HQ

Overview

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Plasmid Preparation and Validation

To investigate the role of CYP450-mediated hydroxyquinone (HQ) synthesis in termite attraction, two CYP450 variants (wild type and double mutation) were cloned into distinct plasmid backbones, alongside control plasmids for expression validation.

All plasmids were custom-synthesized by VectorBuilder (a commercial gene synthesis and plasmid construction service), extracted using a standard plasmid isolation kit (Plasmid Midi Preparation Kit for All Purpose, Beyotime D0020), and quantified via UV spectrophotometry (dsDNA setting) to ensure suitability for downstream experiments.

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

Plasmid Construction and Expression Validation

Experimental Controls for Fluorescence Background Validation

To ensure the specificity of fluorescence signals and confirm cell viability, all samples were imaged using two microscopy techniques: fluorescence microscopy 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 cell morphology and confirm cellular presence. To rule out background interference, two fundamental control groups (PBS buffer control and E. coli BL21 control) were established. Individual images of these controls are shown in Figure 1A–C, with each panel containing both fluorescence and TL-DIC channels. 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 plasmids using uniform settings (excitation: 540–580 nm, dichroic mirror: 585 nm, emission: 592–668 nm). All samples, including E. coli BL21 harboring T7-CYP450-mCherry following IPTG induction, E. coli BL21 harboring T7-CYP450 DM-mCherry following IPTG induction, E. coli BL21 harboring T7-CYP450-mCherry without IPTG induction, and E. coli BL21 harboring T7-CYP450 DM-mCherry without IPTG induction, were imaged in both transmitted light-differential interference contrast (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; Western blotting was not conducted due to technical challenges in transferring the large ~143 kDa fusion proteins. IPTG induction entailed growing cultures to mid-logarithmic phase (OD₆₀₀ ≈ 0.6), adding isopropyl β-D-1-thiogalactopyranoside 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 throughout the cytoplasm, verifying functional 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 without IPTG induction, the T7-CYP450-mCherry sample (Fig. 1d) showed healthy cells in the TL-DIC channel, affirming viability in the absence of IPTG, but no detectable red fluorescence in the fluorescence channel, thereby validating the IPTG dependency of the T7 promoter. Likewise, the T7-CYP450 DM-mCherry negative control (Fig. 1e) exhibited viable cells in the TL-DIC channel, indicating normal physiology without IPTG, with negligible red fluorescence in the fluorescence channel, further confirming the IPTG-dependent nature of T7-driven expression for the double-mutant fusion protein.

IPTG-Induced Expression of T7-CYP450-mCherry Fusion Proteins in E. coli BL21:

Caption: Fluorescence validation of CYP450-mCherry fusion proteins, demonstrating IPTG-dependent expression and enhanced signal in the double mutation variant.

Western Blot Analysis

Western blot was used to validate expression of flag-tagged constructs (lacking mCherry) and to confirm mCherry expression in FNR-mCherry. Blots were probed with anti-flag or anti-mCherry antibodies and visualized via chemiluminescence.

T7-CYP450-flag and T7-CYP450 DM-flag (transformed into BL21)

Western blot analysis detected a clear band at ~115 kDa in the IPTG-induced T7-CYP450-flag sample (Fig. 2a) using anti-flag antibodies, indicating stable expression of the wild-type CYP450. In the IPTG-induced T7-CYP450 DM-flag sample (Fig. 2b), a more intense band at ~115 kDa was observed relative to the wild-type construct, suggesting elevated expression levels of the mutated variant, consistent with enhanced stability in engineered CYP450 mutants.

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:

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 (Fig.3)

To quantify hydroxyquinone (HQ) production, the maximum absorption wavelength of HQ was first determined to be 290 nm via spectral scanning. A standard curve was generated by plotting HQ concentration (mg/mL) against absorbance at 290 nm. Linear regression analysis yielded a calibration equation of Y=4.200X+1.286 (where Y is absorbance and X is HQ concentration), with a high coefficient of determination (R2 = 0.9936), indicating a strong linear correlation. Statistical analysis confirmed the slope was significantly non-zero (F = 312.8, \(P = 0.0032\)), and the 95% confidence interval for the slope (3.178–5.222) did not include zero. Collectively, this standard curve was validated as a reliable tool for quantifying HQ in subsequent bacterial culture samples.

Time-course of HQ Production (Fig.4)

For functional assays, phenol (substrate for CYP450-mediated hydroxylation) was added to bacterial cultures harboring T7-CYP450-flag (wild-type) or T7-CYP450 DM-flag (double mutation variant). Cultures were incubated with shaking for 4 hours, and 150 μL samples were collected every 30 minutes. After centrifugation, pellets were resuspended in potassium phosphate buffer, and absorbance at 290 nm was measured to assess hydroxyquinone (HQ) accumulation—this wavelength was selected because HQ exhibits a characteristic maximum absorbance (λₘₐₓ) at ~290 nm due to its molecular chromophores (conjugated double bonds and hydroxyl groups), enabling specific and sensitive quantification. Nonlinear regression analysis was performed to characterize the kinetic profiles of HQ production :

•  T7-CYP450 DM-flag: HQ concentration remained low for the first ~2.5 hours but rose sharply around 3 hours, reaching a plateau near 0.25 mg/mL by 4 hours. This kinetic profile was well described by nonlinear regression, with a high coefficient of determination (R2 = 0.9959) indicating excellent model fit. The estimated half-maximal effective concentration (IC₅₀, reflective of substrate utilization kinetics) was 681.6 (95% confidence interval [CI]: 627.7–748.8), and the Hill slope was 3.619 (95% CI: 3.079–4.675), suggesting cooperative behavior in catalysis.

• T7-CYP450-flag: HQ production increased gradually but remained at a low level (< 0.05 mg/mL even at 4 hours). The nonlinear fit for this variant had a lower (R2 = 0.8575), and the estimated IC₅₀ was 527.0 (with incomplete 95% CI due to limited signal magnitude), consistent with substantially weaker catalytic activity compared to the double mutation variant.

4-hour Endpoint Analysis of HQ Concentration (Fig.5)

At the 4-hour time point, a comparative analysis of HQ concentration was performed across three groups (Fig.7):

• Control group (BL21 without plasmid): HQ concentration averaged ~0.029 mg/mL, confirming no intrinsic phenol-hydroxylating activity in the host strain.

• T7-CYP450-flag (wild-type variant): HQ concentration averaged ~0.043 mg/mL, which was slightly higher than the control group, indicating modest wild-type CYP450 activity.

• T7-CYP450 DM-flag (double mutation variant): HQ concentration reached ~0.269 mg/mL—nearly 6.2-fold higher than the wild-type group and >9.2-fold higher than the control—demonstrating drastically enhanced catalytic efficiency of the double mutation variant.

Collectively, these results confirm that the double mutation in CYP450 significantly boosts its ability to convert phenol to HQ, validating the engineered variant’s superior functionality.

References

1. 参考文献一
2. 参考文献二
3. 参考文献三
4. 参考文献四
5. 参考文献五