HUMANPRACTICE

In agricultural production, efficient nitrogen fixation and nematode control are critical issues for boosting legume yields and advancing green agriculture. While traditional chemical nitrogen fertilizers provide short-term nitrogen supplementation, they often lead to soil compaction and fertility decline. Parasitic nematodes, like root-knot nematodes, damage crop roots, disrupting nutrient uptake pathways and inhibiting symbiotic interactions between nitrogen-fixing bacteria and plants. This creates a dual constraint of “nitrogen deficiency-nematode damage,” severely undermining agricultural sustainability. Our team is committed to addressing this challenge by exploring low-cost, environmentally friendly solutions through synthetic biology.

Literature studies indicate that luteolin serves as the core signaling molecule enabling legumes to establish symbiotic relationships with rhizobia. It specifically attracts nitrogen-fixing bacteria in the soil to aggregate around roots and induces nodule formation, significantly enhancing biological nitrogen fixation efficiency. simultaneously, when plants suffer nematode infestation, roots mass-produce salicylic acid as a stress response signal. Cry proteins derived from Bacillus thuringiensis exhibit specific toxic effects against multiple plant-parasitic nematodes while maintaining high safety for beneficial soil organisms. Furthermore, as an environmentally responsive molecular switch, salicylic acid has been demonstrated in synthetic biology systems to enable precise regulation of “stress signal-target gene expression,” offering a viable pathway for dynamic nematode control.

Integrating existing research foundations with agricultural production needs, we decided to focus on the targeted design of engineered bacteria: using synthetic biology to construct dual-functional engineered bacteria. On one hand, they continuously synthesize luteolin to enhance attraction to nitrogen-fixing bacteria, promoting root nodule colonization and efficient nitrogen fixation; while also endowing them with a salicylic acid sensing module. When nematode feeding on roots elevates salicylic acid concentrations, the engineered bacteria rapidly activate cry protein expression, achieving precise nematode repellency. This approach simultaneously addresses the agricultural challenges of low nitrogen fixation efficiency and nematode damage.

To precisely define the project direction and solidify its theoretical foundation, we made a special visit to Professor Su Chao at Huazhong Agricultural University during the project's initial phase. Professor Su has long been engaged in research on nitrogen fixation through the legume-rhizobium symbiosis. We conducted in-depth interviews centered on three core topics: “Current Practices in Agricultural Nitrogen Fertilizer Application,” " Nitrogen Fixation Molecular Mechanisms of Rhizobia,“ and ”Regulatory Characteristics of Flavonoids on Nitrogen-Fixing Bacteria." These discussions provided critical academic references for project design.

Regarding pain points in the agricultural nitrogen fertilizer market and application, Professor Su highlighted, based on his team's years of field survey data: In current global agricultural production, traditional chemical nitrogen fertilizers account for over 70% of nitrogen inputs, yet crop utilization rates remain only 30%-40%. Unabsorbed nitrogen leaches or volatilizes into water bodies and the atmosphere, causing environmental issues such as water eutrophication and soil acidification. While biological nitrogen fixation (primarily through the rhizobium-legume symbiosis) enables “green nitrogen supply,” its efficiency in natural conditions is constrained by factors like soil microbial communities and climate. Commercial application coverage remains below 15%, creating a market gap characterized by “high demand and low efficiency”—a reality that further validates the necessity of our project to “optimize biological nitrogen fixation efficiency through synthetic biology.”

Regarding the molecular mechanism of rhizobial nitrogen fixation, Professor Su systematically dissected the core process of “plant-rhizobium signal interaction”: During growth, legumes secrete flavonoids into the soil. These compounds specifically recognize and bind to the NodD protein on the rhizobium cell membrane, activating expression of the nod gene cluster within the rhizobium. This triggers the synthesis and secretion of Nod factors by the rhizobium. Upon binding to receptors on plant root cell membranes, Nod factors induce root epidermal cell deformation and cortical cell division, ultimately forming nodule structures. Within these nodules, rhizobia differentiate into rhizobial cells, converting atmospheric nitrogen into ammonia via nitrogenase for plant uptake. This mechanism provides a clear molecular target for our subsequent work on “optimizing nitrogen fixation by regulating flavonoid secretion.”

Regarding the regulatory effects of luteolin and naringenin (flavonoids) on nitrogen-fixing bacteria, Professor Su particularly highlighted the current “mechanistic blind spot” in research: Although existing studies have confirmed that both can act as signaling molecules to induce nod gene expression in rhizobia, and while it is known that naringin serves as a precursor to luteolin, the core mechanisms by which they regulate nitrogen-fixing bacteria remain incompletely understood. Based on this understanding, we are more inclined to select luteolin as the core signaling molecule in our subsequent work.

In early experimental planning, we interviewed Professor Peilin Li (UCSF) to validate our approach, presenting two core modules—"Luteolin-Mediated Nitrogen Fixation" and "Salicylic Acid-Induced Nematode Repellency"—based on a single engineered microbe. Professor Li noted that while shared use of one bacterium avoids multi-strain compatibility issues, the modules remain isolated: the nematode repellency system only activates cry proteins via nematode-triggered salicylic acid, lacking coordination for "legume-nitrogen-fixing bacteria-nematode" interactions.

This critique revealed a lack of systemic connectivity. Post-interview, literature reviews clarified nodulation-nematode links: nematodes damage root hairs (critical for rhizobial colonization) and secrete effectors suppressing nodulation genes (e.g., *ENOD40*, *NOD25*), inhibiting nodule formation. Conversely, healthy nodules thicken root cell walls and secrete antimicrobials, reducing nematode invasion—creating a "nodule protection-nematode control" feedback loop. These findings validated Professor Li’s assessment, supporting improved "nitrogen fixation-nematode control" synergy and guiding protocol optimization.

During the initial project design phase, Escherichia coli BL21 was provisionally selected as the host organism based on laboratory operational convenience and exogenous gene expression efficiency. However, during discussions with Professor Sylvain Fisson from Paris-Saclay University and Dr. Fengquan TAN, a postdoctoral researcher at the local Institute of Botany, they raised pertinent questions: As a typical intestinal bacterium, can E. coli BL21 stably survive and proliferate in complex soil environments while continuously secreting the target product, luteolin? This question directly addressed the core bottleneck for project implementation—if the chassis organism cannot colonize soil and function effectively, the subsequent dual-function design of “attracting nitrogen-fixing bacteria and responding to salicylic acid synthesis by producing Cry proteins” would be unattainable.

Guided by this question, we conducted targeted research: Literature review revealed that E. coli BL21 natural habitat is the animal gut, with limited tolerance for soil conditions—low oxygen, high osmotic pressure, and competition from indigenous microbes—making it unsuitable for the agricultural requirement of “long-term colonization and sustained activity.” Following this analysis, we ultimately switched the chassis organism to Bacillus subtilis BS168 —a recognized dominant soil microorganism with exceptional soil colonization ability. It forms stress-resistant spores to withstand adverse conditions, possesses a mature secretion system, and is non-pathogenic. This strain perfectly aligns with the project's application scenario of “synergistic interaction with legume root systems and nitrogen-fixing bacteria in soil,” providing critical assurance for the stable operation of the subsequent dual-function system.

While optimizing the chassis organism, Professor Sylvain Fisson and Postdoctoral Fellow Fengquan TAN raised two critical questions regarding the enrichment mechanism and potential risks of the project's core product, luteolin: First, what secretion regulation model should be adopted for luteolin? Should engineered bacteria be allowed to proliferate autonomously in soil after transfer into the carrier and secrete naturally? Or should specific trigger signals be designed to regulate the secretion process? Alternatively, could product release be achieved through cell lysis? Second, as an exogenous synthetic molecule, could luteolin pose toxicity to the engineered bacteria themselves? Furthermore, could its excessive accumulation inversely inhibit the colonization of nitrogen-fixing bacteria and nodule formation in the soil, thereby compromising nitrogen fixation efficiency?

To address the regulation of luteolin secretion, we conducted literature reviews and molecular biology tool screening. Ultimately, we established a controllable synthetic pathway based on the cre-loxp site-specific recombination system: placing the tandem sequence of luteolin synthesis-related enzyme genes between loxp sites. In the initial state, this pathway remains “off” due to promoter silencing, preventing blind secretion by engineered bacteria during non-target phases. Simultaneously, we selected a mild phage as the delivery vector for the cre recombinase gene. When the phage carrying the cre gene infects the engineered bacteria, it integrates the cre gene into the bacterial genome, where the cre recombinase recognizes and cleaves the loxp sites, thereby activating the luteolin synthesis pathway. This achieves precise regulation of “phage-triggered product synthesis,” ingeniously utilizing the phage as a molecular switch.

Regarding the safety risks of luteolin, systematic literature review revealed that concentrations exceeding 50 μmol/L in soil significantly inhibit signal recognition between nitrogen-fixing bacteria and legume roots, reducing nodule formation by over 30%. Additionally, high luteolin concentrations mildly suppress growth in some indigenous soil microorganisms. To address this issue, we further optimized the system design by leveraging the biological characteristics of temperate phages: We screened for temperate phages from soil that, upon infecting engineered bacteria, do not immediately cause cell lysis. Instead, they enter a lysogenic cycle alongside the engineered bacteria's proliferation. However, after approximately ten to twenty generations of bacterial growth, the phage initiates a lytic cycle, causing the engineered bacteria to undergo natural lysis and death. This design ensures that engineered bacteria continuously synthesize and secrete luteolin before lysis, meeting the requirements for attracting nitrogen-fixing bacteria. Simultaneously, it strictly controls the survival period and total population of engineered bacteria in the soil through cell lysis, preventing excessive accumulation of luteolin. It also eliminates potential ecological risks associated with long-term colonization of engineered bacteria, achieving a dual balance between product functionality and environmental safety.

While our experiments progressed steadily according to plan, we participated in the Conference of China iGEMer Community in early August. As a core academic exchange platform independently initiated by domestic iGEM teams, we engaged in targeted discussions with multiple Chinese iGEM teams regarding synthetic biology project development. We shared the design rationale and preliminary experimental progress of our “Luteolin-Mediated Nitrogen Fixation” and “Salicylic Acid-Sensing Nematode Repellency” modules, while also focusing on learning from other teams' practical experiences in functional regulation of engineered bacteria and experimental data optimization. During the event, one team's concept of “multi-signal molecule synergistic response” provided fresh perspectives for refining the linkage mechanisms between our two modules. Simultaneously, we shared preliminary literature reviews and pre-experimental conclusions addressing challenges encountered by some teams in nitrogen fixation-related experiments. This brief yet highly productive exchange not only enriched our project optimization strategies but also accumulated valuable peer insights for preparing for future iGEM competitions.