The widespread use of glyphosate in tea plantations has led to significant residue issues, directly affecting tea quality and exports. Our team’s inspiration came from a news report[1], which mentioned that a tea plantation was closed due to excessive glyphosate residues. This sparked our interest in glyphosate degradation technology. Given the extensive use of glyphosate and its long-lasting residue, developing an efficient and safe biodegradation method became critical. Such a technology would not only improve the quality of agricultural products but also effectively protect the environment.

Glyphosate is a broad-spectrum organophosphorus herbicide[2], widely used in agriculture due to its efficiency, low cost, and non-selective weed control properties. It is typically mixed with surfactant formulations and prepared as water-based sprays, with “Roundup” being a representative product[3]. Pure glyphosate is a white, non-volatile solid powder, with a density of 1.74 g/mL, a melting point of 230°C, and the molecular formula C₃H₈NO₅P. Its molecular structure contains hydrophilic groups like hydroxyl, carboxyl, and amino groups, making it water-soluble but insoluble in most organic solvents. It is relatively stable under normal conditions, which simplifies storage. However, it is difficult to degrade in the environment, leading to persistent residues during use[4].

Although glyphosate is widely used globally, particularly in tea plantations and other crops, concerns about its environmental and health impacts have gradually emerged. Glyphosate decomposes slowly in soil, leading to accumulation and affecting soil microorganisms and ecosystems. Additionally, its primary degradation product, AMPA (aminomethylphosphonic acid), is also toxic[5], further exacerbating environmental pollution. Studies have shown that glyphosate and its metabolites, once they enter the human body through the food chain, may adversely affect the endocrine system, liver, kidney functions, and the reproductive system. Prolonged exposure to glyphosate-contaminated environments may lead to severe health issues, including endocrine disorders and potential carcinogenic risks[6].

The common methods for treating glyphosate include chemical degradation, physical treatment, and biodegradation. While these methods can partially address the glyphosate residue problem under different conditions, they also have significant limitations. For instance, chemical degradation acts quickly but may result in secondary pollution; physical methods offer only short-term removal of glyphosate without fully degrading it; and while biodegradation is eco-friendly, current applications are inefficient, with long degradation cycles, making practical implementation difficult. Below is a detailed comparison of these methods[8].

Table 1: Current Glyphosate Solutions and Their Limitations

Although these methods can partially address the glyphosate residue issue, none can achieve efficient, safe, and pollution-free degradation simultaneously. Therefore, developing an innovative solution that is both efficient and controllable has become an urgent need.

To address the current glyphosate residue issue in tea plantations, there is an urgent need for an innovative and efficient solution. The ideal degradation method should efficiently and safely break down glyphosate in the soil, avoiding secondary pollution. The bio-degradation method using genetically engineered microorganisms can effectively meet this need. By designing E. coli to rapidly absorb and degrade glyphosate, combined with a suicide system to prevent the engineered bacteria from spreading into the environment, this approach represents a promising solution. This innovative biodegradation technology not only improves glyphosate degradation efficiency but also reduces the negative impact on the environment and crops.

To efficiently degrade glyphosate, our project employs synthetic biology to design a multi-module system in E. coli . Glyphosate can be degraded through two pathways: C-N cleavage and C-P cleavage. Under natural conditions, most microorganisms break down glyphosate via C-N cleavage, producing AMPA (aminomethylphosphonic acid) and glyoxylate, but AMPA is highly toxic. It can persist in the soil for extended periods, disrupting soil microbial communities and harming ecosystem health. Additionally, once AMPA enters the human body through the food chain, it may cause endocrine disruption and nervous system disorders, particularly affecting the immune system, liver, kidneys, and reproductive system. Therefore, AMPA poses multiple environmental and health risks.

In contrast, the C-P cleavage pathway breaks the carbon-phosphorus bond, directly producing non-toxic phosphate (Pi) and glycine, a naturally occurring amino acid that can be further metabolized or utilized. This makes the C-P cleavage pathway safer and more efficient, as it degrades glyphosate without generating harmful by-products. Hence, our design incorporates the C-P cleavage pathway to minimize environmental and biological toxicity risks, ensuring safe glyphosate degradation.

Based on literature research and experimental validation, we selected relevant key genes from different bacterial species and introduced them into E. coli , enabling the engineered bacteria to absorb, degrade glyphosate, and further process AMPA. Additionally, we integrated a biosafety suicide system to ensure that the engineered bacteria are safely eliminated after completing their task, preventing environmental contamination.

Effective degradation of glyphosate requires its efficient transport into E. coli cells. We introduced the phnE1 and phnE2 genes from Sinorhizobium meliloti 1021[11], which encode phosphonate acid transporters. Studies have shown that Sinorhizobium meliloti 1021[12] can absorb 85% of glyphosate within less than an hour. By expressing the PhnE proteins, glyphosate is quickly transported across the cell membrane, enabling subsequent degradation.

The critical step in glyphosate degradation is C-P cleavage, which depends on the phnJ gene. This gene encodes the carbon-phosphorus lyase enzyme that breaks down glyphosate into glycine and inorganic phosphate. We selected the phnJ gene from Enterobacter cloacae K7[13], as studies indicate that this strain can degrade 40% of glyphosate within five days. In contrast to the more common C-N cleavage pathway, C-P cleavage avoids the formation of AMPA, reducing environmental toxicity.

AMPA (aminomethylphosphonic acid), a byproduct of glyphosate degradation, is still toxic and must be further processed. We introduced the phnO gene[14], which encodes the enzyme aminoalkylphosphonate N-acetyl-transferase. This enzyme converts AMPA into derivatives that are easier to handle, preventing secondary pollution in the environment. AMPA degradation is a crucial component of the system’s safety, ensuring the entire degradation process remains non-toxic and harmless.

To prevent the engineered bacteria from spreading into the natural environment, we designed a suicide system based on the pCspA cold-inducible promoter and the mazF toxin gene. The pCspA promoter is activated in cold outdoor conditions[15], initiating the expression of the mazF gene[16]. MazF is an mRNA endonuclease that recognizes and cleaves the ACA sequence in mRNA, halting protein synthesis and ultimately causing cell death. This design ensures that E. coli is automatically eliminated after completing its degradation task, preventing any potential biological leakage.

In our design, the glyphosate degradation system and the suicide system work in harmony to achieve both efficient degradation and biosafety. Glyphosate is transported into the E. coli cell via the PhnE transporter proteins. Once inside, the PhnJ enzyme, through the C-P cleavage pathway, breaks down glyphosate into non-toxic glycine and phosphate. Additionally, AMPA, which may be produced, is further degraded by PhnO enzyme, ensuring environmental safety.

Although the suicide system is activated immediately upon the engineered bacteria being released into the environment, glyphosate degradation occurs rapidly—faster than the activation of the suicide system. Therefore, the degradation process remains unaffected. The pCspA cold-inducible promoter is activated in low-temperature environments, triggering the expression of the mazF gene. The MazF toxin takes time to accumulate and disrupt bacterial mRNA, ultimately leading to cell death. Due to this delay, the bacteria will self-destruct only after completing the glyphosate degradation task, ensuring full glyphosate degradation and preventing biological leakage.

First, the engineered bacteria will be produced through fermentation in a microbial factory and preserved as freeze-dried powder. These bacterial products will be sold to professional tea garden biological treatment personnel. The technicians will activate the freeze-dried powder and dilute it to the appropriate concentration before using a pesticide sprayer or other spraying equipment to apply it directly to areas contaminated by glyphosate. The bacteria will then quickly absorb and degrade the glyphosate. Simultaneously, the suicide system will activate to ensure that after completing the degradation task, the bacteria self-destruct to avoid any risk of spreading. The entire process is efficient, convenient, and easy to operate.

In addition to glyphosate pollution management in tea plantations, this engineered bacteria system can be applied widely across other agricultural and environmental fields. It can be used in farmland, orchards, lawns, and other areas contaminated by glyphosate, providing rapid removal of herbicide residues and protecting crop growth. Furthermore, the engineered bacteria can be employed for water treatment, removing pesticide residues from water sources and preventing pollution from spreading downstream. This product has the potential for use in multiple ecological environments, helping to reduce the long-term harmful effects of glyphosate on natural ecosystems and enhancing food and water safety.

Utilizing the C-P cleavage pathway, glyphosate is rapidly degraded into non-toxic phosphate and glycine, avoiding the production of harmful by-products like AMPA, and improving degradation efficiency.

The suicide system ensures that the engineered bacteria self-destruct after completing their task, preventing the spread of genetically modified bacteria into the environment, in compliance with biosafety standards.

The system does not produce toxic or harmful residues after degradation, ensuring environmental safety and allowing subsequent crops to grow without interference.

The freeze-dried bacteria are easy to activate and dilute for use, making the application process simple and efficient, ideal for widespread adoption.

This technology is applicable in tea plantations, farmlands, water bodies, and other glyphosate-contaminated areas, demonstrating significant potential for ecological restoration.

This innovative engineered bacteria system offers a breakthrough solution for global agricultural and environmental management. As the issue of glyphosate pollution becomes increasingly severe, our technology will help global agricultural practices move towards sustainable development. This project not only promotes green agriculture but also provides long-term safety for environmental protection and public health. In the future, our technology will be integrated into global agricultural innovation strategies, aiming to reduce chemical pollution, improve soil quality, and contribute to the restoration and sustainable development of ecosystems. It will also support the achievement of the United Nations’ Sustainable Development Goals, including clean water and sanitation, and responsible consumption and production.

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