Engineering
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.
Figure 1: The metabolic pathway of AAP for the synthesis of chorismic acid
Figure 2: Structural comparison of vanillic acid (left) and chorismic acid (right)
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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.
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Figure 3: Molecular docking simulation results of chorismic acid (left) and vanillic acid (right)
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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.
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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.
Figure 4: Map of the pYB1a-pabABC plasmid
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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).
Figure 5: HPLC detection peak chart of 12h fermentation liquid (p-ABA)
Figure 6: Time curve of p-ABA production after 48 hours of fermentation
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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.
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.
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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.
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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).
Figure 8: Exploring the Optimal Addition Amount of L-Glutamine
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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.
Figure 9: Metabolic pathway from vanillic acid to p-aminophenol
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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.
Figure 10: Maps of the pYB1a-pabABC-MNX1 plasmid and the pYB1a-pabABC-ABH60 plasmid
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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).
Figure 11: Protein Gel Electrophoresis Results of ABH60 and MNX1
Figure 12: HPLC detection peak chart of fermentation liquid (p-AP)
Figure 13: Bar Chart of Products of ABH60 and MNX1
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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 inE. 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.
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.
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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.
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As shown in Figure 15, the sequencing results indicate that the plasmid construction was successful.
Figure 15: Sequencing results of R6K-dCas9-nhoA
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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.
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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.
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Colony PCR screening was performed. The results are shown in Figure 16, with a knockout efficiency of 95%.
Figure 16: PCR results of nhoA gene knockout colonies
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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.
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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.
Figure 17: Comparison Chart of Knockout and Interference
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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.
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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).
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During the fermentation process, samples were taken at 6-hour intervals for HPLC analysis to plot the time-course profile of p-AP production.
Figure 18: Time Curve of p-AP Production
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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.
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.
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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.
Figure 20: Maps of the pSB1c-I38-mCherry plasmid
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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.
Figure 21: Relative fluorescence intensity at 30℃ and 37℃
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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.
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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.
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.
Figure 23: HPLC detection peak chart of fermentation liquid (AAP)
Figure 24: Time curve of AAP production after 96 hours of fermentation
Figure 25: Repeat the experiment to determine the maximum yield of AAP
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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.