Experiments

The primary objective of this study is to elucidate the catalytic role of cytochrome P450 (CYP450) in the enzymatic hydroxylation of phenol to hydroxyquinone (HQ), a key chemical implicated in termite attraction mechanisms. Specifically, this work aims to compare the expression efficiency, catalytic activity, and substrate conversion kinetics between the wild-type CYP450 and its engineered double mutant (A83F/A329F). Both enzymes were heterologously expressed in Escherichia coli BL21 (DE3) using T7 promoter–driven plasmid constructs. The study employs a multi-layered analytical approach: (1) Fluorescence microscopy for monitoring mCherry-tagged protein localization, (2) Western blotting for validation of FLAG-tagged protein expression, and (3) spectrophotometric assays for quantification of HQ formation over time — to determine whether the double mutation enhances enzymatic performance and contributes to more efficient HQ biosynthesis.

1. Plasmid Construction and Preparation

Wild-type and double-mutant (A83F/A329F) CYP450 genes were cloned into T7 promoter–based plasmids, each fused with either FLAG or mCherry tags for protein detection: T7-CYP450-flag (BBa_25HS5FHK), T7-CYP450-mCherry (BBa_25H2URHN), T7-CYP450DM-Flag (BBa_25MIYL3S) and T7-CYP450DM-mCherry (BBa_25MRBB84). Control plasmids without CYP450 insert were used as negative references. All constructs were custom synthesized (VectorBuilder) and purified using a Plasmid Midi Preparation Kit (Beyotime D0020). DNA purity and concentration were assessed by UV spectrophotometry at 260/280 nm, with A260/A280 ratios maintained around 1.8 and A260/A230 > 2.0. Purified plasmids were stored at −20 °C until further use.

2. E. coli Transformation and Protein Expression

Competent E. coli (DE3) cells were transformed with the respective plasmids using a standard heat-shock method. Following recovery in antibiotic-free LB medium, transformants were selected on antibiotic-containing agar plates and incubated overnight at 37 °C. Single colonies were inoculated into liquid LB medium and grown to mid-log phase (OD₆₀₀ = 0.5–0.8) before induction with 0.5 mM IPTG for 4–6 h at 37 °C. Cells expressing mCherry-tagged constructs were directly subjected to fluorescence microscopy, whereas those carrying FLAG-tagged constructs were harvested for protein extraction and Western blot analysis.

3. Fluorescence Detection

To verify successful expression of mCherry-tagged CYP450 fusions, induced and uninduced cultures were examined under a fluorescence microscope (excitation: 540–580 nm; emission: 592–668 nm) using consistent exposure parameters. Controls included untransformed E. coli BL21 cells and PBS/LB only background slides. Both bright-field (TL-DIC) and fluorescence images were captured to correlate cellular morphology with protein-localized fluorescence.

4. Western Blot Analysis

Protein expression of FLAG-tagged CYP450 variants was analyzed by Western blotting following standard protocols. Briefly, IPTG-induced E. coli BL21 (DE3) cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with phosphatase and protease inhibitors (Roche). Lysates were centrifuged at 16,000 × g for 20 min at 4 °C, and protein concentrations were determined by BCA assay.

Equal amounts of protein (20 µg) were denatured in 2× Laemmli buffer, separated by SDS–PAGE, and transferred onto PVDF membranes in Tris/Glycine buffer containing 20% methanol. Membranes were blocked with no fat milk in distilled water and incubated with anti-FLAG primary antibodies overnight at 4 °C, followed by rabbit-derived secondary antibody for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL).

5. HQ Production Experiment

Cell suspensions were prepared by washing induced E. coli BL21 cultures with 100 mM potassium phosphate buffer (pH 8.0). Reactions (4 mL per tube) were supplemented with glucose (0.05 g mL⁻¹) and phenol (10 mM) and incubated at 30 °C and 220 rpm. Samples (150 µL) were withdrawn at different time points (0, 0.5, 1, 2, 2.5, 3, and 4 h), and enzyme activity was quenched by methanol addition. After centrifugation, HQ concentrations in the supernatants were determined spectrophotometrically at 290 nm based on a pre-established standard curve. Kinetic parameters were analyzed via nonlinear regression to evaluate the catalytic efficiency of wild-type and mutant enzymes.

Plasmid Construction and Preparation

To investigate the role of CYP450-mediated hydroxyquinone (HQ) synthesis in termite attraction, CYP450 and its variant (a double mutation at position A83F and A329F) were cloned into distinct plasmid backbones, alongside control plasmids for expression validation. All plasmids were extracted using a standard plasmid isolation kit (Plasmid Midi Preparation Kit for All Purpose, Beyotime D0020), and the DNA concentration were quantified by UV spectrophotometry. The concentrations of the extracted plasmids were as follows:

Plasmid Name	Concentration (ng/μL)
T7-CYP450-flag	65.1
T7-CYP450 DM-flag	46.1
T7-CYP450-mCherry	47.4
T7-CYP450 DM-mCherry	145.9

Protein Expression Validation

Fluorescence Results

Experimental Controls for Fluorescence Background Validation

Fluorescence microscopy was used for detecting mCherry, a red fluorescent protein, with uniform settings (excitation: 540–580 nm, dichroic mirror: 585 nm, emission: 592–668 nm), and transmitted light-differential interference contrast (TL-DIC) microscopy to visualize. To rule out background interference, two fundamental control groups (PBS buffer control and E. coli BL21 control) were established. The E. coli BL21 (no plasmid) sample (Fig. 1a) served as a host-specific control for T7-driven constructs (all transformed into BL21); the TL-DIC channel revealed well-defined cells, verifying viability, whereas the fluorescence channel showed no detectable red fluorescence, eliminating BL21 as a background source.

Fluorescence Microscopy for mCherry-Tagged Constructs

Fluorescence imaging was performed to validate the expression of mCherry-tagged protein using the following section: excitation wavelength 540–580 nm, and emission wavelength 592–668 nm). All E. coli BL21 samples, transformed with different constructs: T7-CYP450-mCherry T7-CYP450 DM-mCherry and T7-CYP450-mCherry (wild-type) and with or without IPTG induction, were imaged in both TL-DIC and fluorescence channels to correlate cell morphology with fluorescence signals. For the T7-CYP450-mCherry and T7-CYP450 DM-mCherry constructs transformed into BL21, cultures were subjected to IPTG induction prior to fluorescence microscopy analysis. PTG induction entailed growing cultures to mid-logarithmic phase (OD₆₀₀ ≈ 0.6), adding IPTG to a final concentration of 0.5 mM, and incubating at 37°C for 4 h to induce protein expression. In the IPTG-induced T7-CYP450-mCherry sample (Fig. 1b), the TL-DIC channel revealed healthy BL21 cells, confirming that observed fluorescence was not attributable to debris, whereas the fluorescence channel exhibited strong red fluorescence, conformed the expression of the wild-type fusion protein. Similarly, for the IPTG-induced T7-CYP450 double-mutant-mCherry sample (Fig. 1c), the TL-DIC channel confirmed viable cells, and the fluorescence channel displayed intense red fluorescence, aligning with higher expression levels observed in flag-tagged mutants. For the negative controls of the bacteria without IPTG induction, both the T7-CYP450-mCherry (Fig. 1d) and T7-CYP450 DM- mCherry (Fig. 1e) samples showed healthy cells in the TL-DIC channel, but no detectable red fluorescence in the fluorescence channel, thereby validating the IPTG dependency of the T7 promoter protein expression.

Figure 1: IPTG-Induced Expression of T7-CYP450-mCherry and CYP450-DM-mCherry Fusion Proteins in E. coli BL21. a E. coli BL21 (no plasmid control). b T7-CYP450-mCherry with IPTG-induction. c T7-CYP450 DM-mCherry with IPTG-induction. d T7-CYP450-mCherry negative control (without IPTG induction). e T7-CYP450 DM-mCherry negative control (without IPTG induction).

Western Blot Analysis

Western blot analysis was employed to validate the expression of flag-tagged CYP450 constructs. After SDS-PAGE and transferred the protein to the PDVF membranes. Membranes were probed with anti-flag primary antibodies, followed by visualization through ECL chemiluminescence. For the T7-CYP450-flag constructs transformed into E. coli BL21, a distinct band at approximately 115 kDa was detected in the IPTG- induced T7-CYP450-flag sample using anti-flag antibodies (Fig. 2a), confirming the expression of the wild-type CYP450 enzyme. The expression levels are in similar level after 4 hours IPTG induction, with the strongest expression at 6 hours. For the IPTG- induced T7-CYP450 DM-flag sample (Fig. 2b), a pronounced band at ~115 kDa was observed after 6 hours of IPTG induction, which is similar to the wild-type protein.

a

b

Figure 2: Detection was performed using anti-flag primary antibody (specifically targeting the flag tag fused to CYP450 variants) and a rabbit-derived secondary antibody (for signal amplification), with visualization via chemiluminescence. a T7-CYP450-flag (wild type; ~115 kDa band in IPTG-induced BL21). bT7-CYP450 DM-flag (intense ~115 kDa band in IPTG-induced BL21), indicating higher expression of the mutant.

Note: "Mock" in the blot represents the empty vector control (i.e., E. coli BL21 harboring only the empty plasmid backbone, without the T7-CYP450-flag insert), used to rule out non-specific binding of antibodies to the vector or host strain’s intrinsic proteins.

CYP450 Function and Hydroxyquinone (HQ) Detection

Standard Curve for HQ Quantification

Figure 3: Standard curve for hydroxyquinone (HQ) quantification at 290 nm.

To enable accurate quantification of hydroxyquinone (HQ) production, the maximum absorption wavelength (λ_max) of HQ was determined to be 290 nm through full-spectrum scanning. A standard calibration curve was subsequently constructed by plotting HQ concentrations (mg/mL) against corresponding absorbance values at 290 nm. Linear regression analysis produced the equation Y = 4.200X + 1.286 (where Y denotes absorbance and X represents HQ concentration), accompanied by a high coefficient of determination (R² = 0.9936), signifying robust linearity. Further statistical evaluation affirmed the significance of the non-zero slope (F = 312.8, P = 0.0032), with the 95% confidence interval for the slope (3.178–5.222) excluding zero. These metrics collectively validate the standard curve as a dependable method for HQ quantification in ensuing bacterial culture analyses (Fig. 3).

Time-course of HQ Production

Figure 4: Time-course of phenol-to-HQ conversion by CYP450 variants.

To assess the functional activity of CYP450 variants in converting phenol (substrate for CYP450-mediated hydroxylation) to HQ, bacterial cultures expressing either the wild- type (T7-CYP450-flag) or double-mutant (T7-CYP450 DM-flag) constructs were supplemented with phenol and incubated under shaking conditions for 4 hours. Samples (150 μL) were collected at 30-minute intervals, centrifuged, and the pellets resuspended in potassium phosphate buffer for spectrophotometric measurement of HQ accumulation at 290 nm. This wavelength was selected according to the HQ characteristic λ_max attributable to conjugated double bonds and hydroxyl groups, facilitating precise quantification.

The double-mutant strain exhibited a distinct kinetic pattern: HQ levels remained low for the first ~2.5 hours, followed by a sharp increase after 3 hours, reaching approximately 0.25 mg mL^-1 at 4 hours. In contrast, the wild-type enzyme showed only a gradual increase, with HQ concentration remaining below 0.05 mg mL^-1. Nonlinear regression confirmed a strong fit for the mutant (R² = 0.9959) but a weaker one for the wild type (R² = 0.8575), reflecting greater catalytic efficiency.

Statistical analysis supported these observations: although the mean difference between the two curves was not significant (unpaired t-test, p = 0.0615), an F-test revealed a highly significant difference in reaction variance (F(8, 8) = 35.04, p < 0.0001), indicating markedly different kinetic behaviors. The mutant displayed a more cooperative and consistent catalytic trajectory, achieving significantly higher HQ accumulation by the end of the assay.

These results collectively demonstrate that the A83F/A329F double mutation substantially enhances catalytic performance and reaction stability in CYP450- mediated phenol hydroxylation.

4-hour Endpoint Analysis of HQ Concentration

Figure 5: 4-hour endpoint analysis of HQ production

At the 4-hour endpoint, HQ concentrations were quantified across experimental groups to evaluate catalytic performance (Fig. 5). The control strain (BL21 without plasmid) produced 0.029 mg mL⁻¹ HQ, confirming negligible endogenous activity. The wild- type T7-CYP450-flag yielded 0.043 mg mL⁻¹, while the double-mutant T7-CYP450 DM-flag reached 0.269 mg mL^-1—representing ~6.2-fold and ~9.2-fold increases over the wild- type and control, respectively.

A one-way ANOVA revealed a highly significant difference among the three groups (F(2,6) = 1882, p < 0.0001, R² = 0.9984), with Tukey’s post-hoc test confirming that the double-mutant group was significantly higher than both the wild-type and control (p < 0.05). Brown–Forsythe (p = 0.4823) and Bartlett’s (p = 0.349) tests verified homogeneity of variances, validating the ANOVA assumptions.

Collectively, these results demonstrate that the A83F/A329F double mutation substantially enhances CYP450’s catalytic efficiency in phenol hydroxylation, confirming the superior functionality of the engineered variant.

This study examined the cytochrome P450 from Bacillus megaterium (CYP102A1, P450-BM3) and its engineered double mutant (A83F/A329F) for the bioconversion of phenol to hydroquinone (HQ). Both constructs were expressed in E. coli BL21(DE3), and the mutant produced significantly higher HQ levels (0.269 mg mL^-1) after 4 hours compared with the wild type (0.043 mg mL^-1), indicating improved catalytic efficiency resulting from rational residue substitutions.

Expression stability revealed by Western blot

Western blotting revealed that the double mutant accumulated rapidly during the first 6 hours of IPTG induction but declined after overnight expression. This suggests that the mutant folds efficiently in the early phase but becomes unstable over extended induction, likely due to aggregation or proteolytic degradation. Aromatic substitutions such as phenylalanine can enhance folding through hydrophobic interactions but may also expose aggregation-prone surfaces [1].

Overall, these results suggest a trade-off between high short-term expression and reduced long-term stability in the mutant—a phenomenon frequently observed in enzyme engineering, particularly among hyperactive P450 variants [2][3]. Indeed, in CYP450 BM3-based biocatalysts, highly active mutants have been reported to lose activity rapidly during prolonged reactions, reflecting a general stability–activity trade-off [4].

Catalytic performance and possible mechanisms

The improved HQ formation by the mutant may arise from multiple, non-exclusive factors. On one hand, the higher HQ yield observed for the mutant may result from increased levels of soluble, catalytically competent enzyme at 6 h, providing more active protein for the reaction. On the other hand, the A83F/A329F substitutions could intrinsically enhance catalytic efficiency—by influencing substrate orientation or electron transfer within the active site—similar to the effects reported for the A82F/A328F variant of P450-BM3 [5].

While this mechanistic rationale may also apply to A83F/A329F, direct kinetic evidence (e.g., kcat, Km, and coupling efficiency) will be required to disentangle contributions from increased enzyme abundance versus intrinsic catalytic enhancement.

Physiological relevance and potential applications

HQ acts as a phagostimulating and aggregation cue in termites, effective at nanogram surface loads (ng cm^-2), while higher concentrations can reduce attractiveness [6]. The HQ level achieved in this study (~0.269 mg mL^-1) exceeds typical behavioral thresholds, suggesting that dilution or controlled-release formulation would be required for practical application. Nevertheless, this system provides a green and biosafe route for HQ synthesis suitable for pheromone-based termite management.

Implications for the “Attraction–Killing” termite control system

The target species, Coptotermes formosanus Shiraki, uses HQ and 2-phenoxyethanol as foraging and recruitment cues [7, 8]. HQ produced via CYP450 catalysis could be incorporated into toxic baits to attract termites while ensuring biosafety, as only purified HQ—not engineered bacteria—is used. However, the optimal concentration range must be determined to avoid overdosage or conversion of HQ to quinones, which may reduce attractiveness [6].

Stabilization methods such as microencapsulation, slow-release polymers, or antioxidants can further improve field stability and minimize environmental impact.

Limitations and future outlook

While the A83F/A329F variant demonstrated higher short-term expression and improved catalytic performance, its reduced stability upon extended induction indicates that future optimization is needed. Approaches such as co-expression with chaperones, modulation of induction temperature, or directed evolution targeting structural rigidity may further enhance enzyme robustness. Additionally, expanding substrate scope studies will clarify whether this mutation generally improves aromatic hydroxylation or is specific to phenol. Integrating computational modeling with experimental screening could provide deeper mechanistic insights and guide the next generation of CYP450 engineering.

1. [1] Bertelmann, C., & Buhler, B. (2024). Strategies found not to be suitable for stabilizing high steroid hydroxylation activities of CYP450 BM3-based whole-cell biocatalysts. PLoS One, 19(9), e0309965. https://doi.org/10.1371/journal.pone.0309965

2. [2] Cornelius, M. L., & Grace, J. K. (1997). Effect of termite soldiers on the foraging behavior of Coptotermes formosanus (Isoptera: Rhinotermitidae) in the presence of predatory ants. Sociobiology, 29(3), 247-253. ://WOS:A1997WR54200003

3. [3] Diallo, S., Kasparova, K., Sulc, J., Johny, J., Krivanek, J., Nebesarova, J., Sillam-Dusses, D., Kyjakova, P., Vondrasek, J., Machara, A., Luksan, O., Grosse-Wilde, E., & Hanus, R. (2025). Identification of the trail-following pheromone receptor in termites. Elife, 13. https://doi.org/10.7554/eLife.101814

4. [4] Dodhia, V. R., Fantuzzi, A., & Gilardi, G. (2006). Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego. J Biol Inorg Chem, 11(7), 903-916. https://doi.org/10.1007/s00775-006-0144-3

5. [5] Reinhard, J., Lacey, M. J., Ibarra, F., Schroeder, F. C., Kaib, M., & Lenz, M. (2002). Hydroquinone: a general phagostimulating pheromone in termites. J Chem Ecol, 28(1), 1-14. https://doi.org/10.1023/a:1013554100310

6. [6] Stimple, S. D., Smith, M. D., & Tessier, P. M. (2020). Directed evolution methods for overcoming trade-offs between protein activity and stability. Aiche Journal, 66(3). https://doi.org/ARTN e1681410.1002/aic.16814

7. [7] Teufl, M., Zajc, C. U., & Traxlmayr, M. W. (2022). Engineering Strategies to Overcome the Stability-Function Trade-Off in Proteins. ACS Synth Biol, 11(3), 1030-1039. https://doi.org/10.1021/acssynbio.1c00512

8. [8] Zhou, H. Y., Wang, B. J., Wang, F., Yu, X. J., Ma, L. X., Li, A. T., & Reetz, M. T. (2019). Chemo- and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase. Angewandte Chemie-International Edition, 58(3), 764-768. https://doi.org/10.1002/anie.201812093

From a biological perspective, Enterolobium Contortisiliquum Trypsin Inhibitor (EcTI) is a plant-derived trypsin inhibitor. Its mechanism of action involves targeting and inhibiting the activity of trypsin, a key serine protease. EcTI is capable of binding to the active site of trypsin, a property that makes it a potential tool in termite control research. This is because trypsin plays a crucial role in termites’ ability to hydrolyze dietary proteins into absorbable nutrients. Focusing on EcTI, this project aims to verify its inhibitory effect on trypsin related to termites and lay the foundation for its application in termite control. The practical application potential of EcTI lies in its specific mechanism of action targeting digestive enzymes. Unlike broad-spectrum toxic agents, EcTI targets trypsin, a core component of the termite digestive system. This means it may interfere with the physiological functions of termites in a more targeted manner [1]. The purpose of studying EcTI in this project is not only to confirm its in vitro activity of inhibiting trypsin, but also to establish a theoretical basis for its application in termite control.

2. Plasmid Preparation and Validation

To investigate the role of Enterolobium Contortisiliquum Trypsin Inhibitor (EcTI) in inhibiting the trypsin activity in the termite killing process, the EcTI-His tag sequence (BBa_25N3Q748) was cloned into certain plasmid backbones with J23100 promoter (BBa_J23100) and ampicillin resistance gene to harvest EcTI. The plasmid was custom-synthesized by VectorBuilder and extracted using a standard plasmid isolation kit (Medium-Speed Plasmid Extraction Kit (Universal Type), Beyotime D0020). The DNA concentration was quantified via UV spectrophotometry (dsDNA setting) to ensure suitability for downstream experiments.

The concentration of the extracted plasmid is listed as follows:

• J23100-EcTI (BBa_251SK4RC): 207.3 ng/μL

3. Plasmid Construction and Expression Validation

To provide an oxidative environment more conducive to the formation of disulfide bonds of EcTI, we chose to express the constructed plasmid in the Shuffle T7 strain (NEB lab) [2]. For complex proteins that require the formation of disulfide bonds (such as enzymes or secreted proteins containing multiple cysteines), the environment in Shuffle T7 can significantly reduce misfolded folding and increase the yield of active proteins. While J23100 acts as a strong constitutive promoter [3], its continuous and efficient drive of gene expression may lead to excessive protein accumulation and increased folding pressure. Shuffle T7 precisely alleviates this problem.

4. Preparation of bacterial supernatant

• Control group: Shuffle T7 strain without plasmid culturing overnight
• EcTI group: Shuffle T7 carrying the J2310019-EcTI-His tag plasmid culturing overnight

The bacterial culture was collected after overnight incubation and then centrifuged and washed with PBS buffer to remove LB Broth. Subsequently, the bacteria were subjected to ultrasonic lysis.

5. EcTI Function Analysis

After obtaining the EcTI extract from the Shuffle T7 strain, a trypsin activity assay using the Colorimetric Trypsin Activity Assay Kit (Beyotime P0324S) was conducted to analyze the inhibition effect of our product. The kit involves using a substrate labeled with pNA. Trypsin hydrolyzes this substrate to release free pNA, whose concentration can be detected by measuring absorbance at a specific wavelength. The change in pNA concentration reflects trypsin activity, and by comparing pNA concentration changes, the inhibition effect of EcTI on trypsin activity can be assessed.

Both the control group and EcTI group extraction were diluted 10-fold using Assay Buffer (P0324S-1) for further detection. The EcTI group was also diluted 1000-fold and 100,000-fold, as the trypsin activity was unknown.

To quantify the amount of pNA produced in the reaction system, a pNA concentration standard curve for the trypsin activity detection is constructed (Fig. 1). The absorbance at 405 nm (OD405) of each well was measured. Linear regression was performed with the pNA concentration (nmol) as the abscissa and the OD405 value as the ordinate, yielding the equation Y = 0.02249X + 0.01803 (where Y represents the OD405 value and X represents the pNA concentration). In subsequent steps, the OD405 value of the sample was substituted into this equation to calculate the pNA production, and then the trypsin activity was computed according to the activity formula provided in the kit.

Figure 1: pNA concentration (nmol) Standard curve

As shown in Fig. 2a, the pNA concentration change of the control group (Control (D10) 10-fold dilution) rose gradually over time, reaching nearly 6 nmol at 60 minutes and suggests the trypsin activity. In contrast, the pNA concentration change is relatively low with different dilution EcTI treatment (EcTI (D10) 10-fold dilution, EcTI (D1000) 1000-fold, EcTI (D100000) 100000-fold). The data suggested the reactions were suppressed when compared to the control group. Fig. 2b demonstrated that the trypsin activity of the control group (Control (D10) 10-fold dilution) was about 6 nmol/hour, while the trypsin activities of different dilution EcTI treatment groups were significantly lower than that of the control group and a dose-dependent inhibition effect was found, which proved EcTI could effectively inhibit trypsin activity.

a

b

Figure 2: Inhibitory effect of EcTI extract to trypsin activity. (a) Time-course change of pNA concentration (nmol) with EcTI group and control group (b) Enzyme activity of trypsin with different dilution factors of EcTI group and control group

To assess EcTI's inhibitory effect on trypsin activity, one-way ANOVA and Tukey's multiple comparisons were performed. ANOVA showed a significant overall group difference (F = 6.460, P = 0.0049), and the Brown-Forsythe test confirmed homogeneous variances (P = 0.2474). Tukey's test revealed that compared to the Control Plasmid group, remaining EcTI-treated groups (D10, D1000, and D100000) had significantly lower trypsin activity (P < 0.05 for D1000 and D100000; P < 0.01 for D10), indicating potent inhibition. Even at a high dilution (D100000), EcTI was effective.

Mechanistically, the inhibitory effect of EcTI on trypsin conforms to the functional characteristics of plant-derived trypsin inhibitors (PTIs). Trypsin is a key serine protease in the termite digestive system, responsible for hydrolyzing dietary proteins into absorbable peptides and amino acids. By binding to the active site of trypsin, EcTI blocks its catalytic activity and disrupts the protein digestion process in termites. This mechanism suggests that long-term exposure to EcTI may lead to nutrient deficiency, growth retardation, or even death in termites, providing a theoretical basis for its application in termite control.

The current experiments have only verified the trypsin-inhibitory activity of EcTI in vitro (using bacterial extracts). However, the termite digestive system is a complex microenvironment involving multiple influencing factors. The termite midgut is alkaline (pH 8.5-10.0), while the in vitro assay was conducted in a standard buffer. The stability and inhibitory activity of EcTI may change under alkaline conditions; therefore, subsequent experiments need to simulate the pH of the termite midgut to evaluate its actual effect. In addition, termites secrete other proteases (such as chymotrypsin and elastase) and carbohydrate enzymes. EcTI may interact with these enzymes (e.g., being degraded by other proteases), reducing its effective concentration in the gut. Future studies should analyze the stability of EcTI in termite gut homogenates and its cross-inhibitory effect on other proteases.

1. [1] Ferreira, R. D., Napoleo, T. H., Silva-Lucca, R. A., Silva, M. C. C., Paiva, P. M. G., & Oliva, M. L. V. (2019). The effects of serine protease inhibitor on the survival of the termite Nastitermes corniger, and its use as affinity adsorbent to purify termite proteases. Pest Management Science, 75(3), 632-638. https://doi.org/10.1002/ps.5154

2. [2] Bayer, C. N. (2022). Engineering bacterial genomes throughout the central dogma of molecular biology. Technical University of Denmark. https://backend.orbit.dtu.dk/ws/portalfiles/portal/311756128/ PhD_Thesis_Carolyn_Nicole_Bayer.pdf

3. [3] Ting, W. W., Huang, C. Y., Wu, P. Y., Huang, S. F., Lin, H. Y., Li, S. F., Chang, J. S., & Ng, I. S. (2021). Whole-cell biocatalyst for cadaverine production using stable, constitutive and high expression of lysine decarboxylase in recombinant Escherichia coli W3110. Enzyme and Microbial Technology, 148, 109811. https://doi.org/10.1016/j.enzmictec.2021.109811

Peptides, by biological definition, are short chains of amino acids that are fundamental to many physiological processes. Their relatively small size, compared to larger proteins, makes them versatile tools in therapeutic and research applications. This project focuses on two specific lytic peptides—Hecate and Melittin—which possess the ability to disrupt cell membranes. Hecate is a synthesized lytic peptide known for its antimicrobial effects. In contrast, Melittin is a natural lytic peptide and the primary toxic component in European honeybee venom, also renowned for its potent, non-specific ability to rupture cell membranes. Our project will utilize the core lytic function in a general manner to conduct a preliminary toxicity assessment on termites.

The real-world applications of these peptides highlight their distinct mechanisms. Hecate is typically designed for targeted disruption, making it a valuable asset in pre- clinical studies for diseases like cancer [1]. Melittin, however, acts as a powerful, broad-spectrum blowtorch against cells. The purpose of employing these lytic peptides in our project is not to directly kill termites. Instead, we aim to leverage their membrane-disrupting properties to target and impair the symbiotic protozoa that reside within the termite's gut. These protozoa are essential for the termite's ability to digest cellulose, its main food source. By disrupting this critical symbiosis, we can explore a novel approach to termite control.

Plasmid construction

We constructed the plasmids with pET28a backbone, using the inducible anaerobic promoter FNR promoter to control the expression in low oxygen conditions where glucose is present; and added the kanamycin resistance gene for later transformation screening. Our Hecate and Melittin sequences are fused with a secretion signal NSP4 at the C-terminus of the secretion peptide.

Important to state that NSP4 secretion signal was modified:

NSP4 Amino Acid Sequence Modification

Stage	Sequence	Note
Original NSP4	MKKITAAAGLLLLAAQPAMA	Ends with A
Modified NSP4	MKKITAAAGLLLLAAQPAMK	Last A changed to K
Finalized NSP4-K	MKKITAAAGLLLLAAQPAMKK	One more K is added

This modification introduces a trypsin cleavage site, enabling targeted separation of the peptide from the secretion signal if needed. It also supports inducible gene expression and secretion of the peptide. Pure peptide can be obtained by enzymatic removal of the signal sequence. The plasmid was synthesized by VectorBuilder and DNA extracted using the Medium-Speed Plasmid Extraction Kit (Beyotime D0020), with concentration measured by UV spectrophotometry at 260 and 280 nm.

Totally, three plasmids were designed:

(1) pET28a-FNR-mCherry: Serve as the control to verify the expression of genes under anaerobic expression that controlled by FNR promoter.

Figure 1: The illustration of pET28a-FNR-mCherry

(2) pET28a-FNR-Hecate: The plasmid encoded the NSP4-K-Hecate sequence, and the expression is controlled by the FNR promoter.

Figure 2: The illustration of pET28a-FNR-NSP4-K-Hecate

(3) pET28a-FNR-Melittin: This plasmid expresses the NSP4-K-Melittin peptide using the FNR promoter.

Figure 3: The illustration of pET28a-FNR-NSP4-K-Melittin

After extracting the plasmids, we introduced them into competent cell E. coli MG1655 cells, which are compatible with the FNR promoter. The transformed cells were plated on LB agar containing kanamycin and incubated overnight at 37°C. Single colonies were then picked and cultured in liquid LB broth with kanamycin overnight. The bacteria were either harvested for DNA extraction or used to make frozen stocks by adding glycerol to reach a final concentration of 50%. The stocks were stored at -80°C.

After plasmid DNA extraction, the concentration of the plasmids is as follows:

Table 1: The Plasmid Name and Concentrations

Plasmid Name	Concentration
pET28a-FNR-mCherry	36 ng/μL
pET28a-FNR-NSP4-K-Hecate	11.2 ng/μL
pET28a-FNR-NSP4-K-Melittin	23.8 ng/μL

1. Preparation of the mCherry plasmid carrying Escherichia coli MG1655

The plasmid is introduced into MG1655 competent cells by the heat shock method. After transformation, the cells are plated on LB agar containing kanamycin using the streak plate technique and incubated overnight at 37°C. Only cells that have successfully taken up the plasmid will survive and grow on the selective medium.

The use of the mCherry reporter gene offers the advantage of enabling detection in live bacteria. This approach eliminates the need to extract protein or perform additional analyses, such as Western blotting, to confirm protein expression.

2. Preparation of bacterial supernatant:

2.1 Culture the bacteria

• Control: MG1655 strain without plasmid
• Hecate: MG1655 strain carrying the FNR-Hecate
• Melittin: MG1655 strain carrying the FNR-Melittin

The bacteria are cultured in liquid LB broth medium supplemented with glucose at a final concentration of 1 mg/mL. The cultures are grown in tubes placed on a shaker and incubated at 37°C under aerobic conditions until the OD₆₀₀ reaches 0.4–0.6. At this point, the tube lids are sealed tightly to initiate anaerobic conditions and induce gene expression via activation of the FNR promoter.

2.2 Induce expression in anaerobic condition and collect the supernatant

When the OD₆₀₀ of the cultures reaches 0.4–0.6, the tube lids are sealed tightly to induce gene expression by activating the anaerobic FNR promoter. As the oxygen is gradually consumed, samples are collected at various time points: 4 hours, 6 hours, and overnight (O/N). After collection, cultures are centrifuged at 5,000 rpm for 5 minutes. The supernatant is then transferred to clean Eppendorf tubes and stored at - 80°C.

3. Preparation of the pure peptides:

In addition to extracting peptides from the bacterial expression system, we also obtained a small amount (1 mg) of each pure synthetic peptide from a commercial supplier (China Peptide) for comparison with the extracted samples

3.1 Stock solutions

• Control: Pure DMSO
• Hecate: Pure DMSO is added to 1mg of peptide to make up 1mg/mL stock solution.
• Melittin: Pure DMSO is added to 1mg of peptide to make up 1mg/mL stock solution.

The solutions of pure peptides are aliquoted into 100 μL portions and stored at –80°C. Since repeated freeze-thaw cycles can degrade the peptides, aliquoting into separate tubes allows individual portions to be used as needed, minimizing damage to the remaining samples.

4. Toxin assay

We performed testing on E. coli to confirm the functionality of lytic peptides, Hecate and Melittin, which are known to have antimicrobial effects that suppress bacterial growth. The general procedure involved mixing both the bacteria-synthesized peptides and pure peptides with a culture of the E. coli strain DH5α. Bacterial growth was then monitored by measuring the optical density at 600 nm (OD₆₀₀) over a 12- hour period, with an additional data point collected at 24 hours.

Second, we assessed the effect of the lytic peptides on the multicellular organism C. elegans. This worm is a suitable model for preliminary toxicity assessment due to the ease of observing morphological and behavioral changes. We first tested the peptides' effect on motility by exposing the worms to the peptides and counting the number of thrashes within a 30-second interval in M9 buffer. Furthermore, we used the act- 5p::GFP strain to enable direct observation of the gut morphology following peptide exposure.

The detailed protocols for these experiments are provided in the LAB PROTOCOL.

1. The FNR mediated protein expression by fluorescence detection of mCherry under anaerobic conditions

We introduced the pET28a-FNR-mCherry plasmid into E. coli strain MG1655 competent cell and induced gene expression. The result of functional analysis of the FNR promoter demonstrated successful, though non-specific, induction of mCherry expression. While the expected anaerobic induction was observed, fluorescence was also detected in transformed cells under aerobic conditions. The absence of autofluorescence in the plasmid-free MG1655 control confirms that the signal was plasmid-derived.

Figure 4: The gene expression of the mCherry reporter protein after introducing the pET28a-FNRmCherry plasmid into E. coli strain MG1655.

2. Pure Peptides have Antimicrobial Effect on Escherichia coli strain DH5α in the presence of DMSO

Our results identify the most effective antibacterial concentrations for each peptide: 0.025 μg/μL for Hecate and both 0.025 and 0.05 μg/μL for Melittin (Table 2 and 3). However, interpreting this activity required careful consideration of the DMSO solvent. We confirmed that DMSO itself exerts a time-dependent inhibitory effect, which is most pronounced at a 5% concentration.

Therefore, to isolate the specific contribution of the peptides, we directly compared each peptide treatment to its respective DMSO vehicle control. This analysis, supported by two-way ANOVA and Area Under the Curve (AUC) comparisons, clearly demonstrates that the antibacterial effect in the 1.25% and 2.5% DMSO conditions is significantly stronger when the peptide is present. This confirms that the observed growth inhibition is a direct result of peptide activity and not merely an artifact of the solvent.

Table 2: AUC Measurements and Percent Inhibition of Hecate and Melittin with Varying DMSO Concentrations

Treatment	Description	OD600	SD	Inhibition Rate
Blank	No Treatment	0.4456	0.002376	0%
Kana	Positive control	0.2078	0.005093	100%
5% DMSO	DMSO only	0.3663	0.002194	33.3%
2.5% DMSO	DMSO only	0.4220	0.002241	9.9%
1.25% DMSO	DMSO only	0.4427	0.004446	1.2%
5% DMSO + H	DMSO + Hecate	0.3991	0.006559	19.5%
2.5% DMSO + H	DMSO + Hecate	0.2609	0.005410	77.6%
1.25% DMSO + H	DMSO + Hecate	0.3595	0.003503	36.1%
5% DMSO + M	DMSO + Melittin	0.2997	0.006343	61.3%
2.5% DMSO + M	DMSO + Melittin	0.2993	0.002194	61.4%
1.25% DMSO + M	DMSO + Melittin	0.3633	0.003382	34.6%

Footnote: The inhibition percentage is calculated using the following equation, assuming that Kana AUC is the maximum inhibition percentage that can be achieved: % Inhibition = [(Blank AUC - Sample AUC) / (Blank AUC - Kana AUC)] × 100

Table 3: The Respective Peptide Concentrations in each Treatment group

Group	DMSO (%)	Hecate (μg/μL)
1.25% DMSO	1.25	0
2.5% DMSO	2.5	0
5% DMSO	5.0	0
1.25% DMSO + H	1.25	0.0125
2.5% DMSO + H	2.5	0.025
5% DMSO + H	5.0	0.05
1.25% DMSO + H	1.25	0.0125
2.5% DMSO + H	2.5	0.025
5% DMSO + H	5.0	0.05

2.1 The Effect of DMSO

To ensure that the solvent DMSO did not confound our results, we evaluated its impact on bacterial growth. As shown in Figure 5, a high concentration of DMSO (5%) significantly inhibited the growth of DH5α compared to the untreated control (Blank). In contrast, lower concentrations (2.5% and 1.25%) had no observable effect on growth, as the optical density was comparable to the Blank control. As expected, the kanamycin (Kana) control completely suppressed growth.

A two-way ANOVA revealed that both time and Treatment were significant factors influencing the outcome, but their interaction was the most critical (p < 0.0001 for all factors). This significant Time x Treatment interaction indicates that the effect of the DMSO and Kanamycin depended heavily on the time point measured, rather than having a consistent effect over the entire experiment. Time accounted for the largest proportion of variance in the model, suggesting the measured variable changes considerably throughout the duration of the experiment, regardless of Treatment.

Figure 5: The effect of DMSO on the growth of DH5α

Table 4: Two-way ANOVA results for the effects of DMSO and Kana Treatment.

Source of Variation	% of Total Variation	P Value
Time	70.63	< 0.0001
Treatment	23.29	< 0.0001
Time × Treatment	5.56	< 0.0001

Footnote: Data are from a two-way repeated measures ANOVA with Geisser-Greenhouse correction. The Treatment factor (4 degrees of freedom) compares the Blank, Kana, 5% DMSO, 2.5% DMSO, and 1.25% DMSO groups (n=12 per group).

2.2 The Effect of Pure Hecate (dissolved in DMSO)

Hecate exhibited potent, concentration-dependent antibacterial activity against DH5α (Fig. 6a). The antibacterial effect was attributable to the peptide itself, as growth in all Hecate-treated groups was significantly reduced compared to their corresponding vehicle controls (DMSO alone at the same concentration) and the blank control (Figs. 6b-d). The intermediate Hecate concentration (0.025 μg/μL) showed efficacy comparable to the kanamycin control.

The growth inhibition caused by Hecate was highly dependent on both the peptide concentration and the duration of exposure. A two-way ANOVA confirmed that Time was the dominant source of variation, but a highly significant Time x Treatment interaction (p < 0.0001) was the most critical finding (Table 5). This indicates that the differences between the Hecate Treatment groups and their effects on bacterial growth became more pronounced and distinct as the experiment progressed, rather than being constant over time.

Figure 6: Concentration-dependent antibacterial activity of Hecate against E. coli DH5α. (a) Bacterial growth curves treated with varying concentrations of Hecate. (b-d) Comparison of Hecate activity against vehicle controls at specific DMSO concentrations. The final test concentrations for each group are listed in Table 2. The "Blank" group represents untreated cells, and "Kana" (kanamycin) serves as the positive antibiotic control.

Table 5: Two-way ANOVA results for the effects of Hecate Treatment.

Source of Variation	% of Total Variation	P Value
Time	58.66%	< 0.0001
Hecate Treatment	18.87%	< 0.0001
Time × Treatment	21.43%	< 0.0001

Footnote: Data are from a two-way repeated measures ANOVA with Geisser-Greenhouse correction. The Treatment factor (4 degrees of freedom) compares the Blank, 5% DMSO+M, 2.5% DMSO+M, and 1.25% DMSO+M groups (n=12 per group).

2.3 The Effect of Pure Melittin (dissolved in DMSO)

Melittin exhibited strong, dose-dependent antibacterial activity against DH5α, with higher concentrations (0.025 and 0.05 μg/μL) showing a similar and significant inhibitory effect after 4 hours of Treatment ,(Fig. 7a). This effect is directly attributable to melittin, as growth in all melittin-treated groups was substantially reduced compared to their corresponding vehicle controls containing DMSO alone (Figs. 7b-d). The effect of the Melittin Treatment was highly dynamic over time. A two-way ANOVA revealed a highly significant Time x Treatment interaction (p < 0.0001), which accounted for a substantial portion of the total variance ,(Table 6). This indicates that the differences in antibacterial efficacy between the groups were not constant but changed significantly throughout the experiment.

Figure 7: Concentration-dependent antibacterial activity of Melittin against E. coli DH5α. (a) Bacterial growth curves treated with varying concentrations of Melittin. (b-d) Comparison of Melittin activity against vehicle controls at specific DMSO concentrations. The final test concentrations for each group are listed in Table 2. The "Blank" group represents untreated cells, and "Kana" (kanamycin) serves as the positive antibiotic control.

Table 6: Two-way ANOVA results for the effects of Melittin Treatment.

Source of Variation	% of Total Variation	P Value
Time	58.66	< 0.0001
Treatment	18.87	< 0.0001
Time × Treatment	21.43	< 0.0001

Footnote: Data are from a two-way repeated measures ANOVA with Geisser-Greenhouse correction. The Treatment factor (4 degrees of freedom) compares the Blank, 5% DMSO+M, 2.5% DMSO+M, and 1.25% DMSO+M groups (n=12 per group).

3. Bacterial Supernatant Shows No Acute Toxicity to C. elegans After Two-Hour Exposure

To assess the effects of toxins on C. elegans, we exposed the worms to bacterial supernatant for 2 hours. Hecate and Melittin peptides, which primarily disrupt cell membranes, are ingested into the gut; therefore, we hypothesized they could potentially harm the worm’s gut. However, under brightfield microscopy, no clear phenotypic changes were observed. Observations using Green-Fluorescent Protein (GFP) fluorescence revealed the gut remained linear and smooth, without twists, indicating the worms were healthy after 2 hours of toxin exposure. This suggests these peptides may exhibit selectivity for certain cell membranes, as eukaryotic membranes differ from protozoan membranes.

Figure 8 The stage L4 Caenorhabitis elegans strain act-5p::GFP, which contains GFP gene in the gut cells, observed under the Nikon SMZ18 stereomicroscope at 90× magnification, to take the brightfield images and combined with Nikon Intensilight C-HGFI to observe the GFP signal and photograph. Note: The images taken by the software only presents in white and black.

4. Hecate Bacteria Supernatant Shows Impact on Caenorhabditis elegans Thrashing Ability

While bacterially-synthesized Hecate and Melittin were not lethal to C. elegans, Hecate specifically induced a significant paralytic effect. Treatment with Hecate resulted in a statistically significant reduction in thrashing rate compared to the control (p=0.0124), whereas Melittin had no significant effect on motility (p > 0.9999). This suggests that Hecate's designed affinity for eukaryotic membranes may allow it to disrupt neuromuscular function in the nematode more effectively than the general cytolysin Melittin.

Figure 9: Scatter bar graph comparing the effect of feeding bacteria with Hecate or Melittin expression plasmids on C. elegans.

Table 7: The statistics of Bonferroni's post-hoc analysis for reference.

Comparison	Mean Difference	95% CI	P-value
Control vs. Hecate	16.05	3.26 to 28.84	0.012
Control vs. Melittin	2.21	-11.88 to 16.30	>0.99

Footnote: ns = not significant; * = statistically significant (p < 0.05 after Bonferroni adjustment for 2 comparisons).

1. The Non-Specific Gene Regulation of the FNR promoter

The FNR promoter, traditionally characterized as an anaerobic promoter in prior studies and documented in the iGEM registry, demonstrated unexpected gene expression activation under both aerobic and anaerobic conditions in our experiment. This observation suggests that the FNR promoter may not be exclusively anaerobic. A related finding was reported by the iGEM team NCKU Tainan 2019, which characterized the same FNR promoter part [2]. They noted significantly higher fluorescence signals in aerobic conditions compared to anaerobic ones, though their analysis focused on GFP expression, while our study utilized the mCherry protein.

Further investigation revealed a potential issue with the mCherry reporter gene, which may obscure promoter regulation. The codon-optimized mCherry exhibits constitutive background fluorescence due to aberrant translation initiation. This phenomenon arises from an internal alternative translation initiation site (ATIS) at the 10th methionine residue (M10). An upstream Shine-Dalgarno-like sequence enhances ribosome recruitment to this site, resulting in the production of a truncated protein isoform that retains functional chromophore properties and fluoresces red [3]. Consequently, significant background fluorescence arises even under repressive conditions, complicating the interpretation of promoter activity, particularly for the oxygen-sensitive FNR promoter in aerobic environments.

Looking for future research, the immediate priority is to refine the genetic system by validating the FNR promoter with an alternative reporter like GFP and exploring other tightly regulated inducible promoters to ensure precise control.

2. The Better Antimicrobial Efficacy of Lytic Peptide Hecate

A comparative analysis of the 2.5% DMSO and 1.25% DMSO conditions, as presented in Table 2, reveals that the inhibition percentage in the Hecate Treatment group consistently surpasses that observed in the Melittin group. These findings indicate that Hecate demonstrates an enhanced antimicrobial efficacy relative to Melittin, especially in the 2.5%DMSO condition.

It is well-established that DMSO functions as a commonly used vehicle solvent and possesses inherent cytotoxic properties [4].

Consequently, rigorous experimental controls were implemented to ensure that the measured inhibitory effects primarily originate from the peptides, rather than from DMSO itself. Parallel vehicle control groups were included to facilitate accurate assessment. The data unequivocally demonstrate that the observed inhibition cannot be attributed solely to DMSO. Although DMSO displays some degree of inhibitory activity, which appears to be potentiated in the presence of lytic peptides, this trend is clearly evidenced in Table 2. Notably, at a concentration of 1.25% DMSO, the solvent alone exerts minimal inhibition on E. coli. However, pronounced inhibitory effects are observed upon administration of Hecate and Melittin, highlighting the significant contribution of the peptides to the overall antimicrobial activity.

Experimental data show that Melittin remains stable at both 5% and 2.5% DMSO, while Hecate’s stability decreases dramatically under higher DMSO concentrations. At 5% in water, DMSO acts as a mild denaturant by competing for and breaking the hydrogen bonds that stabilize peptide secondary structures [5], particularly alpha-helices, through strong hydrogen bonding with the peptide backbone. Melittin resists DMSO denaturation due to its relatively stable, cooperative hydrogen-bond network, a strong hydrophobic core that makes it harder for DMSO to disrupt, and clustering of positive charges away from the helix core [6], reducing solvent accessibility to charged residues. In contrast, Hecate’s weaker hydrogen bonding, high charge density (lysine-rich sequence), and insufficient hydrophobic core can make it more prone to DMSO attack. These features collectively destabilize the peptide and allow easier access for DMSO to disrupt its structure.

In vivo testing on E. coli, Heacte demonstrated superior antimicrobial activity over Melittin when delivered in a 1.25% and 2.5% DMSO solvent, highlighting how a peptide's inherent lytic potential is critically modulated by its stability in the delivery formulation.

3. Hecate and Melittin Show No Acute Toxicity in the Non-Target Organism Caenorhabditis elegans

In our experiment, Caenorhabditis elegans were exposed to both purified lytic peptides and peptides synthesized by bacteria. The nematodes were fully immersed in a liquid medium containing the respective peptides; however, no mortality was observed. We hypothesize that this outcome is primarily due to the protective cuticle of C. elegans, a well-documented, rigid barrier that protects against diverse environmental toxins and pathogens []. This robust exoskeleton likely prevents the penetration of lytic peptides like Hecate and Melittin, which are known to exert their effects through direct interaction with cell membranes [8, 9]. This feature could account for the observed resistance to Hecate and Melittin.

Alternatively, lytic peptides may only exert their action upon ingestion, which occurs through the rhythmic pharyngeal pumping of the medium into the digestive tract. Nevertheless, microscopic examination of gut tissue revealed no visible cellular damage; the intestinal linings appeared smooth and undistorted. These findings support the hypothesis that Hecate and Melittin may selectively target unicellular organisms, consistent with our objective to avoid toxic effects on non-target organisms.

4. The Greater Impact of Bacterially Synthesized Hecate on C. elegans

The observation that bacterial supernatant containing Hecate markedly impairs C. elegans thrashing motility more effectively than Melittin might indicate the difference in the interactions of these antimicrobial peptides with a complex multicellular organism.

Although Hecate was derived from melittin, key amino acid substitutions have rendered it a more potent disruptor of C. elegans motility in our assay. While both peptides are membranolytic, we hypothesize that Hecate's uniformly cationic and highly amphipathic alpha-helical structure facilitates a more potent interaction with the anionic phospholipids prevalent in neuronal and muscle cell membranes. This targeted disruption of the neuromuscular system would manifest as a rapid and severe loss of motility. Alternatively, the difference may be kinetic; Hecate may induce a pronounced paralytic state at sub-lethal concentrations more effectively than melittin, which may cause more immediate generalized lysis. This aligns with Hecate's designed mechanism of carpet-like membrane disruption, which could be more effective on the complex multicellular systems of a nematode compared to melittin's toroidal pore formation.

In future research, to fully leverage the intriguing bioactivity of Hecate, its mechanism should be investigated in C. elegans using fluorescently tagged tissues to confirm the site of action and distinguish between paralysis and lethality.

5. Key Conclusions

The FNR promoter showed activity in both aerobic and anaerobic conditions, likely due to mCherry reporter background fluorescence, necessitating alternative promoters. Hecate exhibited superior antibacterial activity against E. coli, especially at lower DMSO concentrations, but is less stable at higher DMSO. Neither peptide showed acute toxicity to C. elegans, likely due to the nematode cuticle, indicating selective toxicity. Hecate uniquely induced non-lethal paralysis in C. elegans, suggesting specific neuromuscular disruption not seen with Melittin.

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