Overview
This year, to replace chemical pesticides, we developed an efficient and safe fungal biopesticide. We improved the virulence of 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. We have achieved the following contributions.
Characterization of KillerRed and SuperNova
We characterized KillerRed (BBa_K1184000) and SuperNova (BBa_K4882008). M. anisopliae expressing KillerRed and SuperNova produced significantly fewer spores under sunlight than WT, indicating KillerRed and SuperNova’s phototoxicity. We proved that KillerRed and SuperNova can be a part of a suicide switch, and future teams can use them.
Pmcl1 part improvement
Pmcl1(short) (BBa_K4882000) is an improved version of Pmcl1 (BBa_K2040100). Pmcl1 is a hemolymph inducible promoter that turns on only in insect hemolymph. Our results showed that suicide switches consisting of Pmcl1(short) led to significantly fewer spores than Pmcl1(full-length) in M. anisopliae. We proved that a shorter version of this part (1586bp) can lead to a higher expression of the downstream genes.
Built a potent suicide switch for M. anisopliae
We constructed four vectors, pBARGPE1-Pmcl1-SuperNova-SV40-BenA, pBARGPE1-Pmcl1(short)-SuperNova-SV40-BenA, pBARGPE1-Pmcl1-KillerRed-SV40-BenA, and pBARGPE1-Pmcl1(short)-KillerRed-SV40-BenA, to find the best suicide switch for M. anisopliae. Our results showed that Pmcl1(short)-SuperNova was the best suicide switch, leading to significantly fewer spores. See Engineering Success for more details.
Created an efficient Fungal biopesticide
By constructing and transforming pBARGEP1-Pmcl1(short)-Mcl1ss-LqhIT2-BenA into M. anisopliae, we significantly increase its virulence. Our results showed the LC50 of the LqhIT2 strain was 25.26-fold lower than that of the WT strain. As a result, we successfully built an efficient engineered fungal biopesticide. See Engineering Success for more details.
Proved the safety of LqhIT2 in genetic engineering
Previous studies showed that LqhIT2 is an insect-specific toxin and is safe for humans. However, genes can mutate, and we wanted to ensure the safety of LqhIT2, especially in the context of genetic engineering. We checked similar sequences of LqhIT2 and results showed that all similar genes encode for insect-specific toxins, which showed the biosafety of LqhIT2. See Safety for more details.
| Gene | Query Cover | E value | Per. Ident | Accession | Toxicity |
| LqqIT2 | 98% | 4.00E-33 | 88.52% | P19855.2 | Insect-specific |
| LqhIT5 | 98% | 9.00E-23 | 73.77% | P81240.1 | Insect-specific |
| BotIT4 | 98% | 8.00E-34 | 95.08% | P55903.1 | Insect-specific |
| BotIT5 | 98% | 3.00E-33 | 93.44% | P55904.1 | Insect-specific |
| BotIT6 | 98% | 1.00E-18 | 63.93% | P59864.1 | Insect-specific |
| BjIT2 | 98% | 1.00E-28 | 78.69% | P24336.1 | Insect-specific |
| BmKITa | 98% | 4.00E-29 | 81.97% | Q9XY87.1 | Insect-specific |
| BmKITb | 98% | 3.00E-28 | 80.33% | Q95WX6.1 | Insect-specific |
| BmKITc | 98% | 2.00E-24 | 75.41% | Q9Y1U3.2 | Insect-specific |
| BmKAEP | 98% | 2.00E-29 | 80.33% | P15228.2 | Insect-specific |
| BmKAEP2 | 98% | 1.00E-28 | 81.97% | Q86M31.1 | Insect-specific |
| BmKIT2 | 98% | 1.00E-28 | 81.97% | P68727.1 | Insect-specific |
| BmKIT3 | 98% | 8.00E-29 | 83.61% | Q17231.2 | Insect-specific |
Clarified the approval process for GM microorganisms in China
We did literature research and checked all the regulations related to GMOs in China. The most important one is the Administration of the Safety Evaluation of Agricultural Genetically Modified Organisms (2016 Revision). We listed the roles and tests and showed how to get the Agricultural Genetically Modified Organisms Safety Certificate step-by-step. See Human Practices for more details. We hope our work gives future teams some clues about how to get their GM products approved by the Chinese authorities.