DESIGN
OVERVIEW

Exogenous bacterial diseases in plants cause significant harm. Diseases such as bacterial wilt and rice bacterial blight spread through multiple pathways, impairing crop yield and quality. Traditional chemical control methods tend to induce drug resistance in pathogenic bacteria and cause environmental pollution, creating an urgent need for green control approaches.
Erucamide emerges as a promising solution. It can directly inhibit pathogenic bacteria by disrupting their cell membranes and blocking the expression of virulence genes. Additionally, it activates plant immunity, forming an antimicrobial barrier and enhancing broad-spectrum resistance.
This project involves modifying Escherichia coli (E. coli) to transform it into a "cell factory" capable of secreting erucamide. Relevant genes are introduced to enable the synthesis of erucic acid and its subsequent conversion into erucamide. The secretion system is optimized to ensure the release of erucamide to the rhizosphere or surface of plants.
Erucamide secreted by this engineered bacterium can inhibit the movement, attachment, and colonization of bacterial wilt pathogens towards plant roots, while also activating plant immunity to strengthen defense mechanisms. Experiments have demonstrated that it reduces the incidence of bacterial wilt and the mortality rate of infected plants. Furthermore, the engineered bacterium naturally degrades in soil, ensuring safety and environmental friendliness. This model can be extended to the control of other plant diseases, holding great potential for reducing the use of chemical pesticides.
1. SYNTHETIC PATHWAY (GLUCOSE → ERUCAMIDE)
The synthetic process consists of four key stages: carbon metabolism modification, fatty acid synthesis, amidation, and secretory release:
  • Carbon metabolism modification: Glucose → Acetyl-CoA (mediated by PtsG)
  • Fatty acid synthesis: Acetyl-CoA → Fatty acids → Erucic acid (mediated by FabH/KCS)
  • Amidation: Erucic acid → Erucamide (mediated by GlnA)
  • Secretory release: Extracellular secretion of the product
STAGE 2: CARBON CHAIN EXTENSION (CORE "WORKSHOP")
Driven by the synergistic action of the T7 promoter and RBS, the β-ketoacyl-CoA synthase encoded by the KCS gene exhibits strong carbon chain extension capabilities. Using intermediate products of fatty acid synthesis as raw materials, it gradually extends the carbon chain length through repeated enzymatic reactions, ultimately generating erucic acid—a very-long-chain fatty acid with 22 carbon atoms. Erucic acid serves as both a key intermediate in the metabolic process and a "bridge molecule" leading to the target product (erucamide).
STAGE 3: ERUCAMIDE SYNTHESIS
The T7 promoter and RBS initiate the expression process again. The enzyme encoded by the glnA gene acts as a "chemical modifier," adding an amino group to erucic acid through an amidation reaction, thereby converting it into bioactive erucamide. This transformation not only endows the molecule with new biological functions but also allows the product synthesized by microorganisms to cross species barriers and play a role in the plant disease resistance system.
Throughout the process, the expression regulatory system composed of the T7 promoter and RBS acts as a precise "flow controller," ensuring the timing and intensity matching of gene expression in each stage. Enzymes encoded by genes such as PtsG, FabH, KCS, and glnA function like a set of coordinated "molecular machines," gradually converting the chemical energy of glucose into the biological activity of erucamide. This process perfectly embodies the core concept of "design-construction-regulation" in synthetic biology. Finally, the synthesized erucamide is released outside the cell through extracellular secretion, completing the metabolic pathway.
KEY GENES INVOLVED IN SYNTHESIS
  • PtsG: Regulates glucose uptake and carbon metabolism flux
  • The role of PtsG is particularly critical—it is necessary to optimize glucose utilization to support the non-natural metabolic pathway. Under high glucose uptake conditions, PtsG promotes the rapid accumulation of acetyl-CoA through glycolysis and initiates subsequent fatty acid synthesis.
  • FabH: Initiator of erucic acid carbon skeleton synthesis
  • Erucic acid (C22:1 ω-9) is a very-long-chain monounsaturated fatty acid. Its synthesis begins with de novo fatty acid synthesis mediated by FabH: FabH initiates the condensation of C2 units (acetyl-CoA + malonyl-ACP → C4-ACP). Subsequent extensions (mediated by FabB/FabF) form palmitic acid (C16:0), which is then extended to erucic acid by elongases. Thus, FabH plays a role in initiating fatty acid synthesis and providing a precursor (C16:0) for erucic acid.
  • KCS: "Architect" of carbon chain extension
  • The β-ketoacyl-CoA synthase encoded by the KCS gene acts as an "architect" in the fatty acid synthesis pathway. Using intermediates in fatty acid synthesis as substrates, it gradually extends the carbon chain through a series of enzymatic reactions, ultimately promoting the formation of erucic acid.
  • GlnA: Key nitrogen source provider for amide groups
  • The synthesis of erucamide relies on an amidation reaction: Erucic acid + Glutamine → Erucamide + Glutamate. GlnA provides the amide group donor (glutamine) required for the reaction, serving as the direct nitrogen source for erucamide synthesis. It also maintains the intracellular glutamine pool and supports the activity of fatty acid amide hydrolases FAA1/FAA2.
Since E. coli inherently possesses the KCSand KCR genes, no modification of these genes is required. Therefore, the project introduces the other three genes (PtsG, FabH, glnA) into E. coli. The relevant gene sequences of the above three proteins from Aspergillus fumigatus are synthesized, spliced into the pET-28a plasmid using homologous recombination technology, and then the three plasmids are separately introduced into three BL21 (DE3) competent cells for protein purification.
ERUCABEAD: AN INNOVATIVE ENGINEERED BACTERIUM PRODUCT FOR RESISTING EXOGENOUS PLANT PATHOGENS
In current agricultural production, infestation by exogenous plant pathogens—particularly pathogens such as bacterial wilt pathogens—poses a major threat to crop growth:
  • High harm of exogenous pathogens: Exogenous plant pathogens like bacterial wilt pathogens invade plants, causing disease or even death, severely reducing crop yield and quality and leading to significant economic losses for farmers.
  • Limitations of traditional control methods: Traditional pesticides used to control these pathogens either tend to be overused by farmers (causing residues and pollution) or exhibit unstable efficacy, failing to provide sustained and effective control.
2.1 PRODUCT DESIGN CONCEPT: FOCUS ON "PRECISION" AND "EASE OF USE"
To address the infestation of exogenous plant pathogens, ErucaBead—an engineered bacterium product with fixed dosage and simple operation—is designed, drawing inspiration from innovative concepts such as "laundry detergent pods." Its core design concepts are as follows:
  • Fixed-dose packaging: The standardized bead form enables precise dosage of the engineered bacterium, avoiding improper dosage by farmers during use.
  • Simplified operation process: Farmers can easily use the product without cumbersome steps, improving usability.
2.2 CORE COMPOSITION AND FUNCTIONS OF ERUCABEAD
ErucaBead uses high-alkalinity water-soluble polyvinyl alcohol (PVA) as the outer membrane, encapsulating E. coliengineered bacteria capable of secreting erucamide to form a bead-shaped product. Its structure and functions are detailed below:
(1) Outer Membrane: High-Alkalinity Water-Soluble PVA
The PVA outer membrane plays a crucial role in the product and offers numerous advantages:
  • Rapid water solubility: This material contains a large number of hydroxyl groups, resulting in strong hydrophilicity. A 75 μm thick PVA film (100 cm²) can completely dissolve in room-temperature water within approximately 1 minute with stirring. During use, farmers only need to place the beads in a certain volume of water; once dissolved, the solution can be directly sprayed in the field, enabling extremely simple operation.
  • Excellent biocompatibility and degradability: It is the only material that can be used as a carbon source by bacteria. In the natural environment, it can be decomposed by microorganisms and enzymes, achieving a degradation rate of up to 75% within 46 days. Ultimately, it decomposes into CO₂ and H₂O. Biological tests have shown that it is non-toxic to organisms and does not cause environmental pollution.
  • Improved soil conditions: The liquid formed after the PVA film dissolves penetrates into the soil, increasing soil aggregation, aeration, and water retention capacity, which is beneficial for plant growth and development.
  • Strong mechanical properties: It has a tensile strength of 44.1~63.7 MPa and an elongation at break of up to 400%, exhibiting high mechanical strength and good toughness, facilitating product transportation and storage.
  • Effective isolation and protection: It has excellent barrier properties and antistatic properties, isolating the internal E. coli engineered bacteria from the external environment, preventing electrostatic dust from contaminating the product, and ensuring the activity of the engineered bacteria.
  • Low cost and easy processing: Raw materials are easily accessible, and the production and processing technology is simple, making it suitable for mass production and reducing product costs.
  • (2) Internal Component: E. coliEngineered Bacteria Capable of Secreting Erucamide
  • The E. coli engineered bacteria encapsulated within the PVA outer membrane are the core of the product’s functionality. Their design and functional characteristics are as follows:
  • Erucamide secretion: The engineered bacterium can secrete erucamide, which forms a protective film on the plant surface. This film effectively blocks the invasion of exogenous plant pathogens (such as bacterial wilt pathogens), preventing plants from infection and damage.
  • Stable survival: Benefiting from the protection of the PVA outer membrane and a suitable internal environment, the E. coli engineered bacteria maintain good activity during storage, ensuring their ability to secrete erucamide normally when in use.
2.3 Application Scenarios and Outstanding Advantages
ErucaBead is mainly used to prevent plants from infection by exogenous pathogens such as bacterial wilt pathogens. It provides dosage guidelines based on the planting density of different plants to achieve precise application. Its advantages are mainly reflected in the following aspects:

  • Precise dosage, avoiding waste: The fixed-dose bead packaging allows farmers to use a specific number of beads according to planting conditions (following guidelines) without the need for self-estimation. This avoids problems of excessive or insufficient dosage, ensuring control efficacy while preventing waste.
  • Simple and convenient operation: During use, farmers only need to place the beads in a specified volume of water; after dissolution, the solution can be directly sprayed. The entire process is simple and easy to understand, conforming to farmers’ usage habits and making it easy to handle.
  • Stable control efficacy: The PVA outer membrane effectively protects the activity of the engineered bacterium, enabling it to secrete erucamide normally during use. This ensures the continuous and effective formation of a protective film on the plant surface to resist exogenous pathogens.
  • Environmental friendliness and safety: All components of the product are biodegradable, leaving no toxic residues in the environment after use. Additionally, it can improve soil conditions, aligning with the development concept of green agriculture.