Engineering | GreatBay-SCIE

Abstract

Aiming to deal with spider mite infestations, GreatBay-SCIE designed Dienamite, an environmentally-friendly acaricide that integrates extermination, prevention and recovery. To achieve these effects, we incorporated venom peptides (MVP/SVP), both of which target ion cannels of spider mites and lead to death or praralysis of the pests; zingiberene, specifically 7-epi-zingiberene (7epiZ), 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxy zingiberene (9H10epoZ), repels spider mites and prevents infestation; lastly, harpin protein, which enhance disease resistance and defense response. For our sprayed acaricide, GNA is fused to venom peptides to enhance oral and contact toxicity. MVP-GNA fusion proteins achieve soluble expression, and display high lethality against spider mites in a spraying toxicity assay. Aiming to suppress potential development of drug resistance, we also incorporate SVP-GNA fusion proteins, which target a diverse array of receptors and ion channels in the pest nervous system.[1] According to our test assay, SVP-GNA fusion proteins also display significant contact efficacy. 7epiZ, the repellent in Dienamite, is synthesized through introducing a dual-plasmid system to E. coli , achieving an exceptionally high yield. 9HZ, a terpene with enhanced repellent effect, is synthesized through co-culture of S. cerevisiae with E. coli. Lastly, Harpin protein was also successfully expressed and purified, achieving a high protein yield according to the BCA assay conducted.

Cycle 1

Cycle 2

Cycle 3

Dry design

To eliminate spider mites in our project, we went for their natural antagonist: predatory mites. With some research, we discovered that predatory mites employ venom peptides to paralyze, deter, and kill their prey. As proteins from millions of years of evolution, such venom peptides present themselves as an efficient, specific, and biodegradable solution for extermination of spider mites.

We soon found a 2024 research by Chen et al.[2] describing two venom peptides from a predatory mite species, Neoseiulus barkeri. However, being polyphagous [3], mite venom peptides (MVP) from N. barkeri do not display an ideal lethality in the research (at only 58% and 22% lethal rate in 48 hours against T. cinnabarinus). We therefore seek to find more potent venom peptides against spider mites from more specific predatory mites.

Hence we aligned mRNA sequences [4] of the two N. barkeri venom peptides (NbVP1F and NbVP2F) against genome of Phytoseiulus persimilis, a predatory mite that preys predominantly on spider mites and is commonly used in biological control of the pest[5]. We then identified two orthologous venom-peptide-encoding loci [Fig.1] in the P. persimilis genome. Filtering out the introns, we obtained protein sequences of orthologous venom peptides in P. persimilis , of which are named PpVP1F and PpVP2F.

**Fig.1**| BLAST results of NbVP1F and NbVP2F mRNA (GenBank: OR995725.1 and OR995726.1) against *P. persimilis* chromosome 1 (GenBank: CM075101.1), visualized using Kablammo [6]

Dry build

With further research into biology and molecular characteristics of the obtained MVPs, we realized they are homologous to the diverse and well-studied class of spider venom peptides (SVP): both are small, cysteine-rich peptides involving numerous disulfide bonds[1]. We then discovered that the SVPs' toxicity depends on a core venomous domain, which has inhibitory effects on its molecular targets. Other regions of a SVP, however, are not involved in paralyzing/lethal effects directly [7].

Thus, we realized that the full, in vivo forms of the MVPs obtained by BLASTing genomes directly can be truncated down to the core venomous domain for more efficient heterologous expression in unicellular chassis such as E. coli or P. pastoris while retaining or achieving an even higher toxicity.

Matching structural prediction results of the MVPs with a truncated (to only core venomous domain) SVP, rCtx-4 [Fig.2], we identified core venomous domains and thus truncated the full-length versions of the four venoms (NbVP1, NbVP2, PpVP1, and PpVP2) to give their short versions composed only of the venomous domain: NbVP1S, NbVP2S, PpVP1S, and PpVP2S.

**Fig.2**| AlphaFold structural prediction results of PpVP2(solid blue/white) superimposed against core venomous domain of rCtx-4(transparent grey), the non-core regions of PpVP2S is colored in white

Dry test

To understand how MVPs achieve their potential lethal and paralyzing effects, we constructed a phylogenic tree with protein sequences of core venomous domain of SVPs with known molecular target sites [8][9][10] and of the MVPs. The results suggest the MVPs possibly target voltage-gated calcium ion channels, CaV [Fig.3A].

We therefore used AlphaFold Server and HDock to predict effects of the venom peptide against Tetranychus urticae voltage-gated calcium ion channels (sequences from KEGG BRITE [2]). The results show that the venom peptide interferes with alpha 2 subunit of the calcium ion channel, blocking the flow of calcium ions[Fig.3B]. While the prediction is likely to be valid, experiments will still need to be carried out to determine the exact molecular actions[Fig.3C].

Dry learn

From this cycle of engineering, we obtained coding sequences for eight MVPs: NbVP1S, NbVP1F, NbVP2S, NbVP2F, PpVP1S, PpVP1F, PpVP2S, and PpVP2F from predatory mites.

While the MVPs will likely become potent venom peptides against spider mites, numerous disulfide bonds in MVPs makes its expression in E. coli difficult, prompting us to investigate an appropriate expression system.

Therefore, to parallel our dry research on MVP and wet lab preparations for MVP expression, we employed SVP as a representative for our vector exploration due to their molecular similarities with MVPs. Through additional research we realised a lectin, Galanthus nivalis agglutinin (GNA), could enhance the oral and contact toxicity of fused venom peptides [10]. Thus, we also planned to integrate this into our next cycle.

Aiming to make the proteins expressed solublem, we explored expression systems using signal peptides in E. coli.
Click on the title below to see more details.

E. coli expression Systems for Cysteine-Rich Peptides

Design

Due to the high homology and molecular similarities between SVPs and MVPs, and the fact that SVP expression systems are well-explored, we launched engineering of SVP simultaneously with further dry research on MVP. [7] [11]

Under the recommendation of Professor Glenn King, signal peptides (SP) such as MalE and PelB is attached to the N-terminus. The SPs allow newly synthesized proteins to be secreted into the periplasm for a more efficient folding due to a more oxidative environment. [12] A SUMO tag, proven to be able to increase protein solubility, is also integrated into our design, in an attempt to prevent incorrect folding that leads to inclusion bodies. [12] [Fig.4A] GNA is fused to the C-terminus of the venom peptides to improve contact efficacy. [13] [Fig4.B]

We have designed a total of four plasmids with pET28a as the vector, integrating SUMO and SPs to improve solubility of the SVP, rCtx-4, which is selected as a representative of cysteine-rich peptides. We therefore obtained the plasmids MalE-rCtx-4-GNA, MalE-SUMO-rCtx-4-GNA, PelB-rCtx-4-GNA, and PelB-SUMO-rCtx-4-GNA [Fig.4C].

**Fig.4**| (A) SUMO tag facilitates correct folding of the attached protein; Signal peptides (SP) allows perismatic secretion; SUMO protease can then remove the SUMO tag from the final protein (B) Fusing venom peptide with GNA significantly improved oral and contact toxicity against spider mites (C) Plasmid constructs of SP-SVP-GNA, and SP-SUMO-SVP-GNA, where SP, the signal peptide can be MalE or PelB

Build

The plasmids are assembled using GoldenGate assembly, and are subsequently transformed into E.coli strain DH5α. Colony PCR results and sequencing results then verifies the plasmid constructs. [Fig.5].

**Fig.5**| (A) Colony PCR results of 1: PelB-SUMO-rCtx4-GNA, 2: MalE-SUMO-rCtx4-GNA, 3: MalE-rCtx4-GNA, and 4: PelB-rCtx4-GNA (B) Alignment of sequencing results of colony PCR products against their respective designs

Test

Plasmids were transformed into E.coli strain BL21 (DE3), and is then induced by IPTG. The SDS-PAGE shows that MalE-rCtx-4-GNA, MalE-SUMO-rCtx-4-GNA, and PelB-SUMO-rCtx-4-GNA were expressed in inclusion bodies, whereas PelB-rCtx-4-GNA was not successfully expressed [Fig.6].

**Fig.6**| SDS-PAGE of 1: MalE-rCtx4-GNA, 2: MalE-SUMO-rCtx4-GNA, 3: PelB-rCtx4-GNA, and 4: PelB-SUMO-rCtx4-GNA, with complete proteins of BL21(DE3) as control; S: supernatant, P: precipitate

Learn

The SDS-PAGE results suggest that both signal peptides and the SUMO tag did not facilitate correct folding of SVPs, of which aggregates to form inclusion bodies. This suggests that our current combination of MalE/PelB-SUMO is not working as intended and other strategies must be attempted. We therefore decided to switch for the G1M5 signal tag-SUMO system in the next cycle.

Design

The G1M5 secretion system was incorporated along with the SUMO tag in an attempt to achieve both extracellular expression and correct folding of cysteine-rich venom peptides.[11] [Fig.7A].GNA is fused with the SVPs to enhance oral and contact toxicity.

From this, a total of 8 plasmids with mite venom peptide engineered with G1M5, incorporating the SUMO tag were designed, giving G1M5-His-SUMO-MVP-GNA-his. (In which MVP could be NbVP1S, NbVP1F, NbVP2S, NbVP2F, PpVP1S, PpVP1F, PpVP2S, PpVP2F). [Fig.7B]

**Fig.7**| (A)G1M5 secretion system (B)SUMO protease truncates the protein product down to MVP-GNA-His(In which MVP could be NbVP1S, NbVP1F, NbVP2S, NbVP2F, PpVP1S, PpVP1F, PpVP2S, PpVP2F)

Build

We employed GoldenGate assembly to assemble our plasmids from respective fragments and the vector pET28a; which were subsequently transformed into the E.coli strain DH5α. The colony PCR results and sequencing results together verifies the plasmid constructs [Fig.8].

**Fig.8**| (A) Colony PCR results of G1M5-SUMO-MVP-GNA (B) Alignment of sequencing results of the colony PCR products against the corresponding designs

Extracted plasmids were transformed into E.coli strain BL21(DE3), which is then induced by IPTG. SDS-PAGE results show that apart from PpVP2F and NbVP2F, all other MVPs achieved soluble expression.

We successfully expressed the proteins but encountered challenges in its purification. Thus, we performed SUMO digestion on unpurified supernatant directly. Following overnight SUMO digestion, an SDS-PAGE was run, whose results suggest that PpVP1S, PpVP1F, PpVP2S were successfully digested by the SUMO protease. The digested supernatant is then used for toxicity bioassay [Fig.9].

**Fig.9**| Fig.9 Digestion of PpVP2S, PpVP1S, and PpVP1F’s supernatant with SUMO proteases; S: supernatant, SUMO: SUMO-digested supernatant (for each MVP, the marker and SDS-PAGE result for the sample are from the same SDS-PAGE gel, full image could be accessed at https://static.igem.wiki/teams/5184/parts/mvpsumo-full.webp )

Test

For toxicity assay, Professor Huang of SCAU tested the contact toxicity of venom peptides on females of Tetranychus urticae, which is the most prominent species of the spider mites [14]. The SUMO-digested supernatant is applied to the spider mites using a spraying method. The supernatant portion of BL21(DE3) that underwent the same treatments is used as control. The results reveal that PpVP2S, PpVP1S, and PpVP1F are all effective in terms of eliminating T. urticae [Fig.10]. Moreover, PpVP2S achieved a death rate of 98.25% in the first day and 100% in the next.

**Fig.10**| (A) *T. urticae* before being sprayed with MVP (B) T. urticae after being sprayed with MVP (C) Survival plot of the MVPs along with control(supernatant portion of BL21(DE3)) (D) Lethality data of MVPs with control over 24, 48, and 72 hours; data are the means of ± SD of three parallel replicate experiments.

Learn

The potent efficacy of all MVPs elucidate the exceptional contact toxicity inherent to GNA. Particularly, PpVP2S achieved complete elimination in only two days. The other two MVPs, though slightly weaker, also exhibit desirable lethality of 83.64% in 72 hours.

Meanwhile, we also learnt that all of our MVPs potentially target the CaV channel. In order to suppress the development of drug resistance, we desire to incorporate SVPs, which target a diverse array of ion channels. Structurally similar, and equally safe to beneficial insects, the SVPs serve as an ideal supplement for MVPs. Since we successfully used the G1M5-SUMO secretion system for MVP biosynthesis, we also plan to use the same expression system for SVPs.

Design

To prevent development of spider mites' drug resistance, three SVPs, targeting various ion channels, were chosen to supplement MVP to target a more diverse range of molecular targets [Fig.11A].

As shown in Cycle 1-1, MVP and SVP are structurally similar. Thus, same vector designs were applied to SVP. A total of 3 plasmids with SVP engineered with G1M5, incorporating the SUMO tag were designed, giving G1M5-His-SUMO-SVP-GNA-His. (In which SVP could be rCtx-4, Cs1A, HxTx-Hv1h) [Fig.11B].

**Fig.11**| (A) Table of venom peptides and their corresponding ion channel targets (B) Plasmid constructs of and G1M5-His-SUMO-SVP-GNA-His(In which SVP could be rCtx-4, Cs1A, HxTx-Hv1h)

Build

We employed GoldenGate Assembly for assembly of the plasmids, which were subsequently transformed into the E.coli strain DH5α. Colony PCR and sequencing results then verifies the plasmid construct [Fig.12].

**Fig.12**| (A) Colony PCR of 1: G1M5-His-SUMO-rCtx4-GNA-His, 2: G1M5-His-SUMO-Cs1A-GNA-His,3: G1M5-His-SUMO-hv1h-GNA-His (B) Alignment of sequencing results of colony PCR products against the designs

Plasmids were transformed into E.coli straid BL21 (DE3) and is induced by IPTG. SDS-PAGE shows that Cs1A, rCtx4, HxTx-hv1h were successfully expressed intracellularly in the supernatant portion.

After overnight SUMO protease digestion, the results were assessed by an SDS-PAGE, which suggests all SVPs were successfully treated by SUMO, giving only the SVP-GNA fusion protein for later toxicity assays [Fig.13].

**Fig.13**| Digestion of supernatant samples of HxTx-Hv1h, rCtx-4 and Cs1A with SUMO protease, with supernatant of BL21(DE3) as control S: supernatant, SUMO: supernatant after digestion with SUMO protease

Test

Using the same method described in cycle 1-3, the contact efficacy of SVP-GNA fusion proteins was tested. The results reveal that all spider venom peptides exhibit lethal effects on T. urticae in comparison to the control group. In particular, rCtx-4 displays complete extermination within 2 days [Fig.14].

**Fig.14**| (A) Lethality data of SVPs with control over 24, 48, and 72 hours; data are the means of ± SD of three parallel replicate experiments (B) Survival plot of the SVPs along with supernatant of BL21(DE3) as control

Learn

The remarkable potency of SVPs, exemplified by rCtx-4, substantiates the efficacy of our engineering endeavors. Specifically, rCtx-4 displays a phenomenal 100% lethality of female T. urticae in merely 2 days. In the future, we hope that we will be able to provide a collection of SVPs and MVPs, suppressing drug resistance development.

To further optimize venom peptide production, we revisited the initial objective of extracellular expression, which can reduce production time and costs. Given the limitations of E. coli secretion systems, we therefore initiated engineering efforts using an eukaryotic chassis, P. pastoris.

In addition to expressing the venom peptides in E. coli, we also explored using P. pastoris as chassis for our protein expression. Click on the title below to see more details.

VP expression in P. pastoris

Design

Inspired by the interview we conducted with Professor Elaine Fitches(link hp), we decided to replace E. coli with P. pastoris (strain SMD1168H) as the chassis. With secretory tags, P. pastoris can hopefully secrete venom peptides, which can reduce the time and costs involved in sonication cell lysis.

We opted to synthesize several neurotoxins including Mbla, rCtx-4, HxTx-Hv1h, and Cs1A, which target a diverse range of molecular targets. Fusion proteins between them and GNA with a linker sequence of GGGGSAAA is engineered onto the vector pGAPZaB[15][Fig.15]. At the N-terminus of the toxin sequence, the P. pastoris secretory tag—the α-factor—is annexed to secrete the venoms extracellularly. Meanwhile, a polyhistidine tag is fused at the C-terminus, which could be used for optional protein purification[10].

**Fig.15**| (A)Integration of venom peptide coding sequence into P. pastoris GAP integration locus (B) Integration loci after integration of GNA-SVP-His

Build

Digesting pGAPZaB with endonuclease Bsmb1, we linearized the sequences of rCtx4-GNA and Cs1A-GNA, exposing their homologous wall[Fig.16].

**Fig.16**| Bsmb1-digested DNA sequences for integration into P. pastoris via HR

By electrotransforming these two linearized sequences into competent cells of SMD1168H strain, the sequences are expected to be integrated into the yeast genome. We successfully obtained the yeast cultures. However, due to time limitations, we weren't able to perform colony PCR to verify our results. In the remaining time of October, we will continue exploring P. pastoris as the chassis, including the validation, fermentation, and expression of rCtx-4-GNA and Cs1A-GNA and other designs.

Design

7-epi-zingiberene (7epiZ), having repellent, fecundity-reducing, and toxic effects on spider mites [16], is a crucial ingredient of our acaricide. To produce it, we introduced two plasmids to E. coli: pMVA, which introduces the enzymes of the Mevalonate pathway, and pW1-ZIS-NPPS-Mvan4662, which introduces the subsequent enzymes necessary for the production of 7epiZ [Fig.17A&B].

The enzymes, LA2167ZIS(ZIS), the zingiberene synthase [17], Mvan4662Q93S(Mvan4662), a Z,Z-farnesyl diphosphate (Z,Z-FPP) synthase, and SltNPPS(NPPS) a neryl pyrophosphate synthase (NPP) [18], are introduced to E. coli via plasmid expression [Fig.17C]. The three enzymes are cloned into the vector pW1, giving pW1-ZIS-NPPS-Mvan4662. Coding sequences for ZIS is placed closest to the promoter to increase its expression level, for we believe it will likely be the rate-limiting enzyme of the three.

**Fig.17**| (A) The metabolic pathway of 7epiZ (B) Dual-plasmid *E. coli*, containing pMVA and the zingiberene synthesis plasmid. pMVA consists of a total of 7 coding sequences under two promoters. Zingiberene synthesis plasmid consists of three coding sequences under one promoter. (C) Plasmid construct of zingiberene synthesis plasmid: pW1-ZIS-Mvan4662-SltNPPS

Build

pW1-ZIS-Mvan4662-SltNPPS, via GoldenGate cloning, is transformed into E. Coli strain DH5ɑ. After having sequence verified [Fig.18], plasmids are transformed into pMVA DH5ɑ competent cells. The resultant strain, named SCIE1, is thus capable of producing 7epiZ with glucose as its sole carbon source.

**Fig.18**| (A)Colony PCR results of DH5ɑ strain harboring pW1-ZIS-Mvan4662-NPPS; the coding sequence is amplified by four sets of primers, denoted by A, B, C, and D (B) Alignment of colony PCR sequencing results against the designed plasmid

Test

SCIE1 is induced by IPTG and its fermentation is carried out with dodecane as the solvent. The products are collected, and GC-MS analysis and structure elucidation results [Fig.19A&B] suggest that the desired 7epiZ is produced.

Our products also underwent quantitative analysis using caryophyllene as an internal standard according to the method described in the study of Zhang, Suping, et al.[19] According to the GC-MS analysis, the production of 7epiZ is 740mg/L [Fig.19C&D].

**Fig.19**| (A)Gas-phase chromatography results for the SCIE1 culture with dodecane as solvent (B)Mass spectrometry and structure elucidation results of the sample (C) Standard curve of caryophyllene. (D) Yield of 7epiZ of the SCIE1 strain; data are the means of ± SD of three parallel replicate experiments

We also tested the repellency of 7epiZ against T. urticae using supernatant portion of uninduced SCIE1 culture as control and induced SCIE1 strain as the test sample containing 7epiZ[Fig.20]. Initially, the T. urticae is placed on the middle line. It is observed that T. urticae tends to move towards the side containing control instead of the side containing 7epiZ, proving the repellency of 7epiZ.

Learn

Achieving a high concentration of 740mg/L, the GC-MS results suggest that the three enzymes ZIS, Mvan4662, and NPPS are all functionally expressed in E. coli. The repellency of 7epiZ was also tested and proved through bioassay.

In order to achieve better repellent effects, we decide to synthesize 9HZ and 9H10epoZ from further oxidations of 7epiZ. This requires introduction of the oxidase ShZPO and the reductases SlCPR2/AtCPR1 as its redox partner.

We investigated the synthesis of the 9HZ and 9H10epoZ and the expression of enzymes found in eukaryotic cells using E. coli as chassis through truncating the N-terminus anchor region and introducing a SpyTag-SpyCatcher system.
Click on the titles below for more details.

9HZ and 9H10epoZ Biosynthesis in E. coli

Design

After successfully producing 7epiZ, we aim to produce 9HZ and 9H10epoZ, two terpenes that are even more efficient than 7epiZ. [Fig.21A] [16] To enable our product to achieve enhanced repellent effects, we explored further on the basis of cycle 2-1 and introduced the oxidase ShZPO and the reductases AtCPR1 and SlCPR2 as its redox partners [Fig.21B].[20] The oxidase and reductase were originally found in eukaryotic organisms, immobilized on the endoplasmic reticulum (ER) in eukaryotic plant cells [Fig.21C]. [21]

**Fig.21**| (A) Compared to 7epiZ, 9H10epoZ shows better repellency and fecundity-reducing effects against spider mites (B) Metabolic pathway for 9H10epoZ, a zingiberene polyoxidase, ShZPO catalyzes the two sequential oxidation of 7epiZ (C) In plant ER, cytochrome P450 reductases (SlCPR2) and cytochrome P450 oxidases (ShZPO) cluster together; the reductase oxidizes NADPH and transfers the high energy electrons to its corresponding oxidase for the oxidase’s catalysis

We aim to synthesize 9HZ and 9H10epoZ in E. coli, a prokaryotic organism without an ER. Thus, we optimize the oxidase and reductases for production in E. coli through cutting the N-terminal anchor regions of the three enzymes. Specifically, 25 amino acids at the N-terminal of ShZPO, 76 of SlCPR2 and 55 of AtCPR1 were cut according to the tag analysis results [Fig.22].

**Fig.22**| (A) Protein transit signal peptide prediction using DeepLoc 2.1 for AtCPR1, ShZPO, and SlCPR2. Only results for the first 100 amino acids are shown, a higher Y-value of a letter denotes a higher chance of the amino acid being part of the signal peptide (B) Structural prediction results of AtCPR1, ShZPO, and SlCPR2 using AlphaFold Server, the first 55, 25, and 76 amino acids are colored in gray while rest of the enzymes in blue.

Build

We employed GoldenGate Assembly to construct the plasmids pW1-ZIS-NPPS-Mvan4662-t25ShZPO-t76SlCPR2 and pW1-ZIS-NPPS-Mvan4662-t25ShZPO-t55AtCPR1. The colony PCR results reveal successful plasmid construction. The sequencing result confirms that the fragments are successfully linked with no mutations. [Fig.23]

**Fig.23**| (A) Colony PCR results of pW1-ZIS-Mvan4662-NPPS-t25ShZPO-t76SlCPR2 and pW1-ZIS-Mvan4662-NPPS-t25ShZPO-t55AtCPR1 in DH5ɑ (B) Alignment of sequencing results of pW1-ZIS-Mvan4662-NPPS-ShZPO-SlCPR2 (top) and pW1-ZIS-Mvan4662-NPPS-ShZPO-AtCPR1 (bottom) against the design

Test

Fermentation of pW1-ZIS-NPPS-Mvan4662-t25ShZPO-t76SlCPR2 and pW1-ZIS-NPPS-Mvan4662-t25ShZPO-t55AtCPR1 in DH5α was induced by IPTG and lasted 24 hours using dodecane as solvent. After the products were collected and underwent GC-MS analysis, we discovered that 9HZ and 9H10epoZ were not detected. Instead, only 7epiZ was produced. [Fig.24]

**Fig.24**| (A) Gas-phase chromatography results for the pW1-ZIS-Mvan4662-NPPS-t25ShZPO-t76SlCPR2 culture with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample

Learn

Since only 7epiZ is detected, we hypothesize that the problem lies in the expression of the oxidase and reductases. The expression level of oxidase and reductases in E.coli may be too low, or the enzymes could only be expressed as inclusion bodies instead of maintaining their functions after expression. To confirm our hypothesis and enhance the activity of the enzymes, we must test the expression of our enzymes and introduce an approach that can increase the efficacy of the oxidase and reductases.

SpyTag-SpyCatcher and P450 Enzyme Verification

Design

To increase the expression level and functionality of oxidase and reductases in E. coli, we introduced the SpyTag-SpyCatcher system in addition to truncating the N-terminal anchor regions [Fig.25A]. Through the formation of an isopeptide bond between the tag and the catcher, introduction of this system links the oxidase and reductases together, thus facilitating efficient electron transfer.[22]

We constructed the plasmid pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2, aiming to test whether the enzymes can maintain their functions after expression and whether the SpyTag can be linked to the SpyCatcher [Fig.25B]. Also, we incorporated the plasmids sc t25ShZPO, st t76SlCPR2 and sc t55AtCPR1 to test whether the enzymes can be successfully expressed in E. coli.[Fig.25C]

**Fig.25**| (A) Attachment of SpyTag and SpyCatcher to two cytochrome P450 enzymes; the formation of isopeptide bond between lysine on SpyCatcher and aspartic acid on SpyTag can link the two enzymes together, this proximity allows electron transfer and thus catalysis of the CYP (t25ShZPO) enzyme to occur (B) Plasmid construct of pW1-p-ZIS-Mvan4662-NPPS-T-P-SpyTag-t25ShZPO-SpyCatcher-t76SlCPR2-t (C) Plasmid constructs of SpyTag-t76SlCPR2, SpyCatcher-t25ShZPO, and SpyCatcher-t55AtCPR1

Build

We employed GoldenGate Assembly to build the plasmids pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2, sc t25ShZPO, st t76SlCPR2, sc t55AtCPR1[Fig.26A&B] and then transformed the built plasmids into the E. coli strain DH5α. The colony PCR results and sequencing results show that the plasmids are successfully constructed with no mutations.[Fig.26C&D]

**Fig.26**| (A) Colony PCR results of sc t55AtCPR1, t76SlCPR2, and sc t25ShZPO (B) Alignment of the sequencing results colony PCR results in Fig26A: sc t55AtCPR1, t76SlCPR2, and sc t25ShZPO against designed plasmids (C) Colony PCR results of pW1-ZIS-Mvan4662-NPPS-st t25ShZPO-sc t76SlCPR2; the insert region is amplified with two sets of primers and their products are designated SCIE22E and SCIE22F (D) Alignment of the sequencing results of colony PCR products in Fig26C, SCIE22E and SCIE22F against designed plasmids

Test

Fermentation of pW1-ZIS-NPPS-Mvan4662-st t25ShZPO-sc t76SlCPR2 in DH5α was induced by IPTG and lasted 48 hours using dodecane as solvent. After the products were collected and underwent GC-MS analysis, we discovered that still, 9HZ and 9H10epoZ were not detected. Instead, only 7epiZ was produced.[Fig.27]

Plasmids sc t25ShZPO, st t76SlCPR2, sc t55AtCPR1 were transformed into E.coli strand BL21(DE3), fermented and induced by IPTG. The SDS-PAGE result shows that only reductases linked with the SpyTag or SpyCatcher could be expressed successfully, and the oxidase was expressed in inclusion bodies.[Fig.28]

**Fig.27**| (A) Gas-phase chromatography results for the t25ShZPO-t76SlCPR2 culture with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample

**Fig.28**| SDS-PAGE of 1: SpyCatcher-t25ShZPO, 2: SpyCatcher-t55AtCPR1, 3: SpyTag-t76SlCPR2, with complete proteins of BL21(DE3) as control S: Supernatant, P: precipitate

Learn

After the verification of enzyme expression in E. coli, and discovering the absence of 9HZ and 9H10epoZ in our products, we confirm our hypothesis that the oxidase ShZPO linked with the SpyTag-SpyCathcer system cannot be expressed successfully in E. coli. The results suggest that using a prokaryotic organism as chassis may not lead to satisfying results.

Design

Due to the fact that 9HZ and 9H10epoZ, which exhibit better repellent and fecundity-reducing properties, can not be successfully synthesized in E. coli, we decided to change the chassis for our terpene synthesis. After communication with Dr. Su from Earlham Institute, we opted for the yeast S. cerevisiae (strain CEN.PK2-1C).[Fig.29] As an eukaryote with endoplasmic reticula, S. cerevisiae enables co-localization of the oxidase with reductases while also ensures efficient enzyme expression.

**Fig.29**| (A) Biosynthesis pathway of 9HZ and 9H10epoZ, starting from IPP and DMAPP that are products of the MVA pathway (B) Integration of the two cytochrome P450 enzymes coding sequences into *S. cerevisae* genome: the pCRCT plasmid, encoding the endonuclease Cas9 and sgRNA for His integration locus leads to restriction at the His locus, of which, after a series of homologous recombination between the yeast genome and insert fragments, leading to integration of the cytochrome P450 enzyme genes into the *S. cerevisae* genome

Build

We inserted DNA fragments to His integration locus of CEN.PK2-1C using lithium acetate transformation. Afterwards, yeast colony PCR was conducted, which shows that the target strands were integrated into the genome successfully. The sequencing result also shows that the fragments are integrated into the yeast genome with no mutation. The constructed strains are named ShZPO-SlCPR2 and ShZPO-AtCPR1 respectively. [Fig.30]

**Fig.30**| (A) Colony PCR results of ShZPO-SlCPR2A in His locus, ShZPO-SlCPR2B in His locus, ShZPO-tCPR1A in His locus, and ShZPO-AtCPR1B in His locus in *S. cerevisae* (B) Alignments of sequencing results of colony CPR products against designed locus

Test

ShZPO-SlCPR2 and ShZPO-AtCPR1 were cocultured with SCIE1 respectively. Fermentation of the cocultures was carried out, lasting 48 hours at 28°C 200 rpm and using dodecane as solvent. After the products were collected and underwent GC-MS analysis, 9HZ, a mid-product of the redox reaction of 7epiZ to 9H10epoZ was detected from the co-culture using ShZPO-SlCPR2 only.[Fig.31]

**Fig.31**| (A) Gas-phase chromatography results for culture of ShZPO-SlCPR2 in His locus with dodecane as solvent (B) Mass spectrometry and structure elucidation results of the sample

Learn

The fact that opting for S. cerevisiae as our chassis enables successful production of 9HZ verifies our hypothesis--compared to E. coli, S. cerevisiae produces the oxidase and reductases efficiently, maintaining their functions after expression. During fermentation, we explored the method of co-culture for S. cerevisiae and E. coli, which will help the iGEM community through providing a novel fermentation method.

In the future, we will investigate optimizations of ShZPO to enable successful expression of the enzyme in E. Coli via computational modelling of amino acid point mutations and molecular docking.

Design

To allow faster recovery of the plant after an infestation, we introduced harpin protein [Fig.32A]. Harpin protein originates from bacterial plant pathogens that, through evolution, had become a plant immunostimulant that can trigger a range of plant responses leading to enhanced disease resistance and promoted plant growth [22].

A research [13] suggests that the original harpin protein gene leads to formation of secondary structures in its translation initiation region (TIR) of its mRNA and impacts final protein yield.[23] We therefore designed and synthesized two plasmids with different TIRs of the harpin genes: pET28a-hrpN and pET28a-hrpN-ori[Fig.32B]. hrpN is the TIR-optimized version, with a less negative delta G for secondary structure formation in its TIR of the original coding sequence comparing with the unoptimized version, hrpN-ori[Fig.32B].

**Fig.32**| (A) Harpin protein will induce hypersensitive response in plants; this will thus promote growth and a faster recovery of the plant after a spider mite infestation (B) Plasmids constructs for hrpN and hrpN-ori (C) RNA secondary structure prediction and their associated free energy changes in the TIR of hrpN-ori and hrpN using RNAfold

Build

The synthesized plasmids pET28a-hrpN and pET28a-hrpN-ori [Fig32B] are transformed into BL21(DE3), cultured under 37°C, and is subsequently induced with IPTG when OD600 reached 0.8-1.0. Supernatant and precipitate were collected after sonication cell lysis.

Test

The supernatant was purified via 6×His tag, followed by dialyzing the purification product. The results were assessed via SDS-PAGE [Fig.33A], revealing successful expression of all proteins. A BCA assay was carried out then to determine specific protein yield of 152.94 μg/ml for hrpN and 106.90l for hrpN-ori [Fig.33B&C].

**Fig.33**| (A) Purification of hrpN and hrpN-ori, with complete protein of BL21(DE3) as control S: supernatant, P: precipitate, FT: flowthrough, W: wash E: elusion (B) BCA assay of hrpN and hrpN-ori (C) Comparison of protein yields of hrpN(152.94μg/ml) and hrpN-ori(106.89μg/ml), statistical test: unpaired t-test suggests a significant difference ****: p< 0.0001; data are the means of ± SD of three parallel replicate experiments.

Learn

BCA assay suggests that the optimized hrpN results in a significant increase in protein yield comparing to hrpN-ori , signifying the effect of the structure of the TIR on expression rate and thus final yield of the protein.

Aiming to produce a multifunctional acaricide, the terpenes, venom peptides and Harpin proteins are all incorporated into the product, targeting pre-infection, mid-infection and post-infection stages respectively. Being the first to discover the MVPs from P. persimilis, we are determined to fully make use of the novel venom peptide, which will work together with the SVPs, targeting a diverse range of ion channels. Both of them display high lethal effects, with some reaching 100% after 48 hours. Through investigating the zingiberene synthesis pathway, our production of 7epiZ reaches a high yield of 740mg/L. We also successfully synthesized 9HZ, a terpene with higher repellent effects, through exploring the co-culture of S. cerevisiae and E. coli. Harpin proteins, which play an important role in the recovery of plants after infestation, are successfully expressed and achieve a high yield of 152.94μg/mL. Ultimately, we wish to bring prosperity back to gardens and augment agricultural production. We also believe that the emergence of Dienamite provides worldwide users a more eco-friendly substitution, minimizing toxic residues and preserving the environment.

**Fig.34**| Project overview diagram for Dienamite.

Reference

[1]Guo, Ruiyin, et al. ‘Spider-Venom Peptides: Structure, Bioactivity, Strategy, and Research Applications’. Molecules, vol. 29, no. 1, Dec. 2023, p. 35. DOI.org (Crossref), https://doi.org/10.3390/molecules29010035
[2]KEGG BRITE: Ion Channels - Tetranychus urticae (two-spotted spider mite). (n.d.). https://www.kegg.jp/kegg-bin/show_brite?htext=tut04040&option=%2da&highlight=107370320&option=-a&selected=join-brite-kegg-1&extend=B6B8B14
[3]Zhu, T., Li, W., Xue, H., Dong, S., Wang, J., Shang, S., & Dewer, Y. (2023). Selection, Identification, and Transcript Expression Analysis of Antioxidant Enzyme Genes in Neoseiulus barkeri after Short-Term Heat Stress. Antioxidants, 12(11), 1998. https://doi.org/10.3390/antiox12111998
[4]GenBank accession numbers: OR995725.1 and OR995726.1
[5]Phytoseiulus persimilis. (n.d.-b). https://biocontrol.entomology.cornell.edu/predators/Phytoseiulus.php
[6]Salman, S. Y., & Keskin, C. (2019). The effects of milbemectin and spirodiclofen resistance on Phytoseiulus persimilis A. H. (Acari:Phytoseiidae) life table parameters. Crop Protection, 124, 104751. https://doi.org/10.1016/j.cropro.2019.02.027
[7]Windley, M. J., Herzig, V., Dziemborowicz, S. A., Hardy, M. C., King, G. F., & Nicholson, G. M. (2012). Spider-Venom peptides as bioinsecticides. Toxins, 4(3), 191–227. https://doi.org/10.3390/toxins4030191
[8]Yu, N., Yan, Y., Han, Q., Zhang, L., & Liu, Z. (2023). Insecticidal toxicity of ω‐Atypitoxin‐Cs1a and its inhibitory effects on insect voltage‐gated calcium channels. Pest Management Science, 79(12), 4879–4885. https://doi.org/10.1002/ps.7689
[9]Vásquez-Escobar, J., Benjumea-Gutiérrez, D. M., Lopera, C., Clement, H. C., Bolaños, D. I., Higuita-Castro, J. L., Corzo, G. A., & Corrales-Garcia, L. L. (2023c). Heterologous Expression of an Insecticidal Peptide Obtained from the Transcriptome of the Colombian Spider Phoneutria depilate. Toxins, 15(7), 436. https://doi.org/10.3390/toxins15070436
[10]Sukiran, N. A., Pyati, P., Willis, C. E., Brown, A. P., Readshaw, J. J., & Fitches, E. C. (2022). Enhancing the oral and topical insecticidal efficacy of a commercialized spider venom peptide biopesticide via fusion to the carrier snowdrop lectin (Galanthus nivalis agglutinin). Pest Management Science, 79(1), 284–294. https://doi.org/10.1002/ps.7198
[11]Chen, L., Adang, M. J., & Shen, G. (2024a). A novel spider venom peptide from the predatory mite Neoseiulus barkeri shows lethal effect on phytophagous pests. Pesticide Biochemistry and Physiology, 202, 105963. https://doi.org/10.1016/j.pestbp.2024.105963
[12]Liu, C., Yan, Q., Yi, K., Hu, T., Wang, J., Zhang, Z., Li, H., Luo, Y., Zhang, D., & Meng, E. (2022). A secretory system for extracellular production of spider neurotoxin huwentoxin-I in Escherichia coli. Preparative Biochemistry & Biotechnology, 53(8), 914–922. https://doi.org/10.1080/10826068.2022.2158473
[13]Mirzadeh, K., Shilling, P. J., Elfageih, R., Cumming, A. J., Cui, H. L., Rennig, M., Nørholm, M. H. H., & Daley, D. O. (2020). Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region. Microbial Cell Factories, 19(1). https://doi.org/10.1186/s12934-020-01339-8
[14]Tetranychus urticae (two-spotted spider mite). (2022b). [Dataset]. In CABI Compendium. https://doi.org/10.1079/cabicompendium.53366
[15]Fitches EC, Pyati P, King GF, Gatehouse JA. Fusion to snowdrop lectin magnifies the oral activity of insecticidal ω-Hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system. PLoS One. 2012;7(6):e39389. doi: 10.1371/journal.pone.0039389. Epub 2012 Jun 22.
[16]Dawood, M. H., & Snyder, J. C. (2020). 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, 11. https://doi.org/10.3389/fpls.2020.00035
[17]Bleeker, P. M., Mirabella, R., Diergaarde, P. J., VanDoorn, A., Tissier, A., Kant, M. R., Prins, M., De Vos, M., Haring, M. A., & Schuurink, R. C. (2012). Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative. Proceedings of the National Academy of Sciences, 109(49), 20124–20129. https://doi.org/10.1073/pnas.1208756109
[18]Lei, M., Qiu, Z., Guan, L., Xiang, Z., & Zhao, G. (2023). Metabolic Engineering for Efficient Production of Z,Z-Farnesol in E. coli. Microorganisms, 11(6), 1583. https://doi.org/10.3390/microorganisms11061583
[19]Zhang, Suping, et al. ‘De Novo Biosynthesis of Alpha-Zingiberene from Glucose in Escherichia Coli’. Biochemical Engineering Journal, vol. 176, Dec. 2021, p. 108188. DOI.org (Crossref), https://doi.org/10.1016/j.bej.2021.108188
[20]Choi, Won, et al. ‘Heterologous Expression in E. Coli and Functional Characterization of the Tomato CPR Enzymes’. Applied Biological Chemistry, vol. 66, no. 1, Dec. 2023, p. 92. DOI.org (Crossref), https://doi.org/10.1186/s13765-023-00850-x
[21]Li, Yikui, et al. ‘De Novo Biosynthesis of P-Coumaric Acid in E. Coli with a Trans-Cinnamic Acid 4-Hydroxylase from the Amaryllidaceae Plant Lycoris Aurea’. Molecules, vol. 23, no. 12, Dec. 2018, p. 3185. DOI.org(Crossref), https://doi.org/10.3390/molecules23123185
[22]Chen, Y., Tan, S., Yang, F., Chen, Z., Wu, Z., & Huang, J. (2017). Soluble expression and purification of a functional harpin protein in Escherichia coli. Process Biochemistry, 57, 200–206. https://doi.org/10.1016/j.procbio.2017.03.010
[23]Cai, Zengying, et al. ‘Efficient Expression and Purification of Soluble HarpinEa Protein by Translation Initiation Region Codon Optimization’. Protein Expression and Purification, vol. 188, Dec. 2021, p. 105970. DOI.org (Crossref), https://doi.org/10.1016/j.pep.2021.105970

Cycle1-1: MVP Genome Mining & Optimization

Cycle1-2: MVP Expression and Toxicity Assay

Cycle1-3: SVP Expression and Toxicity Assay

Cycle2-1: 7epiZ Biosynthesis

Cycle2-2: 9HZ and 9H10epoZ Biosynthesis co-culture

Cycle3: Harpin protein expression and yield determination

Conclusion