Part collection
Abstract
Our project aims to incorporate three major components--mite & spider venom protein, zingiberene and harpin protein—for control of infestation by spider mites (acariformes: Tetranychidae ). This novel approach not only exterminates the pests, but also takes plant recovery and pest prevention into consideration. Mite venom peptides (MVP), with high stability and specifity, functions through targeting the ion channels of spider mites and exhibits satisfactory toxicity. Since all the MVPs we incorporated potentially target CaV channels, we incorporated spider venom peptides (SVP). SVPs target a diverse array of receptors and ion channels in the pest nervous system, thus suppressing the development of drug resistance in spider mites. According to our test results, SVPs also display significant contact efficacy. The terpenes 7-epi-zingiberene (7epiZ), 9-hydroxy-zingiberene (9HZ), 9-hydroxy-10,11-epoxy zingiberene (9H10epoZ), with their significant repellent and fecundity-reducing efficacy, can be used as a preventive approach against infestations. Lastly, Harpin protein, the third component of Dienamite, is able to foster plant immunity and improve plant conditions. With a combination of terpenes and proteins targeting multiple aspects of pest control, Dienamite acts as an efficient and eco-friendly acaricide.

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Extermination

For the extermination of spider mites, we incorporated two types of venom peptides: mite venom peptides (MVP) and spider venom peptides (SVP).

  1. The first type of venom peptide we discovered is MVPs. MVPs are novel venom peptides that lead to the death or paralysis of spider mites through blockage of ion channels. We aim to obtain highly specialized venom peptides through genome mining in P. persimilis, a specialized predator of spider mites. Specifically, we first investigated PpVP1 and PpVP2. With structural prediction results using AlphaFold, we modified the two full length peptides to give more potent, shorter recombinant PpVP1S and PpVP2S.

  2. Due to the fact that all the MVPs we discovered potentially target CaV channels, we aim to incorporate venom peptides with more diverse molecular targets to suppress potential development of drug resistance in spider mites. Thus, we decide to incorporate SVPs, proteins with numerous disulfide bonds which also functions through binding with ion channels and rapidly modifying ion conductance. Specifically, we incorporated rCtx-4 (targeting NaV channels), Cs1A (targeting CaV channels), and HxTx-Hv1h (targeting both CaV and KV channels). As our toxicity assays suggest, all the venom peptides display significant contact efficacy against spider mites.

  3. In order to enable the successful and effective expression of the venom peptides, we introduced the annex of G1M5-SUMO tag, which increases the solubility of the proteins expressed. Galanthus nivalis agglutinin (GNA), a lectin which enhances both oral and contact toxicity of the fusion protein, is also introduced. pET28a is chosen as the vector for our protein expression in E. coli.
All the venom peptides described above are successfully expressed and tested, showing significant contact efficacy.

Part Numbers Name Type Part Description
BBa_K5184038 PpVP1-F Protein coding sequences MVP potentially targeting insect CaV channels, leading to paralysis and death.
BBa_K5184042 PpVP1-S Protein coding sequences MVP potentially targeting insect CaV channels, leading to paralysis and death.
BBa_K5184043 PpVP2-S Protein coding sequences MVP potentially targeting insect CaV channels, leading to paralysis and death.
BBa_K5184021 rCtx-4 Protein coding sequences SVP targeting the insect NaV channels, leading to death and paralysis.
BBa_K5184032 Cs1A Protein coding sequences SVP targeting the insect CaV channels, leading to death and paralysis.
BBa_K5184033 HxTx-Hv1h Protein coding sequences SVP targeting the insect CaV and KCa channels, leading to death and paralysis.
BBa_K5184022 G1M5-SUMO-tag Protein coding sequences A recombinant tag that allows soluble expression of cysteine-rich venom peptides
BBa_K1974020 GNA Protein coding sequences A plant derived mannose-specific lectin that enhances oral and contact toxicity of SVPs.
BBa_K5184071 G1M5 tag-PpVP2S-GNA Composite part Allows soluble expression of the fusion protein PpVP2S-GNA in E. coli strain BL21(DE3). Fusion of GNA allows improved oral and contact toxicity.
BBa_K5184072 G1M5 tag-PpVP1S-GNA Composite part Allows soluble expression of the fusion protein PpVP1S-GNA in E. coli strain BL21(DE3). Fusion of GNA allows improved oral and contact toxicity.
BBa_K5184073 G1M5 tag-PpVP1F-GNA Composite part Allows soluble expression of the fusion protein PpVP1F-GNA in E. coli strain BL21(DE3). Fusion of GNA allows improved oral and contact toxicity.
BBa_K5184074 G1M5 tag-rCtx4-GNA Composite part Allows soluble expression of the fusion protein rCtx4-GNA in E. coli strain BL21(DE3). Fusion of GNA allows improved oral and contact toxicity.
BBa_K5184075 G1M5 tag-Cs1A-GNA Composite part Allows soluble expression of the fusion protein Cs1A-GNA in E. coli strain BL21(DE3). Fusion of GNA allows improved oral and contact toxicity.
BBa_K5184076 G1M5 tag-HxTx-Hv1h-GNA Composite part Allows soluble expression of the fusion protein HxTx-hv1h-GNA in E. coli strain BL21(DE3). Fusion of GNA allows improved oral and contact toxicity.


Repulsion

To repel spider mites, we decided to synthesize the terpenes 7epiZ, 9HZ and 9H10epoZ, with the latter two having higher repellency than the first. In addition, the terpenes also have fecundity-reducing and lethal properties, contributing to the efficacy of our pesticide.

  1. Using glucose as the raw material, we incorporated the enzyme SltNPPS (BBa_K5184000), which transforms IPP and DMAPP produced through the MVA pathway into NPP; Mvan4662 (BBa_K5184001), catalyzing the formation of Z,Z-FPP from NPP; and ShZIS (BBa_K5184002), synthesizing 7epiZ from Z,Z-FPP. Thus, we construct a complete biosynthesis pathway in E. coli that is able to produce 7epiZ using glucose as the iniital substrate. Through GC-MS analysis, it is verified that 7epiZ is successfully produced.

  2. In order to produce 9HZ and 9H10epoZ, the oxidase ShZPO is used collaboratively with the reductase SlCPR2 or AtCPR1. The collection of the oxidase and reductase can oxidise 7epiZ, leading to the formation of 9HZ and 9H10epoZ. However, these enzymes were originally found in eukaryotic plants. Thus, to express the enzymes in prokaryotic E. coli, we truncated the N-terminus ER transit peptide and added the SpyTag-SpyCatcher system, which will imitate the co-localization of the oxidase with the reductase in ER, thus allowing efficient electron transfer between the P450 enzymes. After testing through protein expression, we discovered that the oxidase ShZPO cannot be expressed successfully in E. coli.

  3. Aiming to improve our production method, we changed our chassis from E. coli to S. cerevisiae, constructing a pathway for the transformation of 7epiZ into 9HZ and 9H10epoZ using the enzymes ShZPO, SlCPR2 and AtCPR1. 9HZ is shown to be successfully produced according to the GC-MS analysis.


Part Numbers Name Type Part Description
BBa_K5184000 SltNPPS Protein coding sequences A neryl diphosphate synthase which catalyzes the formation of NPP from DMAPP and IPP.
BBa_K5184001 Mvan4662 Protein coding sequences A cis-farnesyl diphosphate synthase that catalyzes the formation of Z,Z-FPP from NPP.
BBa_K5184002 ShZIS Protein coding sequences A sesquiterpene synthase that catalyzes the formation of 7epiZ from Z,Z-FPP.
BBa_K5184003 ShZPO Protein coding sequences A cytochrome P450 oxygenase that works with a reductase to transform 7epiZ into 9HZ and 9H10epoZ.
BBa_K5184004 slCPR2 Protein coding sequences A NADPH-cytochrome P450 reductase that works with an oxidase to transform 7epiZ into 9HZ and 9H10epoZ.
BBa_K5184005 AtCPR1 Protein coding sequences A NADPH-cytochrome P450 reductase that works with an oxidase to transform 7epiZ into 9HZ and 9H10epoZ.
BBa_K5184054 sc-t25ShZPO Protein coding sequences Truncated version of the oxidase ShZPO, with SpyCatcher added for co-localization. Transforms 7epiZ into 9HZ and 9H10epoZ.
BBa_K5184055 st-t25ShZPO Protein coding sequences Truncated version of the oxidase ShZPO, with SpyTag added for co-localization with a reductase. Transforms 7epiZ into 9HZ and 9H10epoZ.
BBa_K5184056 sc-t55AtCPR1 Protein coding sequences Truncated version of the reductase AtCPR1, with SpyTag added for co-localization with a reductase. Transforms 7epiZ into 9HZ and 9H10epoZ.
BBa_K5184058 sc-t76SlCPR2 Protein coding sequences Truncated version of the reductase SlCPR2, with SpyCatcher added for co-localization with an oxidase. Transforms 7epiZ into 9HZ and 9H10epoZ.
BBa_K5184059 st-t76SlCPR2 Protein coding sequences Truncated version of the reductase SlCPR2, with SpyTag added for co-localization with an oxidase. Transforms 7epiZ into 9HZ and 9H10epoZ.
BBa_K5184015 SltNPPS-Mvan4662-ShZIS Composite part Pathway for transformation of IPP and DMAPP into 7epiZ in E. coli.
BBa_K5184018 SltNPPS-Mvan4662-ShZIS-st-t25ShZPO-sc-t76SlCPR2 Composite part Attempted pathway for transformation of IPP and DMAPP into 7epiZ, then oxidised to 9HZ and 9H10epoZ in E. coli.
BBa_K5184019 pTDH3-ShZPO-tTDH3-pPGK1-slCPR2-tPGK1 Composite part Expressing the enzymes ShZPO and SlCPR2 in S. cerevisiae using the promotors pTDH3, pPGK1 and the terminators tTDH3, tPGK1. Transforms 7epiZ to 9HZ and 9H10epoZ.
BBa_K5184020 pTDH3-ShZPO-tTDH3-pPGK1-AtCPR1-tPGK1 Composite part Expressing the enzymes ShZPO and AtCPR1 in S. cerevisiae using the promotors pTDH3, pPGK1 and the terminators tTDH3, tPGK1. Transforms 7epiZ to 9HZ and 9H10epoZ.


Recovery

In addition to elimination and repulsion of spider mites, we also aim to prevent immunodeficiency and death of plants. Thus, we incorporated Harpin protein, which is able to induce systematic acquired resistance and enhance the defense response against diverse pathogens and insects. BBa_K5184052 and BBa_K5184053 encode for Harpin protein which can be expressed successfully with solubility. HrpN is the optimized version of the original coding sequence, hrpN-ori, signified by less negative delta G for secondary structure formation in its TIR.

Part Numbers Name Type Part Description
BBa_K5184052 HrpN Protein coding sequences Codes for a TIR-optimized version of harpin protein that can stimulate plants to produce hypersensitive response.
BBa_K5184053 HrpN ori Protein coding sequences Unoptimized (in the TIR) version of the harpin protein.


Reference
[1]Dawood, Mohammad H., and John C. Snyder. “The Alcohol and Epoxy Alcohol of Zingiberene, Produced in Trichomes of Wild Tomato, Are More Repellent to Spider Mites Than Zingiberene.” Frontiers in Plant Science, vol. 11, Feb. 2020, p. 35. DOI.org (Crossref), https://doi.org/10.3389/fpls.2020.00035.
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