Design
Overview
Our engineered Escherichia coli (E. coli) system is designed for efficient and reliable bioremediation of aromatic hydrocarbons through four integrated functional modules.
A quorum sensing circuit (based on LuxI/LuxR and pLuxR) ensures energy-efficient, density-dependent regulation of degradation and resistance genes, expressing them only at high cell density to reduce metabolic burden.
The degradation module incorporates catabolic gene clusters (dfd for dibenzofuran and xyl for toluene), enabling the breakdown of these toxic compounds into less harmful metabolites.
The resistance module enhances bacterial survival under harsh conditions via the NhaA antiporter, which maintains intracellular ion balance and pH stability, and the AcrAB-TolC efflux pump, which expels toxic intermediates.
Finally, the biosafety module combines physical containment using polyurethane encapsulation to trap cells within bioreactors and a temperature-sensitive suicide switch that eliminates escaped bacteria in the environment. Together, these modules form a robust, scalable, and secure solution for treating aromatic hydrocarbon pollution.
1. Quorum sensing circuit
The Quorum Sensing System is like the brain of our engineered bacteria. Its main function is to rapidly activate the expression of downstream gene circuits within a very short period of time when it senses the need to cope with pollutants. Therefore, the existence of the Quorum Sensing System enables us to more sensitively control and express the remaining functions of the engineered bacteria, such as their powerful degradation ability and resistance to harsh pollutant environments.
Fig. 1 | Gene circuit 1 of quorum sensing system with LuxI
Fig. 2 | Gene circuit 2 of quorum sensing system with LuxRm
Fig. 3 | Gene circuit 3 of quorum sensing system showing how positive feedback loop is formed
When applied to specific scenarios, LuxI responds first. After being activated by its corresponding promoter plac, LuxI generates AHL signaling molecules. These molecules increase simultaneously with the concentration of the bacterial concentration. As AHL signaling molecules accumulate continuously in the bacteria, they bind to LuxRm and form a complex.
The mutated LuxR that we use is composed of two modules or domains, an amino-terminal regulatory domain and a carboxy-terminal transcriptional activating domain(Dunlap. 1999). The amino-terminal domain of AHL binding regulates the activity of the carboxy-terminal domain, which binds to DNA and activates the transcription of the lux operon. In the absence of AHL, the amino-terminal domain prevents the carboxy-terminal domain from binding to DNA. The binding of the amino-terminal domain to AHL is interpreted as altering this interaction, allowing the carboxy-terminal domain to then bind to the lux regulatory region and activate the transcription of the lux operon. Alternatively, the amino-terminal domain may prevent the proposed multimerization of LuxR in the transcriptional function. In the presence of AHL, this interaction will be changed, allowing LuxR dimers to form and bind to the palindromic symmetric lux box. The LuxR–AHL complex cooperates with RNA polymerase to promote the binding of RNA polymerase to the lux operon promoter and initiate transcription. As a result, the downstream gene circuits are successfully activated.
This is the basic operation process of our quorum sensing system. However, this common quorum sensing mode is unable to fully meet our expectations for its efficiency and sensitivity, it still imposes a considerable burden on the bacteria and cannot quickly degrade pollutants. Therefore, the quorum sensing system we designed is more advanced and efficient. Specifically, in our quorum sensing system, we place the position of pLuxRm before LuxI. In this way, the complex formed by AHL signaling molecules and LuxRm not only initiates the expression of downstream genes circuits but also reactivates the promoter of LuxI, forming a positive feedback loop. In the positive feedback mode, the concentration of the bacterial population can increase several times faster than in the normal quorum sensing system, responding to the requirements more quickly.
Fig.4 | A schematic diagram of the mechanism of the quorum sensing system
Therefore, when the bacterial concentration is low, our quorum sensing system is inactive, and the expressed degradation enzymes and genes expressing anti-toxin and anti-salt proteins will be expressed in relatively small amounts. On the contrary, when the bacterial concentration is high, the quorum sensing system becomes active, that is, the expression of degradation enzymes and genes expressing anti-toxin and anti-salt proteins will be carried out in large quantities. The advantage of this mechanism is that it can be used to balance the growth and degradation work of E. coli, enabling our engineered bacteria to quickly take effect in this work without causing cell growth overload due to a slow increase efficiency.
In summary, this quorum sensing system not only controls the expression of all downstream genes, ensuring that the engineered bacteria can correctly express their functions, but also through the positive feedback formed by LuxI and pLuxRm in the closed circuit, rapidly reaches the threshold of the bacterial population density and activates downstream genes. This mechanism ensures that degradation and resistance genes are only activated at high biomass, balancing metabolic load and optimizing energy utilization. This regulation significantly improves the overall efficiency and scalability of the system, representing a significant improvement compared to previous studies on degrading AH substances.
2. Degradation module
The two targeting AHs that we mainly focused are dibenzofuran (DF) and toluene. These two compounds were selected because they are chemically recalcitrant and thus pose significant challenges for conventional degradation methods. However, our engineered bacteria can easily and efficiently solve these problems.
As mentioned in the quorum sensing section, our quorum sensing system is responsible for activating and provoking all downstream gene expressions, including the gene circuits which expresses degrading proteins. These gene circuits can be further categorized into two parts: the dfd gene cluster, which targets the dibenzofuran (DF) and the Xyl gene cluster, which targets the toluene.
The dfd gene clusters are cloned from a cosmid library of the DBF63 genome. It includes dfdA1234, dfdB and dfdC genes which all work to together for DF degradation(Kasuga, Nitta & Kobayashi. 2013).
Fig.5 | Gene circuit of dfd gene clusters
dfdA1 and dfdA2 initiates the degradation by encoding the large and small subunits of the oxidase respectively.This process generates 2,2′,3-trihydroxybenzene (THB) through the oxidation of DF.
dfdB and dfdC work closely together to convert the THB produced in the previous step into salicylic acid. Specifically, the former acts as a diol dehydratase, preparing for the subsequent hydrolysis by meta-cleavage of THB, while the latter functions as a hydrolase, further converting the product with the methylene position breakage into salicylic acid as a final degrading product.
Additionally, dfdA3 encodes the ferredoxin used for electron transfer and dfdA4 encodes the reductase to assists the above processes.
Compared to DF, salicylic acid is not considered to be a substance of very high concern (SVHC) for the environment according to ECHA(European Chemicals Agency. 2024). Therefore, the degradation is considered to be effective.
Fig. 6 | Degrading mechanism of dibenzofuran by dfd gene circuit
For the Xyl gene circuit, we only adopted a part of the gene cluster of the upper regulatory region on the TOL plasmid pWW0 mentioned in the references. These genes includes XylUWMABC, which we integrated them into the Xyl gene cluster for our own engineered bacteria responsible for degrading toluene(Assinder & Williams. 1990).
Fig.7 | Gene circuit of Xyl gene clusters
XylM and xylA work first in the degradation of toluene. They are both xylene oxidase (XO) which promote the occurrence of the single oxygenation reaction, converting toluene into benzyl alcohol.
XylB then immediately converted benzyl alcohol into benzaldehyde. XylB is a benzyl alcohol dehydrogenase (BADH), which can catalyze dehydrogenation reactions.
XylC works at last to convert benzaldehyde to benzoic acid as a final product also through dehydrogenation process.
Compared to toluene, benzoic acid is lot more stable in the environment and has a relatively low acute toxicity to aquatic organisms(Industrial Chemicals. 2022). Therefore, the degradation is considered to be effective.
It’s true that toluene exists in natural environment and is highly necessary to be seriously dealed with, so we made our engineered bacteria capable of degrading toluene theoretically. However, in China, toluene is classified as a hazardous chemical and its use is subject to strict legal regulations. According to the ‘Regulations on the Safety Management of Hazardous Chemicals’ (Order No. 591 of the State Council), no unit or individual is allowed to use hazardous chemicals in violation of the restrictive provisions(State Council of the People’s Republic of China. 2011). In this case, we are not allowed to test the ability of our engineered bacteria on degrading toluene, but it’s still anticipated in the future.
To sum up, in the degradation part of our work, we mainly utilized the xfd and xyl gene circuits to effectively convert two highly harmful AHs, DF and toluene, into substances with extremely low environmental hazards. The former has been experimentally proven to be effective and efficient, demonstrating the superiority of our method over traditional methods. However, the latter could not be proven through experiments due to some unavoidable factors. But overall, it is clear that this degradation system, regulated by the quorum sensing system, can efficiently and quickly complete the main degradation tasks of the engineered bacteria.
3. Resistance module
In resistance module, our goal is to construct a resistance module regulated by a quorum sensing system that enhances the adaptability and survival of E. coli under high-salt and toxic environments. The core idea is to detect cell density using signaling molecules and only activate resistance genes when it’s actually needed. This can help avoid unnecessary energy consumption and metabolic burden. Each gene is preceded by a ribosome binding site for efficient translation. Meanwhile, each protein is labeled with an LAA-LAA degradation tag which can quickly target proteins for degradation. This tag facilitates the targeted degradation of membrane-associated proteins, preventing their excessive accumulation and maintaining cellular balance. We achieve coordinated resistance to saline and chemical stress through the regulation of ion balance and active efflux in this module.
We integrated NhaA ion exchanger and AcrAB-TolC efflux pump system to provide E. coli with salt tolerance and toxin resistance, respectively.
Fig. 8 | Gene circuit of salt and toxin resistance module
NhaA is located in the inner membrane and functions as an antiporter of Na⁺ and H⁺. It is most active under alkaline stress, especially at pH 8.0 - 8.5, and becomes inactive under acidic conditions. NhaA exports one Na⁺ ion while importing two H⁺ ions, which helps the cell lower its intracellular pH and reduce sodium toxicity. This activity enables cells to maintain internal balance under high-salt environments, contributing to improved osmotic tolerance (Smith, B & Fernando. 2024).
Fig. 9 | The transport function of the NhaA antiporter
In parallel, the AcrAB–TolC system constitutes a continuous transmembrane efflux channel that spans from the inner membrane to the extracellular space. It is composed of three essential components: AcrB, AcrA, and TolC.
AcrB is a proton-motive-force–driven transporter embedded in the inner membrane that actively recognizes and captures diverse toxic substrates from the cytoplasm and periplasm. AcrA, located in the periplasm, functions as an adaptor protein that bridges AcrB and TolC, stabilizing the entire complex and facilitating substrate translocation. TolC forms a cylindrical channel in the outer membrane that serves as the final exit duct for expelled compounds.
Together, they constitute a continuous transmembrane conduit that actively exports harmful metabolites and complex organic pollutants, thereby minimizing intracellular accumulation of toxic compounds and safeguarding cellular homeostasis. According to previous article, under pollutant stress, transcriptional levels of global regulators and efflux components are significantly upregulated, highlighting the central role of efflux pumps in bacterial resilience (Su. 2025). By incorporating these elements, our module enhances E. coli’s robustness in chemically challenging environments.
Fig. 10 | AcrAB-TolC system working as drug efflux pump
4. Biosafety module
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References
1. Dunlap, P. V. (1999). Quorum regulation of luminescence in Vibrio fischeri. Journal of Molecular Microbiology and Biotechnology, 1(1), 5–12.
2. Kasuga, K., Nitta, A., Kobayashi, M., et al. (2013). Cloning of dfdA genes from Terrabacter sp. strain DBF63 encoding dibenzofuran 4,4a-dioxygenase and heterologous expression in Streptomyces lividans. Applied Microbiology and Biotechnology, 97(10), 4485–4498.
3. European Chemicals Agency. (2024). Registered substance – Salicylic acid. Retrieved July 19, 2024.
4. Assinder, S. J., & Williams, P. A. (1990). In A. H. Rose & D. W. Tempest (Eds.), Advances in microbial physiology (Vol. 31, pp. 1–69). Academic Press
5. Industrial Chemicals (Australia). (2022, June). Benzoic acid: Human health tier II assessment. Australian Government Department of Health and Aged Care.
6. State Council of the People’s Republic of China. (2011, March 11). Regulations on the safety management of hazardous chemicals. The State Council of the People’s Republic of China.
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9. Smith, B. L., Fernando, S., & King, M. D. (2024). Escherichia coli resistance mechanism AcrAB-TolC efflux pump interactions with commonly used antibiotics: a molecular dynamics study. Scientific Reports, 14(1), 2742.
10. Su, C., Cui, H., Wang, W., Liu, Y., Cheng, Z., Wang, C., ... & Tang, H. (2025). Bioremediation of complex organic pollutants by engineered Vibrio natriegens. Nature, 1-10.