Our project aims to improve technology, enrich the parts library, promote food safety, and advance the application of synthetic biology. To this end, we have selected Lactobacillus rhamnosus, a probiotic widely used in dairy fermentation, as the carrier for our engineered strain. This Gram-positive bacterium has a thick peptidoglycan layer in its cell wall, allowing it to physically adsorb 80% of aflatoxins present in the environment, thereby concentrating the toxins. However, despite its initial effectiveness in physical adsorption, its binding capacity is limited; over time, the adsorbed aflatoxins gradually detach from the bacterial cells, returning to a free state.

To overcome this limitation, we introduced the gene for laccase (CotAgold), enabling the strain to completely degrade aflatoxins. Laccase effectively breaks down the molecular structure of aflatoxins, converting them into harmless byproducts. Simultaneously, we employed a finely tuned regulation system that allows the engineered bacteria to express laccase only when binding aflatoxins. These modifications not only enhance the strain’s ability to concentrate toxins but also provide an efficient and safe solution for aflatoxin treatment in silage feedS, grain and oil food industries.

In terms of fine regulation, we utilized a special nanobody (Nanobody) derived from the peripheral blood of alpacas, characterized by a unique structure that naturally lacks light chains, representing the smallest unit capable of recognizing and binding to target antigens. The selected nanobody Nb26 specifically binds to aflatoxins. To achieve effective detection and response, we fused Nb26 with Gaussia luciferase (GenBank: BAR71165.1) to form a chimeric protein. When Nb26 binds to aflatoxins, Gaussia luciferase is activated, producing a distinct blue fluorescence signal.

Additionally, we incorporated a light-sensitive protein (VVD) that can form a homodimer upon receiving blue fluorescence. To ensure precise regulation of laccase expression, we split the coding sequence of T7 RNA polymerase into two parts and fused each part with the VVD sequence. When the light-sensitive protein receives blue light and forms a dimer, the split T7 RNA polymerase sequences reassemble, restoring their activity. The activated T7 RNA polymerase will specifically bind to the T7 promoter preceding the laccase gene, thereby initiating the expression of laccase. This blue light-induced system achieves the goal of accurately controlling laccase expression under specific conditions.

To prevent the engineered strains from accidentally escaping and entering the external environment during use, leading to environmental contamination, we designed a glucose operon suicide system. This system uses the difference in glucose concentration between the human gut, dairy environments, and the external environment as a trigger condition. We selected a promoter, PGlu, that responds to changes in glucose concentration, which controls the expression of the suicide gene mazF.

The mazF gene encodes an endoribonuclease that can recognize and cleave RNA at the ACA site, triggering programmed cell death in the microorganisms. Importantly, this process does not cause cell lysis, preventing the leakage of cellular contents during death and reducing potential biosafety risks. When the engineered strain is in a low-glucose environment, the PGlu promoter activates the mazF gene, triggering the suicide mechanism, ensuring that the strain does not survive or spread uncontrollably.

Our project offers a novel approach to the degradation and prevention of aflatoxins based on synthetic biology. Aflatoxins are classified as Group 1 carcinogens by the World Health Organization’s International Agency for Research on Cancer and are widely found in soil, plants, and nuts. According to the Food and Agriculture Organization (FAO), about 25% of edible crops are affected by mycotoxins, primarily AFT. We hope that upon completion, our project will effectively address aflatoxin contamination in agricultural production, promote the safety of grain and oil products, protect public health, and alleviate concerns about aflatoxin contamination in food. Additionally, our research will advance the development of synthetic biology, bringing its methods into public awareness and benefiting our daily lives.