Aflatoxins are a class of colorless, odorless secondary metabolites produced by various fungi, including Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are highly toxic and carcinogenic, capable of entering the human and animal body through the food chain, leading to a series of health issues. Their toxicity mechanism mainly involves binding to DNA, causing gene mutations and subsequently inducing cancer, particularly liver cancer.Globally, a total of 905,677 new cases (corresponding to a crude rate of 11.6 cases per 100,000 people) and 830,180 patient death (corresponding to a crude rate of 10.7 cases per 100,000 people) due to infection with aflatoxins in 2020[1].Therefore, long-term exposure to aflatoxins is considered one of the main causes of hepatocellular carcinoma.

Due to the strong carcinogenicity of aflatoxins, the International Agency for Research on Cancer (IARC) of the World Health Organization classified them as Group I carcinogens in 1993, indicating sufficient evidence of carcinogenicity in humans. To date, researchers have identified more than 20 types of aflatoxins, among which aflatoxin B1 (AFB1) is the most toxic and carcinogenic[2]. AFB1 poses a significant threat, as prolonged low-dose exposure can lead to serious diseases such as embryonic malformations, genetic mutations, and primary liver cancer, and can even suppress the immune system. Additionally, AFB1 can enter the bodies of livestock and poultry through contaminated feed, leaving residues in their meat, dairy products, and eggs, thereby endangering human health through the food chain. Acute ingestion of large amounts of AFB1 can cause acute poisoning, manifested by liver failure, coma, and even death. Therefore, how to efficiently and safely detect and degrade AFB1 has become an urgent issue in the field of food safety.

Figure 1 The structural feature of aflatoxin B1

Biodegradation methods for AFB1 have attracted increasing attention due to their specificity and efficiency. Enzymes or metabolites produced by microorganisms can degrade AFB1 under natural conditions, and the degradation products are usually non-toxic or less toxic, offering great potential for application. Through our literature research, we learned that Bacillus subtilis contains Laccase, an enzyme capable of efficiently degrading AFB1[3]. Laccase is a copper oxidase that can decompose AFB1 through oxidation, producing non-toxic or less toxic products[4]. Additionally, we found that the cell wall of Lactobacillus rhamnosus contains a large amount of peptidoglycan, which can adsorb AFB1, providing an opportunity for degradation. However, Lactobacillus rhamnosus itself lacks the ability to degrade AFB1, and its binding to toxins is relatively unstable. Therefore, we envision using genetic engineering to introduce the laccase gene into Lactobacillus rhamnosus, enabling it to efficiently degrade AFB1 while utilizing its natural adsorption properties to improve degradation efficiency.

Based on the above inspiration, we designed an innovative project. The core of our solution is to introduce the Laccase gene into Lactobacillus rhamnosus via genetic engineering, enabling it to degrade AFB1. To achieve precise toxin response, we also incorporated a fine-tuning mechanism. In our design, when AFB1 are present in the environment, the toxin first binds to a nanobody in the enviroment near the engineered bacteria. Once bound, this nanobody emits a specific Blue fluorescence due to its attached fluorophore[5]. The Blue fluorescence activates a photosensitive protein, VVD, inside the engineered bacteria, which, upon receiving the light signal, undergoes domain changes and restore T7 RNA polymerase activity.

To ensure the specificity of the degradation pathway, we added a T7 promoter upstream of the laccase gene, ensuring that laccase expression is only initiated in the presence of AFB1, thereby avoiding unnecessary material and energy waste[6].

Figure3 The expression of the target gene is initiated after the VVD homodimer restores T7 RNA polymerase.

To ensure efficient extracellular degradation of AFB1 by laccase, we added a signal peptide sequence in front of the laccase gene, guiding the enzyme to be secreted outside the cell for action. Moreover, to ensure biosafety, we introduced a self-destruct mechanism in the engineered bacteria, which triggers a suicide program once the toxin degradation task is completed, preventing the uncontrolled spread of the engineered bacteria in the environment and ensuring biosafety.

The system we designed has potential applications in multiple fields, particularly in silage feeds and grain safety, and food production. We hope that this technology can be widely used in the storage and processing of agricultural and animal husbandry products to help reduce AFB1 contamination and protect public health. In addition, this technology could be applied in the livestock industry to ensure that AFB1 in feed are not transferred to animal products, thus safeguarding the human food chain.

In the future, we plan to expand this technology to other harmful fungal toxins, further optimizing genetic engineering methods to improve the system’s response speed and sensitivity, providing more reliable solutions for food safety. Through our ongoing efforts, we believe that this system can bring health and safety to more people.

[1]Pickova D, Ostry V, Toman J, Malir F. Aflatoxins: History, Significant Milestones, Recent Data on Their Toxicity and Ways to Mitigation. Toxins (Basel). 2021 Jun 3;13(6):399. doi: 10.3390/toxins13060399. PMID: 34205163; PMCID: PMC8227755.

[2]罗自生, 秦雨, 徐艳群, 等. (2015). 黄曲霉毒素的生物合成、代谢和毒性研究进展. 食品科学(03), 250-257.

[3]Marianela Bossa, María Silvina Alaniz Zanon, et al. (2024). Aflatoxin Decontamination in Maize Steep Liquor Using Laccases from Basidiomycota.

[4]乐琛,王晓,谢珂,郑艳丽,杨江科 & 雷磊.枯草芽孢杆菌漆酶的理性设计及在霉菌毒素降解的应用.食品发酵工业1-12.doi:10.13995/j.cnki.11-1802/ts.039316.

[5]陈文星, 王凤华, 谭晓亮, 等. (2024). 基于纳米抗体-荧光素酶的黄曲霉毒素B1检测方法. 中国食品学报(04), 349-360.

[6]韩悌云. (2017). 构建响应外界信号的生物分子开关(博士论文, 中国科学技术大学).