Description

I. Problems Addressed by the Project and Potential Impacts

(I) Problems Addressed

  Resolve the issue of polycyclic aromatic hydrocarbon (PAHs) pollution, particularly the challenge that two simple PAHs—naphthalene (highly toxic) and phenanthrene (difficult to degrade and prone to bioaccumulation)—cannot be efficiently and synergistically degraded by the existing scattered degradation pathways in nature.

  Overcome the functional limitations of degradation genes in single bacterial strains. For instance, the nahC gene in the nah gene cluster of Pseudomonas putida G7 has insufficient affinity for substrates, preventing the efficient completion of the naphthalene degradation cycle.

(II) Potential Impacts

  Environmental Aspect: Contribute to the remediation of soil and water contaminated by PAHs, reduce their accumulation in the food chain, mitigate long-term toxicity to ecosystems, and maintain the balance of the biosphere.

  Application Aspect: Provide a replicable "synergistic degradation" model for solving environmental pollutant degradation using synthetic biology, which may be extended to the treatment of other refractory pollutants in the future.

II. Project Goals and Objectives

(I) Core Goal

  Construct an artificial shared degradation cycle for naphthalene and phenanthrene, enabling the originally "isolated" natural PAH degradation pathways to work synergistically through synthetic biology technologies.

(II) Specific Objectives

  1. Endow engineered bacteria (modified E. coli) with the ability to produce rhamnolipids, allowing weakly polar naphthalene and phenanthrene to detach from soil particles and enter cell membranes.

  2. Address the functional gap in the naphthalene degradation pathway, solve the problem of insufficient substrate affinity of nahC, and ensure the efficient completion of the degradation cycle for both naphthalene and phenanthrene.

III. Reasons for Selecting This Project

  1. Urgency of Pollution Hazards: PAHs enter soil, water, and the biosphere through atmospheric deposition, exhibiting bioaccumulation effects. As the number of aromatic rings increases, their carcinogenicity and persistence intensify, posing significant harm to humans and animals. Effective degradation methods are urgently needed.

  2. Defects in Existing Degradation Pathways: Natural PAH degradation pathways are scattered and isolated. The gene clusters of single bacterial strains (e.g., Pseudomonas putida G7, Burkholderia) have functional limitations, failing to efficiently and synergistically degrade multiple PAHs (such as naphthalene and phenanthrene).

  3. Technical Feasibility: The degradation pathways of naphthalene and phenanthrene share commonalities. Key functional genes (e.g., rhl, nah series, phnC) have been identified and can be integrated into E. coli via genetic engineering, providing a technical foundation for achieving synergistic degradation.

IV. Inspiration Behind the Project

(I) Real-World Problems as Drivers

  PAHs are continuously released during automobile exhaust emissions, waste incineration, wildfires, and petroleum refining processes, leading to their massive accumulation in the environment. As widespread persistent organic pollutants, existing treatment methods are inefficient. This real-world pollution issue serves as the core driving force for the project.

(II) Support from Previous Research

  It is known that Pseudomonas can secrete rhamnolipids to help weakly polar organic compounds detach from soil particles, providing ideas for equipping engineered bacteria with "pollutant capture" capabilities.

  It has been confirmed that the nah gene cluster of Pseudomonas putida G7 can initiate the naphthalene degradation pathway, and the phn gene cluster of Burkholderia can degrade phenanthrene. Additionally, phnC can compensate for the functional defects of nahC. These previous studies provide a scientific basis for the integration of gene combinations.

V. Relevant Scientific Background, Technical Details, and Experimental Methods

(I) Scientific Background

  1. Properties of Polycyclic Aromatic Hydrocarbons (PAHs): Aromatic hydrocarbons containing two or more benzene rings. An increase in the number of rings enhances electrochemical stability, persistence, resistance to biodegradation, and carcinogenicity. Naphthalene is highly toxic to aquatic animals, while phenanthrene is difficult to degrade and easily accumulates in the food chain.

   2. Basics of Biodegradation: Specific gene clusters in certain microorganisms can initiate PAH degradation. For example, the nah gene cluster is responsible for naphthalene metabolism (converting naphthalene to salicylic acid), and the phn gene cluster is responsible for phenanthrene degradation. Biosurfactants (e.g., rhamnolipids) can assist pollutants in entering microbial cells.

(II) Technical Details

  3. Functions of Key Genes
  (1)rhl Gene (from Pseudomonas aeruginosa): Controls the synthesis of rhamnolipids, enabling naphthalene and phenanthrene to detach from soil particles and enter the cell membrane of E. coli.
  (2)nahAB Genes: Initiate the initial steps of naphthalene degradation, laying the foundation for subsequent metabolism.
  (3)phnC Gene (from Burkholderia): Acts on substrates that cannot be processed by nahC, filling the gap in the naphthalene degradation pathway.
  (4)nahD-G Genes: Cooperate with nahAB and phnC to complete the naphthalene metabolism cycle, while adapting to the phenanthrene degradation pathway to achieve shared degradation of naphthalene and phenanthrene.

  4. Logic for Engineered Bacteria Construction: Integrate the rhl, nahAB, phnC, and nahD-G genes into E. coli to combine the full-process functions of "pollutant capture → naphthalene degradation → phenanthrene degradation → cycle completion," forming an artificial synergistic degradation system.

(III) Experimental Methods

  1. Gene Cloning: Clone the rhl gene from Pseudomonas aeruginosa, the nahAB and nahD-G genes from Pseudomonas putida G7, and the phnC gene from Burkholderia respectively.

  2. Vector Construction: Insert the cloned genes into appropriate expression vectors to ensure the normal expression of each gene in E. coli.

  3. Transformation and Screening: Transform the constructed vectors into E. coli, and screen for engineered bacteria capable of normal growth using a selection medium (containing naphthalene or phenanthrene) to verify their degradation ability.

  4. Degradation Efficiency Detection: Measure the concentration changes of naphthalene and phenanthrene in the engineered bacteria culture system to confirm the effective operation of the artificial degradation cycle.

VI. Flow Chart of Naphthalene-Phenanthrene Shared Degradation Cycle

  Step 1: Engineered bacteria secrete rhamnolipids to emulsify naphthalene/phenanthrene in soil.

  Step 2: nahAB initiates the process → formation of metabolic intermediates → phnC acts on substrates that cannot be processed by nahC.

  Step 3: nahG catalyzes the conversion to complete a cycle.

  Step 4: The degradation of phenanthrene shares some intermediate steps with naphthalene, with phnC dominating key reactions. Finally, complete degradation is achieved together, producing harmless substances.

Reference

  [1]Kaur, T., Lakhawat, S. S., Kumar, V., Sharma, V., Neeraj, R. R. K., & Sharma, P. K. (2023). Polyaromatic Hydrocarbon Specific Ring Hydroxylating Dioxygenases: Diversity, Structure, Function, and Protein Engineering. Current protein & peptide science, 24(1), 7–21.

  [2]Soberón-Chávez, G., Lépine, F., & Déziel, E. (2005). Production of rhamnolipids by Pseudomonas aeruginosa. Applied microbiology and biotechnology, 68(6), 718–725.

  [3]Habe, H., & Omori, T. (2003). Genetics of polycyclic aromatic hydrocarbon metabolism in diverse aerobic bacteria. Bioscience, biotechnology, and biochemistry, 67(2), 225–243.

  [4]Laurie, A. D., & Lloyd-Jones, G. (1999). The phn genes of Burkholderia sp. strain RP007 constitute a divergent gene cluster for polycyclic aromatic hydrocarbon catabolism. Journal of bacteriology, 181(2), 531–540.

  [5]Ochsner, U. A., Fiechter, A., & Reiser, J. (1994). Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. The Journal of biological chemistry, 269(31), 19787–19795.

  [6]Soberón-Chávez, G., Lépine, F., & Déziel, E. (2005). Production of rhamnolipids by Pseudomonas aeruginosa. Applied microbiology and biotechnology, 68(6), 718–725.