Mycotoxins are secondary metabolites produced by certain filamentous fungi. They often contaminate plant-derived products such as crops, food, and feed, and pose significant hazards. Fungi growing on food are one of the main sources of food and feed contamination, and the toxins produced by these fungi are called mycotoxins. Currently, it is estimated that approximately 25% of the world's crops are contaminated with mycotoxins each year, which imposes a heavy burden on agriculture and public health. In particular, food, grains, and feed produced from them that are contaminated with mycotoxins will further severely affect the conversion rate during animal breeding and the safety of animal-derived foods such as milk, meat, and eggs.
Post-harvest diseases caused by fungi are a major cause of grain loss and waste. In such cases, fruit rot can be directly attributed to plant pathogenic bacteria and fungi that produce mycotoxins. Post-harvest diseases of agricultural products can lead to severe food loss and waste, accounting for one-third of all food. Among these agricultural products, fruits are extremely vulnerable to post-harvest diseases, especially in cases of mechanical damage or improper handling. Fungal post-harvest diseases in fruits rank first among factors that reduce quality and safety, thereby shortening their storage shelf life. In addition to these diseases, processing fruits with post-harvest diseases into new products also results in economic losses. Furthermore, certain fungal species (Aspergillus, Penicillium, Fusarium, Alternaria) produce harmful secondary metabolites in fruits, which are called mycotoxins. Common examples include zearalenone (ZEN), ochratoxin (OT).
Notably, according to research, for humans or animals, mycotoxins not only exhibit certain pathogenicity or even toxicity, but some of these toxins are also carcinogenic to specific animal species. In this regard, the World Health Organization (WHO) and the International Agency for Research on Cancer (IARC) have evaluated a variety of mycotoxins, and identified some of them as carcinogens with high risks to humans. These mycotoxins are usually classified as Group A (confirmed carcinogens) or Group B (possible or probable carcinogens), as they can cause damage to important organs and tissues of the human body, such as the liver, kidneys, skin, hematopoietic organs, and nervous system. By contaminating grains, nuts, oils, and other foods, mycotoxins may pose threats to multiple important systems of the body after ingestion, including but not limited to the liver, kidneys, skin, and nervous system. Long-term or heavy exposure to these toxins can lead to different forms of mycotoxicosis, whose manifestations range from chronic, progressive health problems to acute poisoning. Acute poisoning incidents are relatively rare, while chronic effects are more common, which may involve various health consequences such as immunosuppression, growth retardation, and increased cancer risk.
Ochratoxin A (OTA) is one of the mycotoxins with the widest pollution range and strongest toxicity worldwide. It extensively contaminates crops such as wheat, corn, coffee, and tea, as well as processed products including wine, dairy products, and nuts. Surveys conducted in multiple countries show that the detection rate of OTA in grain samples exceeds 20%, while the detection rate in snack and breakfast samples in some regions even reaches 52%, with the maximum concentration up to 7.43 ng/g, severely penetrating the food supply chain.
Based on their chemical structures, ochratoxins can be mainly divided into three categories: ochratoxin A (OTA), ochratoxin B (OTB), and ochratoxin C (OTC) [13]. They consist of a coumarin moiety and a phenylalanine moiety linked by an amide bond [14]. Among them, OTA is chlorinated, which is uncommon in natural toxins (Figure 1.), and it is generally recognized as the most notorious and toxic one [20]. Therefore, most studies on ochratoxins focus on OTA.
Figure 1 Chemical structure of ochratoxin A
Meanwhile, we visited the Wheat Experimental Station of the Xinjiang Uygur Autonomous Region Academy of Agricultural Sciences and the grain depots of the Xinjiang Production and Construction Corps for on-site exchanges and investigations. During these visits, we gained a deeper understanding of the situation regarding mycotoxin contamination in crops and also shared the concepts of our project with relevant experts.
OTA exhibits extremely strong thermal stability and acid resistance; conventional cooking and high-temperature processing are barely able to destroy its structure. Moreover, long-term exposure to OTA can cause multi-organ toxicity. As a Group ⅡB carcinogen identified by the International Agency for Research on Cancer (IARC), it can induce nephrotoxicity (e.g., Balkan endemic nephropathy), hepatotoxicity (hepatocyte lysis, inflammation), and neurotoxicity (memory impairment, increased risk of neurodegenerative diseases), posing a significant threat to human health.
In the early stage of the project, we conducted extensive social research activities, aiming to gain a comprehensive understanding of the status of mycotoxin contamination, as well as the drawbacks and shortcomings of current detoxification technologies. Meanwhile, we also sought to understand the public's level of awareness regarding mycotoxins.
We carried out questionnaire surveys and background investigations in locations including Shanggu Mountain Village (Huanggang City, Hubei Province), Wangying Town (Lichuan City), Wuhan Communities, Luodian Guangshui Town (Suizhou City), Baxu Village (Xinzhou District, Wuhan City), and Wulidui Community (Ziyang District, Yiyang City). Through our research, we found that the general public has very little knowledge about mycotoxins.
Furthermore, based on our understanding, the current detoxification technologies have obvious shortcomings: Physical detoxification (adsorption, irradiation) tends to cause nutrient loss in food, while chemical detoxification (ozone, alkali treatment) may lead to residues or the formation of new harmful substances. Biological enzymatic hydrolysis has become a research hotspot due to its safety and environmental friendliness; however, traditional enzymes (such as carboxypeptidase A) have extremely low efficiency—1 hour of treatment with 67 μg/mL carboxypeptidase can only degrade less than 10% of OTA (at a concentration of 50 μg/L), which is far from meeting industrial demands. Although the amidase ADH3 derived from Stenotrophomonas has been discovered previously, with efficiency far exceeding that of carboxypeptidase (it can completely degrade 50 μg/L OTA within 90 seconds), there is still room for further optimization. Therefore, developing OTA hydrolases with higher efficiency and stronger adaptability has become the key to addressing the pain points in this industry.
Throughout the process, we have maintained close communication with experts and scholars in various fields to continuously refine the project. In accordance with HP’s approach, we have advanced the work of the modeling team, experimental team, and descriptional practice team, forming a continuously evolving closed loop.
Our Team Has Established a Complete Technology Chain of "Enzyme Mining - Structural Analysis - Molecular Modification - Application Adaptation" to Break Through the Bottlenecks of Traditional Detoxification Technologies:
The innovation of modeling research lies in the establishment of an effective two-way verification mechanism: the binding modes predicted by molecular docking are verified through mutation experiments; the flexible regions identified by molecular dynamics simulations are tested via functional experiments; and the energy contributions derived from free energy calculations are confirmed by mutation effects. This "computation-experiment" cross-validation significantly enhances the reliability and efficiency of the research.
We have successfully established seamless connection between these two levels and formed a research closed loop:
1.Macroscopic optimization provides the optimal context for microscopic research: The optimal reaction conditions (temperature, pH) determined by the response surface model are directly used as environmental parameters for calculations such as molecular dynamics simulations. This ensures that microscopic simulations are conducted under the most relevant physiological conditions, making the calculation results more biologically meaningful.
2.Microscopic mechanisms provide molecular explanations for macroscopic phenomena: The interaction mechanism between ADH and OTA has been revealed. Results from molecular simulations, including enzyme-substrate binding modes, residue functions, and dynamic stability, explain at the atomic level why enzymes exhibit specific optimal temperatures, optimal pH values, and thermal inactivation characteristics at the macroscopic level. For instance, the sharp loss of enzyme activity at high temperatures is intuitively reflected in molecular dynamics (MD) simulations as severe unfolding and destabilization of the protein structure. The proposed subsequent modification suggestions provide direct guidance for enzyme engineering applications.
Meanwhile, we have simulated the changing trends of global aflatoxin contamination over the next 50 years.
By exploring natural high-efficiency enzyme resources (ADH3, LlADH) and conducting rational modification of ADH3 and LlADH based on structural biology, this project provides a "green biological tool" for OTA detoxification. It not only fills the gap in the industrial application of ultra-high-efficiency OTA hydrolases but also offers a reference framework of "structural analysis - directed modification" for the biodegradation research of other mycotoxins (such as aflatoxins and zearalenone).
In the future, the team will further optimize the heterologous expression processes of enzymes (e.g., Pichia pastoris and Aspergillus niger expression systems) and immobilization technologies, promoting the translation of research achievements from the laboratory to industrial applications. We will contribute iGEM's strength to addressing global grain contamination issues, safeguarding human health, and realizing the sustainable development of food safety.
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