35 mM, Tris·HCl (pH = 8.0) is 25 mM, MgCl₂ is 2.5 mM, L-glutamine is 15 mM, and anhydrous glycerol is 2 mL.

## Sample Preparation

1. Suck 20 µL of bacterial culture from the shake flask and mix it with 180 µL of water. Transferred to EP tubes and centrifuged at 10000 x g for 10 min.

2. After centrifugation, remove the EP tube and use a 1 mL syringe to aspirate the entire supernatant.

3. Attach the syringe to a 0.22 µm filter membrane, and push the syringe to filter the supernatant into a new EP tube. Repeat the process if necessary to ensure complete filtration.

4. Use a pipette to transfer the filtered supernatant into a brown liquid-phase vial. The sample is now prepared and can be stored at -20°C for short-term use.

### Vanillic Acid Detection

HPLC Detection

1.Use a C18 column with a mobile phase of 0.1% phosphoric acid aqueous solution : pure methanol = 70:30.

2.Detect the absorbance peak at a UV wavelength of 254 nm with a column temperature of 30°C.

### Attempt to Produce AAP Using Vanillic Acid as Substrate

Preparation of vanillic acid as production matrix

1.Weigh 10mM vanillic acid (standard compound) and put it into a 100ml conical flask.

2.Add 40ml of water to the conical flask and use a magnetic stirrer until the vanillic acid is completely dissolved.

3.Filter with 0.22 micron filter membrane to remove bacteria.

Production of AAP

1.Suck 5 ml of fermentation broth into a 10 ml centrifuge tube containing 125OD volume of engineered E. coli strain (BW25113- ΔnhoA carrying plasmids pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT). Resuspend the cell pellet evenly to initiate biotransformation.

2.pipette 20 μL of resuspended culture and mix it with 180 μL of water and put it into a 1.5 ml EP tube as a 0-hour control sample.
The remaining resuspended culture was transferred to a 100ml Erlenmeyer flask containing 5mm l-glutamine and 5mm vanillic acid (final concentration). The flask was sealed with silica gel plugs and shake at 220 rpm for 36 hours at 30 ° C, and then shake at 220 rpm for 60 hours at 37 ° C for fermentation and AAP production.
Every 6 h during the fermentation process, 20 μL of sample was taken from the culture solution and transferred to a 1.5 ml EP tube for subsequent HPLC sample preparation.

Sample Preparation for AAP Detection

1.Take samples at 17 fermentation time points in a 100ml Erlenmeyer flask (including the 0-hour control), take 20 μL of each and add 180 μL of water for dilution, mix with a pipette gun.

2.Centrifuge the diluted sample at 10000 g for 10 min to pellet cells and any debris.

3.After centrifugation, carefully aspirate all supernatants using a 1 ml syringe to avoid precipitation.

4.Connect the yellow filter membrane (pore size 0.22 μ m) to a 1 ml syringe, and filter the supernatant into a new clean Brown HPLC vial. If necessary, repeat aspiration and filtration from the same sample to ensure complete transfer and clarification.

5.Seal the vial with a cap. Samples are now prepared for HPLC analysis. (if immediate analysis is not possible, store the sample at -20 ° C to prevent degradation.)

HPLC Detection

1.Use a C18 reverse-phase column for separation. The mobile phase consists of 0.1% phosphoric acid aqueous solution : pure methanol = 70:30(v/v).

2.Detect the absorbance of the sample at a UV wavelength of 254 nm (optimized for AAP detection). Maintain the column temperature at 30°C throughout the analysis.

3.Identify and quantify the AAP peak in the samples by comparing its retention time and peak area to a standard curve generated using pure AAP standard.

# Results

Module 1: Exploration of Pathway Feasibility

Module 2: Enzyme Optimization and Bypass Optimization

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

New Composite Part

## Module 1: Exploration of Pathway Feasibility

1. Molecular Docking Predicted Feasibility

![](https://static.igem.wiki/teams/5974/results/result-1.webp)

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).

![](https://static.igem.wiki/teams/5974/results/result-2.webp)

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

![](https://static.igem.wiki/teams/5974/results/result-3.webp)

Fig 3. Plasmid pYB1a-pabABC.

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

![](https://static.igem.wiki/teams/5974/results/result-4.webp)

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).

![](https://static.igem.wiki/teams/5974/results/result-5.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-6.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-7.webp)

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%.

![](https://static.igem.wiki/teams/5974/results/result-8.webp)

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

![](https://static.igem.wiki/teams/5974/results/result-9.webp)

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

1. Plasmid Construction

![](https://static.igem.wiki/teams/5974/results/result-10.webp)

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).

![](https://static.igem.wiki/teams/5974/results/result-11.webp)

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

![](https://static.igem.wiki/teams/5974/results/result-12.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-13.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-14.webp)

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

2. Efficiency comparison between ABH60 and MNX1

![](https://static.igem.wiki/teams/5974/results/result-15.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-16.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-17.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-18.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-19.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-20.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-21.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-22.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-23.webp)

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

![](https://static.igem.wiki/teams/5974/results/result-24.webp)

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

1. Testing of Temperature-Sensitive Promoter

![](https://static.igem.wiki/teams/5974/results/result-25.webp)

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

![](https://static.igem.wiki/teams/5974/results/result-26.webp)

Fig 26. Plasmid pSB1c-I38-nhoA

![](https://static.igem.wiki/teams/5974/results/result-27.webp)

Fig 27. Plasmid pSB1c-I38-PANAT

![](https://static.igem.wiki/teams/5974/results/result-28.webp)

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

![](https://static.igem.wiki/teams/5974/results/result-29.webp)

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

![](https://static.igem.wiki/teams/5974/results/result-30.webp)

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.

![](https://static.igem.wiki/teams/5974/results/result-31.webp)

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).

![](https://static.igem.wiki/teams/5974/results/result-32.webp)

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

![](https://static.igem.wiki/teams/5974/results/result-33.webp)

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.