Global food security is increasingly challenged by climate change, land degradation, and pesticide pollution. In North China, the soybean–wheat rotation improves resource use efficiency, enhances soil fertility, and increases yield; however, the widely used herbicide chlorimuron-ethyl is recalcitrant and persists in soil, posing serious threats to wheat germination in the following season and to the ecosystem. Existing remediation strategies—phytoremediation, microbial degradation, and engineered/chemical remediation—often suffer from low efficiency, high cost, or limited applicability.

To address this, we developed a synthetic-biology solution based on engineered E. coli. We first constructed a strain expressing carboxylesterase PnbA and implemented ice nucleation protein (INP)-mediated cell-surface display to achieve efficient degradation of chlorimuron-ethyl. Next, we introduced the tryptophan–indole-3-acetamide (IAM) pathway to biosynthesize indole-3-acetic acid (IAA), thereby promoting root development and restoring soil fertility. In parallel, a temperature-controlled biosafety mechanism was established using the CspA cold-inducible promoter to drive the T4 lysis system, ensuring environmental controllability in field applications. Finally, the product is formulated as lyophilized bacterial powder for storage and transport; before use, it is reconstituted and sprayed 7–10 days between soybean harvest and wheat sowing, delivering a dual benefit of pesticide-residue degradation and crop growth promotion.

This approach has the potential to provide rotation agriculture with a novel biological tool that is efficient, safe, and scalable for pesticide-residue remediation and yield enhancement.

Inspiration

While scrolling on Douyin, our teammates learned that many regions still face a food crisis. On one hand, climate change, environmental pollution, and water scarcity threaten agricultural production, leaving food output insufficient for local populations. On the other hand, food waste and inequitable distribution exacerbate the problem, particularly in some developed countries and impoverished areas. Moreover, as the global population continues to grow, food demand keeps rising, whereas current agricultural practices have yet to respond effectively to this challenge.

Figure 1. Global Hunger Map [1]

According to a WHO report released on July 28, 2025, global hunger has improved overall, yet the situation remains severe in Africa and Western Asia. Worldwide declines are attributed to higher production efficiency, international aid, and policy support, but Africa and Western Asia show an upward trend in hunger, especially in regions affected by conflict, climate change, and political instability (see Fig. 1). The report underscores that food security challenges remain complex, calling for stronger international cooperation and more robust measures to ensure the sustainability of food systems, with a particular focus on vulnerable regions. It also highlights food system reforms and strengthened social safety nets as key strategies[2].

In China, rising food demand—driven by economic growth and population increase—is challenged by environmental pressures, limited arable land, and uncertainties from climate change. In several traditional grain-producing regions, land degradation, pollution, and water scarcity threaten sustainable production. Consequently, both the government and farmers have adopted measures such as crop rotation to tackle food security challenges[3].

Crop Rotation

Figure 2. Soybean–wheat–maize rotation schematic [5]

The soybean–wheat rotation shows clear scientific and practical value (Fig. 2). It improves resource-use efficiency, prevents land idling, and leverages seasonal complementarity between summer soybean and winter wheat to fully utilize annual heat–light resources[4]. It also improves soil health: biological nitrogen fixation by soybean reduces nitrogen fertilizer needs for wheat. Studies report wheat yield increases of 2.87%–41.21% over maize–wheat rotations, alongside significant gains in soil organic carbon and available nitrogen. In Yanzhou District, annual economic returns were 34% higher than maize–wheat rotations. With optimized sowing, fertilization, water management, and pest control, benefits can be maximized[6]. Environmentally, rotations enhance soil health, reduce GHG emissions, and increase resilience. Case studies across the North China Plain and Jiangsu show higher yields, greater system stability, and mitigated climate impacts.

Chlorimuron-ethyl

Pesticide residues in rotation systems can adversely affect rooting of subsequent crops[7]. In the soybean–wheat rotation, farmers widely apply chlorimuron-ethyl and other sulfonylurea herbicides to stabilize soybean yields. Although low-cost and fast-acting, chlorimuron-ethyl persists in soil for extended periods (up to >6 months)[8].

Chlorimuron-ethyl inhibits acetohydroxyacid synthase (AHAS/ALS), depleting branched-chain amino acids (valine, leucine, isoleucine), thereby disrupting protein and enzyme synthesis. As these amino acids are essential for plant growth, their deficiency suppresses cell division—particularly in meristems—ultimately causing plant death[9].

Prolonged use of chlorimuron-ethyl harms both the environment and agricultural production. It disrupts soil microbial communities and soil enzyme activities, constraining crop rotation and degrading soil fertility[10]. Once in water bodies, its high solubility and low partitioning to organics facilitate contamination of surface and groundwater, impairing aquatic plant growth[11]. In crops, it suppresses chlorophyll and superoxide dismutase, reducing photosynthesis and nutrient uptake, thereby limiting growth[12]. Long-term exposure further poses ecosystem risks, including DNA damage in aquatic and amphibian species (e.g., the Chinese toad), threatening biodiversity and ecological balance[13].

Figure 3. Chemical structure of chlorimuron-ethyl

The poor degradability of chlorimuron-ethyl stems from its stable chemical structure (notably the sulfonylurea group and chlorine atom), low solubility, strong soil adsorption, limited microbial degradation capacity, and environmental influences. These factors together lead to slow degradation of chlorimuron-ethyl in soil and aquatic systems, resulting in long-term contamination. Current remediation strategies can be categorized into natural remediation and artificial remediation.

Natural Remediation

Phytoremediation and natural microbial degradation are environmentally friendly methods for soil pollution control.

Phytoremediation can significantly reduce pollutant residues in soil and is suitable for moderate to low contamination levels or as a supplementary treatment. However, under high contaminant concentrations, plant growth becomes inhibited, limiting its remediation efficiency[14].

Figure 4. Microbial degradation pathway of chlorimuron-ethyl [15]

Microbial degradation employs specific bacteria or fungi to decompose pollutants, offering distinct advantages. These microbes are highly adaptable in natural environments, can utilize contaminants as energy sources, and operate with low energy demand and long-term stability.

However, the method has limitations:

• First, efficiency depends heavily on strain characteristics—different microbes exhibit variable degradation abilities, leading to inconsistent outcomes.
• Second, environmental conditions such as temperature, humidity, and pH affect microbial activity, limiting degradation.
• Finally, under high contamination, the process becomes slow, making it insufficient for rapid remediation[15] (see Fig. 4).

Artificial Remediation

Table 1. Artificial remediation methods for chlorimuron-ethyl and their comparison

Artificial remediation mainly employs adsorption or alkaline degradation, but these methods are generally costly, inefficient, and prone to undesirable side effects (see Table 1).

Selection of Chassis Microorganism

Among various candidate chassis organisms, we selected Escherichia coli as the engineering host, based on its ease of manipulation, high biosafety level, and strong compatibility with synthetic biology tools.

Compared with native soil microorganisms, E. coli possesses a well-characterized regulatory system and better controllability, minimizing potential pathogenic risks[18].

Moreover, E. coli has been extensively and successfully used in previous iGEM projects, providing a solid foundation for our engineering design.

Chlorimuron-ethyl Degradation System

Figure 5. Schematic illustration of the degradation pathway of chlorimuron-ethyl

Through literature screening, we identified three enzymes capable of efficiently degrading chlorimuron-ethyl:

the esterase SulE from Hansschlegelia zhihuaiae, the carboxylesterase PnbA from strain CHL1, and the glutathione S-transferase (GST) from Klebsiella jilinsis 2N3[19].

Among them, SulE and PnbA degrade sulfonylurea herbicides such as metsulfuron through carboxylester hydrolysis activity[20,21], while GST cleaves the sulfonylurea bridge of chlorimuron-ethyl to achieve degradation[22] (see Figure 5).

We expressed each enzyme in E. coli and compared their catalytic performance; PnbA was selected as our core degradation enzyme due to its superior catalytic efficiency.

Cell Surface Display

During experimental validation, repeated enzyme expression and purification proved laborious and time-consuming. To enhance efficiency, we employed a cell surface display strategy, anchoring PnbA on the outer membrane of E. coli to enable whole-cell catalysis of chlorimuron-ethyl, eliminating the need for complex extraction and purification procedures. The cell surface display system fuses the target protein with an anchoring motif, enabling stable presentation on the microbial surface.

Its performance depends on the carrier protein, fusion orientation, and host characteristics.

This technique has been widely applied in biocatalysis, vaccine development, and bioadsorption[23] (see Figure 6).

Figure 6. Representative cell surface display systems in E. coli.(a) Representative outer membrane protein display system: OmpA; (b) Representative surface appendage display system: pili and flagella; (c) Lipoprotein-derived system: Lpp–OmpA; (d) Representative autotransporter display system: AIDA-I; (e) Representative fluorescent protein display system: sfGFP[23]

Based on the findings above, we selected ice nucleation protein (INP) as the anchoring carrier. INP, originating from plant-associated bacteria such as Pseudomonas syringae, is an anchoring membrane protein initially discovered for its ability to induce ice crystal formation in supercooled water. It has since been widely used in surface display studies in E. coli. INP consists of three structural domains—the N-terminal domain, central repetitive domain (CRD), and C-terminal domain:

• the N-terminal domain is hydrophobic and responsible for anchoring the protein to the outer membrane;
• the CRD comprises tandem repeat sequences that serve as a structural template for ice nucleation;
• and the C-terminal domain is often used as a fusion site for displaying heterologous proteins[24].

Accordingly, we fused INP to the N-terminus of PnbA, enabling its anchoring to the outer membrane via the surface display system, thereby achieving efficient degradation of chlorimuron-ethyl (see Figure 7).

Figure 7. Schematic representation of PnbA expression via the INP surface display system for chlorimuron-ethyl degradation.

IAA Production System

Long-term crop rotation can significantly deplete soil fertility, thereby restricting plant growth and development.

To address this issue, our team—after consulting relevant researchers—designed an IAA expression system that reduces pesticide-related stress while supplying nutrients to promote seed germination.

Indole-3-acetic acid (IAA) is the primary natural auxin in plants and plays a central regulatory role in plant growth and development.

IAA promotes cell elongation and division, controls apical dominance, and stimulates the formation of lateral and adventitious roots, maintaining a balanced root–shoot ratio and facilitating complete root system development.

Moreover, its polar transport establishes concentration gradients that guide organogenesis and vascular differentiation, serving as a key signal in leaf, floral, and fruit development.

IAA also acts synergistically with other phytohormones, enhancing stress adaptability in plants.

Thus, IAA functions as a crucial regulatory molecule across multiple developmental levels, from cellular to whole-plant scale[25].

IAA biosynthesis proceeds via the tryptophan–indole-3-acetamide (IAM) pathway, which involves two enzymatic steps:

first, tryptophan is converted into indole-3-acetamide (IAM) by tryptophan-2-monooxygenase (IaaM) encoded by the iaaM gene;

then, IAM is hydrolyzed into IAA by IAM hydrolase (IaaH) encoded by the iaaH gene[26] (see Figure 8).

Figure 8. Schematic diagram of the heterologous IAA biosynthesis pathway in E. coli

Biosafety System

The soybean–wheat rotation is mainly practiced in North China, typically from September to October. During this period, the region’s temperature gradually decreases: daytime temperatures range from 23–28 °C, while nighttime temperatures fall to 12–19 °C, with an increasing diurnal temperature difference. The beginning of the month remains warm, whereas the end becomes noticeably cooler. Slight variations exist among cities—Beijing and Tianjin average 19–20.5 °C, while Hohhot (Inner Mongolia) averages around 14 °C. Based on these climatic conditions, we selected a cold-inducible promoter to trigger suicide gene expression, ensuring the environmental biosafety of our engineered strain[27].

The CspA promoter (Cold Shock Protein A promoter) originates from the gene region of cold shock protein CspA in E. coli and functions as a cold-inducible promoter. At low temperatures (below 15 °C), its transcriptional activity is strongly upregulated, making it suitable for cold-responsive gene regulation or the low-temperature production of stable target proteins[28] (see Figure 9).

Figure 9. Mechanism of the CspA cold-inducible promoter under low-temperature conditions[28]

Accordingly, we employed the CspA promoter to drive the expression of the T4 lysis system, which includes T4 holin and T4 lysozyme. T4 holin is a small membrane protein containing two transmembrane domains, with its N-terminus and C-terminus located in the cytoplasm and periplasm, respectively. This structure enables holin to form pores on the cell membrane, leading to membrane disruption[29]. T4 lysozyme is a 164-amino-acid globular protein that binds to the bacterial cell wall and cleaves the bond between N-acetylglucosamine and N-ethylmuramic acid, thereby compromising wall integrity, disturbing osmotic balance, and ultimately inducing cell lysis[30]. The synergistic action of these two proteins causes the complete breakdown of the membrane and cell wall, resulting in programmed self-lysis of the engineered cell.

Figure 10. Gene circuit diagram of the temperature-controlled suicide system regulated by the CspA promoter.

We designed a practically applicable formulation and usage strategy for our engineered system (see Figure 11).

The formulation is prepared as lyophilized bacterial powder, applied through aqueous reconstitution and field spraying, with an optimized timing schedule for maximum effectiveness.

Figure 11. Schematic representation of the proposed field implementation process.

Formulation: Lyophilized Bacterial Powder

After cultivation of the engineered E. coli in a fermentation bioreactor, the cells are processed into lyophilized powder through freeze-drying. This formulation allows for long-term storage and transportation, maintaining cell viability and stability at ambient temperature. Farmers can store and apply the product without relying on a cold chain, making it practical for large-scale agricultural use.

Application: Field Spraying after Rehydration

For use, the lyophilized bacterial powder is rehydrated in water and evenly sprayed onto farmland soil. Once in the soil environment, the engineered bacteria activate their functional modules: on one hand, they degrade residual chlorimuron-ethyl, reducing herbicidal toxicity to wheat seeds; on the other hand, they secrete indole-3-acetic acid (IAA), which enhances germination and root development, thereby improving overall emergence rate and seedling quality.

Application Timing: 7–10 Days between Soybean Harvest and Wheat Sowing

We recommend applying the bacterial formulation 7–10 days before wheat sowing, immediately after soybean harvest.This timing allows sufficient duration for degradation of chlorimuron-ethyl residues in soil, preventing germination inhibition in wheat, while IAA secretion concurrently creates a favorable microenvironment for seed germination, achieving dual benefits of early soil recovery and simultaneous growth enhancement.

This project exhibits several notable advantages:

1. Dual Functionality – Capable of efficiently degrading chlorimuron-ethyl while simultaneously promoting plant growth via IAA, combining remediation with yield enhancement.
2. Efficiency and Safety – Enhanced degradation efficiency through esterase surface display, coupled with an environmentally controllable temperature-regulated lysis system.
3. Ease of Operation – The lyophilized bacterial powder formulation allows easy storage, transport, and rehydration for field spraying, enabling wide-scale implementation.
4. Rotation Compatibility – Specifically designed for the soybean–wheat rotation system, aligning with real-world agricultural practices.
5. Sustainability – Effectively reduces reliance on chemical remediation, improves soil health, and contributes to the advancement of green agriculture.

Figure 12. Infographic illustrating the five major advantages of the project

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