Abstract
For the high-yield demand of crops, pesticides are indispensable necessities. Because of the shortcomings of chemical pesticides, which include the destruction of biodiversity and the harm to human beings, we should use biopesticides instead. Biopesticides are generally safer and provide benefits for both people and the environment. However, existing biopesticides are not faultless, as they are less efficient than most chemical pesticides. In response to the environmental damage and health damage caused by the overuse of chemical insecticides, we improved the efficiency of a fungal biopesticide, Metarhizium anisopliae, by introducing an insect-specific neurotoxin LqhIT2. We also introduced a phototoxic suicide switch Pmcl1(short)-SuperNova to improve safety and reduce the risk of gene leakage.
Part 1 Chemical pesticides
Practically speaking, agriculture does not work without pesticides. As the demand for food increases annually due to the huge population, the invention of new pesticides and their extensive use is necessary. Without the use of pesticides, there would be a 78% loss in fruit production, a 54% loss in vegetable production, and a 32% loss in cereal production (Tudi et al., 2021).
Over 95% of pesticides used in the world are chemically synthesized (Šunjka et al., 2022). As chemical pesticides being powerful tools for the good of human beings, they can be toxic to the farmers or workers applying them. Yearly, more than 26 million people suffer from pesticide poisoning with nearly 220,000 deaths (Ansari, Moraiet, and Ahmad, 2013). Since the pesticide residues in food cannot normally break down and still stay on the food during ingestion, chemical pesticides can cause potential health problems for everyone.
Figure 1. Pesticide use by each country in 2020 from Our World in Data.
It is shown that only 1% of the used insecticides reached their target animals, and the rest are disseminated in the environment (Zhang et al., 2011). According to the Food and Agriculture Organization in the United States, there were more than 2.5 million tons of insecticides used in the US and in China in 2020 and millions of tons of insecticides are left in the ecosystem, non-target animals, and food residue (Faostat). Chemical pesticides cause ecological disasters around the world.
Part 2 Biopesticides
Biopesticides are pesticides derived from natural materials such as animals, plants, bacteria, and certain minerals. Biopesticides have multiple advantages over chemical pesticides. Biopesticides can alter the course of pest resistance. They are safer for human beings and other non-target animals. They are relatively more eco-friendly. And they cause little or no problem with post-harvest contamination (Fenibo et al., 2021). China has published a new standard of pesticide residue: GB2763-2021, which restricts the use of some recent pesticides and increases the requirement. However, it remits the max residue standard of 33 biopesticides, proving their safety. It is high time that we should replace chemical pesticides with biological pesticides.
However, certain limitations make biopesticides unpopular compared to chemical pesticides for farmers. Some of the limitations of biopesticides include:
1. Variable effectiveness: The efficacy of biopesticides can vary depending on environmental conditions, such as temperature, humidity, and sunlight. They may be less effective under certain conditions, which can limit their consistent performance.
2. Slow action and slower results: Biopesticides may have a slower onset of action compared to chemical pesticides. They often require more time to act on pests or diseases, which means the results may not be immediate or as rapid as desired.
3. Limited availability and choice: The range of available biopesticides is more limited compared to chemical pesticides. There may be fewer options for specific pest or disease problems, limiting the choices for farmers and growers.
The disadvantages limit the use of biopesticides. As a result, less than 5% of pesticides used in the world are biopesticides (Šunjka et al., 2022). To overcome these limitations, genetic modification has become one promising method to improve the effectiveness and efficiency of biopesticides.
Part 3
Modification of Metarhizium
anisopliae
Part 3.1 Metarhizium anisopliae
Metarhizium anisopliae grows naturally in soils throughout the world. M. anisopliae is a widely used entomopathogenic fungus. M. anisopliae can kill more than 200 kinds of insect pests by actively invading the insect body wall. M. anisopliae can produce destuxin (non-ribosomal polypeptide toxoid) and other insecticidal toxins to kill pests. In the absence of an insect host, M. anisopliae can form rhizosphere symbiosis with plant roots and persist for a long time.
Because of its wide host range, strong pathogenicity, harmlessness to humans, livestock, and crops, easy large-scale production, and sustainable control effect, it has broad application prospects for biological pest control (Kim et al., 2020). We used the M. anisopliae ACCC30104, a strain native to China.
Part 3.2 Leiurus quinquestriatus hebraeus and LqhIT2
Leiurus quinquestriatus hebraeus is a species of scorpion native to Israel and other Middle East countries (Ross, 2008). L. quinquestriatus hebraeus has very potent venom, which contains toxin peptides.
Figure 2. L. quinquestriatus hebraeus.
LqhIT2 is a 61 amino acid-long scorpion depressant toxin from L. quinquestriatus hebraeus. Although the scorpion is dangerous to humans, LqhIT2 is not. LqhIT2 shows a high preference for insect voltage-gated sodium channels and is totally harmless to mammals. This interesting property makes it a good choice for pesticides.
Figure 3. The 3D structure of LqhIT2 (Karbat et al., 2007).
To improve the efficiency and effectiveness of M. anisopliae as a fungal biopesticide, we chose to introduce LqhIT2 into the fungus. LqhIT2 significantly improved the virulence of M. anisopliae ACCC30104 against pests.
Our product's intended use is in agriculture, where our engineered fungus will inevitably be released into the environment. However, our project has a strong focus on safety and we want to prevent the unintended spread of our fungus.
The concerns about biosafety have hindered the commercialization and public acceptance of engineered biopesticides. To address this issue, the 2016 NYMU_Taipei iGEM Team designed a light-induced suicide switch for M. anisopliae by ligating a KillerRed gene after a hemolymph inducible promoter Pmcl1. KillerRed is a red fluorescent protein that can generate reactive oxygen species (ROS) upon absorption of photons (Onukwufor et al., 2020). When the fungi invade the insect body, they will cumulate KillerRed protein. And when the fungi grow out of the insect body and try to spread spores, their tissues are killed by sunlight.
We plan to improve their suicide switch. We switched KillerRed to SuperNova, a stronger phototoxic protein that can produce three times as much ROS as KillerRed can (Onukwufor et al., 2019). Furthermore, Kanjo et al. proved that a truncated version of Pmcl1 is stronger than the full-length version, so we will turn to the short Pmcl1 promoter. As a result, our improved suicide switch made the fungi more sensitive to light and reached a higher death rate under light.
Summary
As discussed above, misuse of chemical pesticides poses a serious threat to the environment and human health. Biopesticides are good substitutes, but low efficiency is a problem. In this context, we introduced an insect-specific neurotoxin, LqhIT2, to an entomopathogenic fungus, M. anisopliae. This approach not only improves the efficiency and effectiveness of the fungal biopesticide but also reduces the impact on the environment because LqhIT2 is only toxic to insects and is not harmful to other animals. At the same time, to improve safety, we introduced a suicide switch, Pmcl1(short)-SuperNova. With this phototoxic suicide switch, our genetically modified fungi should not be able to reproduce in nature, as their spores will be killed by sunlight.
Compared with chemical pesticides and conventional biopesticides, our engineered fungal biopesticide should have higher efficiency and better effect. It should greatly reduce farmers' pesticide application and labor input, significantly increase crop yield, and improve the quality of agricultural products.
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