Describe how and why you chose your iGEM project.
Describe how and why you chose your iGEM project.
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1 Aspergillus flavus and its aflatoxins contamination
1.1 Aspergillus flavus and aflatoxin
Aspergillus flavus belongs to the class of semi fungi and is a common saprophytic fungus. It is commonly found in moldy grains, grain products, and other organic matter with moldy decay. Its colony grows rapidly, has a loose structure, and the surface is grayish green, while the back is colorless or slightly brown. The mycelium consists of many complex branching hyphae (Luo et al., 2014).
Among numerous metabolites of A. flavus, aflatoxins (AF) are a group of structurally similar mycotoxins a highly toxic substance, which are exceedingly toxic natural poisons, being 68 times more toxic than arsenic. Aflatoxins exhibit teratogenic, carcinogenic, and mutagenic properties, and among the A. flavus-derived ones, the most representative are AFB1 and AFB. Because of its ability to induce liver cancer in humans and animals, AFB1 is the most toxic and carcinogenic among them, classified as a Group 1 carcinogen by the World Health Organization's cancer research agency in 1993 (Wang et al., 2010).
Figure 1 Chemical structures of class B1, B2, G1, G2, and M1 aflatoxin (Jallow et al., 2021)
Aflatoxins predominantly contaminate moldy foods such as peanuts, grains, tree nuts, and rice. Consuming food contaminated with aflatoxins can cause harm to the body and even threaten life. The detrimental effects of aflatoxins on humans and animals are linked to its interference with protein synthesis, as A aflatoxins inhibits this vital process. Research has shown that the cytotoxic effect of aflatoxins stems from its interference with the synthesis of RNA and DNA, which in turn disrupts cellular protein synthesis, leading to systemic damage (Wang et al., 2010).
1.2 Curren status of Aspergillus flavus /aflatoxin contamination
A. flavus is the main species that is mainly responsible for aflatoxin production and crop contamination because it is the most copious molds found in soil and possesses the saprobe character that enables it to grow on many organic nutrient substrates including compost piles, plant debris, cotton, dead insects, stored grains, field crops, animal corpses and animal fodder (Kakde et al., 2012).
Aflatoxin contamination occurs in a wide range of regional crops and food commodities. Food and feed like corn, rice, spices, dried fruits, nuts, and figs are mostly contaminated by aflatoxins. The two major A. flavus-derived aflatoxins, AFB1 and AFB2 are commonly found in a wide range of food commodities. The post-harvest crops are more likely to be contaminated if the storage conditions are optimum for fungus growth. It was found that about 67.9% of maize samples, 92.9% of millet samples, and 50% of sorghum samples that were obtained from a storage room are contaminated by aflatoxins (Kumar et al., 2021)
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2 Current Status of the Prevention and Control of Aspergillus flavus/Aflatoxin Contamination
Given that aflatoxin contamination predominantly arises from A. flavus infestation in crops, the control of aflatoxin contamination and management of A. flavus infection are inherently intertwined. In accordance with the harvest process and subsequent food processing procedures, strategies to combat A. flavus and aflatoxin contamination are generally categorized into pre-harvest and post-harvest approaches, encompassing a range of methodologies including physical, chemical, and biological treatments.
2.1 Physical treatments and chemical treatments
Physical treatments and chemical treatments are among the most conventional control methods, usually used in post-harvest control. The physical treatments include heating, irradiation, microwaves, pulsed light, high water pressure and ultrasound, and chemical treatments include oxidants, antibiotics, fungicides, and organic acids (Agriopoulou et al., 2020). However, some physical methods have disadvantages such as impact on quality and loss of nutrients, long treatment time, low detoxification ability, and potential environmental pollution, while chemical methods may leave behind residuals that are difficult to eliminate.
2.3 Biological treatments
Biological ways to deal with aflatoxin (AF) contamination can be used in both pre-harvest and post-harvest control.
Before harvest, competitive exclusion is a cost-effective and environmentally friendly method, and it involves introducing non-aflatoxigenic strains to outcompete aflatoxigenic strains. Microbial biofungicides, such as some Trichoderma harzianum strains, are also widely used in pre-harvest control and show promise in restricting A. flavus contamination in crops (Agriopoulou et al., 2020).
After harvest, bacteria such as Lactic acid bacteria like Lactobacillus acidophilus and Lactococcus lactis subsp. lactis can inhibit AF production or even remove AFs from feed and food products. Similarly, certain yeasts like Candida and Debaryomyces can significantly inhibit AF production – to improve AFB1 detoxification in the rumen and decrease the AFM1 content of milk. Moreover, enzymes and antifungal proteins have demonstrated usefulness in decreasing AF levels and enzymatic degradation of mycotoxins is a simple method for decontaminating food (Agriopoulou et al., 2020).
These existing biological methods are capable of suppressing A. flavus infections to a certain degree and effectively reducing aflatoxin levels during food processing, but still fail to tackle the fundamental issue of aflatoxin contamination at its source.
3 Application of Terpenoids in the Prevention and Control of A. flavus/Aflatoxin Contamination
3.1 About terpenoids
Terpenoids, consisting of "isoprene" units, are one of the largest classes of natural products. Terpenoids are synthesized from two five-carbon structural units (i.e. the isoprene unit) (Thoppil et al., 2011) Depending on the number of structural units, the monoterpenes include linalool, limonene and geraniol. These terpenoids can be used as preservatives for stored agricultural products due to their insecticidal properties (Theis et al., 2003).
3.2 Inhibitory Effects of Terpenoids on Aspergillus flavus
(1) Linalool
Linalool shows potential as a biofumigant to control aspergillus flavus in post-harvest cereals (Li et al., 2022). Linalool disrupts the integrity of the plasma membrane, causing intracellular leakage of macromolecules such as nucleic acids and proteins. It also inhibits respiratory chain dehydrogenase activity, thereby disrupting cellular respiration and metabolic activity (Guo et al., 2021).
(2) Limonene
Limonene shows significant inhibitory effects on the growth of aspergillus flavus hyphae and reduces aflatoxin production, making it a potential comprehensive control agent. D-limonene increases the fluidity of fungal membranes, leading to high non-specific permeability and loss of membrane integrity, resulting in leakage of vital intracellular components and cell death (Segura-Palacios et al., 2021). In addition, limonene inhibits key enzymes in the aflatoxin biosynthetic pathway, reducing toxin production and A. flavus virulence.
(3) Geraniol
Geraniol significantly inhibits the germination and growth of aspergillus flavus spores. A study showed that geraniol induced rapid and significant ROS production in aspergillus spp. Excessive ROS attack cellular biomolecules such as lipids, proteins and DNA to cause irreversible oxidative damage. Studies have suggested that high levels of ROS deplete intracellular antioxidants, affect DNA and proteins, and thus trigger cell damage or apoptosis (Tang et al., 2018).
4 Applications of Bacillus subtilis in Synthetic Biology
4.1 About Bacillus subtilis
B. subtilis is one of the model strains of Gram-positive bacteria, a common rod-shaped facultative anaerobe capable of forming endospores, widely distributed in natural environments such as soil. it is non-pathogenic and not associated with infection, being recognized by the US Food and Drug Administration (FDA) as Generally Recognized as Safe (GRAS).
Owing to its high safety profiles and well-sequenced genome and elucidated essential genes (Yu et al., 2020), B. subtilis is frequently employed as a model organism in bacterial genetics and cellular metabolism studies.
4.2 Current Status of B. subtilis as a Biological Chassis
Characterized by clear physiological and biochemical traits, relatively simple genetic manipulation, strong secretion and expression capabilities, as well as convenient cultivation and fermentation processes, B. subtilis also serves as an excellent chassis cell and is used in metabolic engineering and metabolic engineering: 1) In metabolic engineering, the strain is systematically optimized through methods such as modulating global regulatory factors, genome reduction and optimization, multi-site and multidimensional regulation, self-biosensing dynamic control, and membrane protein engineering. 2) For protein reagent production enhancement, the production strain is improved by refining gene promoters, optimizing protein signal peptides, leveraging the strain's inherent protein secretion elements, and establishing expression systems that operate without chemical inducers (Kang et al., 2021). https://static.igem.wiki/teams/5525/des/81-des3.png Fig. 2 Modification strategy for optimizing the B. subtilis as chassis cell (Kang et al., 2021).
Engineered into microbial cell factories, it is harnessed to produce industrial enzymes, vitamins, functional sugars, health supplements, and drug precursors, demonstrating formidable capacity for industrial applications (Kang et al., 2021).
Our Project
1 Inspiration and Goal
Aflatoxin contamination poses a significant safety risk to grain and food security. Among the current control methods, physical and chemical approaches give rise to various residue issues, while biological methods fall short of addressing the problem at its source.
We recognize that terpene compounds such as linalool, limonene, and geraniol are efficacious in controlling Aspergillus flavus, and Bacillus subtilis, a bacterium extensively distributed around crop root zones, has already gained wide application as a chassis in synthetic biology. Therefore, we have conceived an idea that we can develop engineered B. subtilis strains with terpene synthesis capability and use them to inhibit the growth of A. flavus in the crop growth stage, so that the aflatoxin contamination can be solve at its source.
2 Our Strategy
In our project, we plan to integrate genes responsible for the synthesis of compounds such as linalool, limonene or geraniol, which are known to inhibit A. flavus growth, into B. subtilis. We will construct engineered B. subtilis strains capable of producing these inhibitory substances. Prior to seeding like peanuts, we will mix the engineered bacterial with the seeds to ensure that the bacteria adhere to the seed surface. Later, the engineered B. subtilis will proliferate and colonize the epidermal surfaces of the plant's root system as the crop germinates and grows, thereby effectively safeguarding against A. flavus infections.
3 Advantage
Compared to existing methods for controlling aflatoxin contamination, our approach presents several advantages:
1) Preventative Control: This method provides a preemptive measure against Aspergillus flavus infection during the field growth stage of crops, thereby addressing aflatoxin contamination at its source. It can significantly reduce the risk of aflatoxin contamination in grain, oilseed, and feed products.
2) Wide Application: The approach can be adapted for use with various crops susceptible to Aspergillus flavus infection, offering broad applicability in agriculture.
3) Non-Toxic Solution: Bacillus subtilis is a safe, non-toxic bacterium that poses no harm to humans or animals. Using it as a biocontrol agent is environmentally friendly and sustainable.
Reference
Luo Z., Qin Y., Xu Y. et al. (2015) Advances in Research on the Biosynthesis, Metabolism, and Toxicity of Aflatoxins. Food Science (3): 8. DOI:10.7506/spkx1002-6630-201503048.
Jallow A, Xie H, Tang X, Qi Z, Li P. Worldwide aflatoxin contamination of agricultural products and foods: From occurrence to control. Compr Rev Food Sci Food Saf. 2021; 20: 2332–2381. https://doi.org/10.1111/1541-4337.12734
Wang L., Hou Y., Hu X. et al. (2010) Progress in Research on the Hazards and Detection Methods of Aflatoxins [J]. Henan Agricultural Sciences 39 (002):123-127. DOI:10.3969/j.issn.1004-3268.2010.02.036
Kakde, U. B. (2012). Fungal bioaerosols: Global diversity, distribution, and its impact on human beings and crops. Bionano Genmics, 5, 323–329.
Kumar, A., Pathak, H., Bhadauria, S. et al. Aflatoxin contamination in food crops: causes, detection, and management: a review. Food Prod Process and Nutr 3, 17 (2021). https://doi.org/10.1186/s43014-021-00064-y
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Li, YN., Zhang, SB., Lv, YY. et al. Mechanisms underlying the inhibitory effects of linalool on Aspergillus flavus spore germination. Appl Microbiol Biotechnol 106, 6625–6640 (2022). https://doi.org/10.1007/s00253-022-12172-x
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Tang, X.; Shao, Y.-L.; Tang, Y.-J.; Zhou, W.-W. Antifungal Activity of Essential Oil Compounds (Geraniol and Citral) and Inhibitory Mechanisms on Grain Pathogens (Aspergillus flavus and Aspergillus ochraceus). Molecules 2018, 23, 2108. https://doi.org/10.3390/molecules23092108
Yu S, Price MA, Wang Y, Liu Y, Guo Y, Ni X, Rosser SJ, Bi C, Wang M. CRISPR-dCas9 Mediated Cytosine Deaminase Base Editing in Bacillus subtilis. ACS Synth Biol. 2020 Jul 17;9(7):1781-1789. doi: 10.1021/acssynbio.0c00151. Epub 2020 Jul 1. PMID: 32551562.
Kang Q., Xiang M. et Zhang D. (2021) Research progress and industrial application of Bacillus subtilis in systematic and synthetic biotechnology (in Chinese). Chinese Journal of Biotechnology., 2021, 037(3): 923-938