This study focuses on the early identification and prevention of postharvest decay in strawberries, constructing a comprehensive synthetic biology system comprising three components: detection, reporting, and response. First, by screening VOC-responsive promoters, we established a biosensing module capable of recognizing volatile organic compounds (VOCs) released during the decay process, enabling sensitive capture of spoilage signals. Second, we integrated the highly responsive promoter PgrpE with the violacein chromogenic pathway (vioABCDE) to develop a detection system that converts invisible spoilage gas signals into visible color changes. Finally, we constructed a synergistic antimicrobial module combining Chitinase and β-1,3-Glucanase to achieve active defense against fungal infection. Through step-by-step validation of these three experimental modules, we realized a closed-loop design spanning from signal perception and colorimetric reporting to active prevention, providing a biological foundation and technical support for the intelligent monitoring and preservation of strawberries and other perishable fruits.
Figure 1. Integrated Synthetic Biology System for Strawberry Spoilage Detection and Inhibition
This experiment aims to screen for VOC-responsive promoters that can be used to detect early spoilage signals in postharvest perishable fruits such as strawberries.
Strawberries are susceptible to fungal infections (e.g., Botrytis cinerea) during harvesting, transportation, and storage. During growth and metabolism, fungi release characteristic volatile organic compounds (VOCs), such as 1-octanol, 1-octen-3-ol, and phenylethyl alcohol. These VOCs can serve as early molecular indicators of fruit spoilage. By evaluating the activation of four VOC-responsive promoters (PsoxS, PlasI, PrecA, PgrpE) in response to these three VOCs, using red fluorescent protein (RFP) as a reporter gene for visual and quantitative analysis, we aim to identify the promoter with the strongest response to spoilage-related VOCs and the lowest background noise. This will provide a foundation for constructing a VOC detection and fruit spoilage early-warning system.
Four VOC-responsive promoters (PsoxS, PlasI, PrecA, PgrpE) were synthesized and cloned into the standard iGEM plasmid pSB1A3 vector using seamless cloning technology. These promoters were placed upstream of the reporter gene mRFP to construct a "promoter-RFP" expression detection system. Escherichia coli DH5α (Beyotime, D0351) was used for plasmid amplification, and positive clones were selected using ampicillin (100 μg/mL, Amp) resistance screening. Single colonies were picked for plasmid extraction and sent for sequencing verification (Tsingke, Beijing). After confirming the correct construction, the plasmids were transformed into the expression strain BL21(DE3) (Beyotime, D1009S) for subsequent VOC response experiments. The final engineered strains were stored at -80°C with 25% (v/v) glycerol as a cryoprotectant.
Figure 2. Plasmid Construction and Transformation Verification
Verified engineered strains were inoculated into LB liquid medium (10 mL, in 50 mL conical flasks) containing ampicillin and cultured at 37°C with shaking at 250 rpm. The OD₆₀₀ was monitored in real-time to ensure the cells were in the logarithmic growth phase. When the OD₆₀₀ reached 0.6, the bacterial culture was aliquoted into a 96-well plate, with 90 μL per well as the detection system. VOC stock solutions (1-octanol, 1-octen-3-ol, phenylethyl alcohol) were prepared as 0.1% (v/v) aqueous solutions and then diluted 10³-fold to the experimental concentration. In the experiment, 10 μL of different VOC solutions were added to each well, ensuring the final system simulated the actual VOC concentrations during strawberry spoilage. A control group without VOCs was included, and the total experimental system volume was 100 μL. The 96-well plate was incubated statically at 37°C for 4 hours to allow full diffusion of VOCs and interaction with the engineered bacteria, activating the promoters and inducing RFP expression.
After incubation, two indicators were measured using a microplate reader:
Figure 3. Screening Process for VOC-Responsive Promoters
We first performed PCR validation on the reporter gene mRFP and the four recombinant plasmids containing different promoters. Target fragments were amplified using specific primers, and the PCR products were analyzed by 1.2% agarose gel electrophoresis, revealing clear single bands (Figure 1). The results showed that the mRFP fragment size matched the expected size (approximately 678 bp), while the amplified fragments of the four "promoter + mRFP" constructs also corresponded to their theoretical sizes: PrecA-mRFP (~802 bp), PgrpE-mRFP (~776 bp), PsoxS-mRFP (~740 bp), and PlasI-mRFP (~835 bp). All bands were at the correct positions with no significant non-specific amplification. To further confirm the accuracy of the constructs, positive clones were subjected to Sanger sequencing, which confirmed that the sequences were fully consistent with the design, with no frameshift or premature termination mutations detected. In conclusion, all four target plasmids were successfully constructed and are suitable for subsequent functional validation experiments.
Figure 4. Verification of PCR results by agarose gel electrophoresis
Figure 5. Construction and Validation of PlasI+mRFP
Experimental results showed that the PlasI reporter strain exhibited differentiated response patterns after 4-hour exposure to different VOCs (Figure 4). The control group (CK) demonstrated the lowest normalized fluorescence value, representing the baseline expression level of the PlasI reporter strain in the non-responsive state.
Under the treatment of 1-octanol and phenylethyl alcohol, the normalized fluorescence value of PlasI significantly increased, approximately 2-fold higher than CK, indicating that PlasI could be markedly induced by 1-octanol and phenylethyl alcohol. The fluorescence value of the 1-octen-3-ol treatment group was about 1.5 times higher than CK, showing a moderate response. Comprehensive comparison revealed that the sensitivity of PlasI to the three VOCs followed the order:
Figure 6. Response of the PlasI reporter strain to different VOCs (4h)
Figure 7. Construction and Validation of PrecA+mRFP
Experimental results demonstrated that the PrecA reporter strain exhibited differentiated response patterns after 4-hour exposure to different VOCs (Figure 2). The control group (CK) showed the lowest normalized fluorescence value, representing the baseline expression level of the PrecA reporter strain in the non-responsive state.
Among the three VOC treatment groups, the PrecA reporter strain displayed comparable response intensities under 1-octanol and phenylethyl alcohol treatments, both approximately 1.5-fold higher than CK, indicating that the PrecA response system could be significantly induced by these two VOCs. The response intensity of the 1-octen-3-ol treatment group was slightly lower than the other two groups, showing a moderate response. Comprehensive comparison revealed that the sensitivity of PrecA to the three VOCs followed the order:
Figure 8. Response of the PrecA reporter strain to different VOCs (4h)
Figure 9. Construction and Validation of PgrpE+mRFP
Experimental results showed that the PgrpE reporter strain exhibited differentiated response patterns after 4-hour exposure to different VOCs (Figure 2). The control group (CK) demonstrated the lowest normalized fluorescence value, representing the baseline expression level of the PgrpE reporter strain in the non-responsive state.
Under the treatment of 1-octanol and phenylethyl alcohol, the normalized fluorescence value of the PgrpE promoter significantly increased, approximately 2.3-fold higher than CK, indicating that PgrpE could be markedly induced by 1-octanol and phenylethyl alcohol. The fluorescence value of the 1-octen-3-ol treatment group was slightly lower than the other treatment groups, but still exhibited relatively strong response intensity. Comprehensive comparison revealed that the sensitivity of PgrpE to the three VOCs followed the order:
Figure 10. Response of the PgrpE reporter strain to different VOCs (4h)
Figure 11. Construction and Validation of PsoxS+mRFP
Experimental results demonstrated that the PsoxS reporter strain exhibited differentiated response patterns after 4-hour exposure to different VOCs (Figure 2). The control group (CK) showed the lowest normalized fluorescence value, representing the baseline expression level of the PsoxS reporter strain in the non-responsive state.
Under 1-octanol treatment, the normalized fluorescence value of the PsoxS reporter significantly increased, approximately 3-fold higher than CK, indicating that PsoxS could be strongly induced by 1-octanol. The fluorescence values of the 1-octen-3-ol and phenylethyl alcohol treatment groups were also higher than CK, with comparable levels between them, showing an increase of approximately 1.2-fold compared to CK. This suggests that PsoxS could also respond to these two VOCs, though the response intensity was significantly lower than that to 1-octanol. Comprehensive comparison revealed that the sensitivity of PsoxS to the three VOCs followed the order:
Figure 12. Response of the PsoxS reporter strain to different VOCs (4h)
To compare the response intensities of VOC-responsive systems constructed with four different promoters and screen for the promoter most sensitive to VOC signals released during spoilage processes. Through this experiment, we aim to identify the optimal responsive element, providing a foundation for subsequent construction of chromogenic reporter systems, thereby enabling efficient detection and visualization of strawberry spoilage signals.
Correctly constructed VOC-responsive strains with four different promoters were inoculated into LB liquid medium containing ampicillin (10 mL, in 50 mL conical flasks) and cultured in a shaker at 37°C and 250 rpm. The OD₆₀₀ value was monitored in real-time to ensure cells were in the logarithmic growth phase. When OD₆₀₀ reached 0.6, the bacterial suspension was aliquoted into a 96-well plate, with 90 μL per well as the detection system. VOC stock solutions (1-octanol, 1-octen-3-ol, phenylethyl alcohol) were first prepared as 0.1% (v/v) aqueous solutions, then diluted 10³-fold to the required experimental concentrations. During the experiment, 10 μL of different VOC solutions were added to each well, enabling the final system to simulate the actual VOC concentrations during strawberry spoilage. A control group without VOCs was established, with a total system volume of 100 μL. The 96-well plate was statically incubated at 37°C for 4 hours to allow sufficient VOC diffusion and interaction with the engineered bacteria, activating the promoters and inducing RFP expression.
After incubation, the 96-well plate was placed in a microplate reader to detect two parameters:
The RFP reporter systems driven by four different promoters were incubated for 4 hours under three VOC conditions simulating strawberry spoilage (1-octanol, 1-octen-3-ol, phenylethyl alcohol), followed by RFP fluorescence measurement with OD₆₀₀ correction. Results were expressed as normalized fluorescence values (Fluorescence/OD) and plotted in Figure 2-6.
To compare the response intensities of four promoters (PsoxS, PlasI, PrecA, PgrpE) to three VOCs (1-octanol, 1-octen-3-ol, phenylethyl alcohol), we calculated response fold changes at the 4-hour incubation time point. This is a commonly used quantitative indicator for evaluating the relative degree of promoter activation by VOCs. Response fold change can reflect promoter sensitivity and specificity - higher fold changes indicate stronger promoter response to specific VOCs.
For each promoter and each VOC:
Response Fold Change = (Normalized fluorescence value of VOC treatment group) / (Normalized fluorescence value of no-VOC control group)
The response intensities of the four promoters to VOCs were ranked and visually represented using corresponding numbers of "+" symbols. The top-ranked promoter is indicated by "++++", while the lowest-ranked promoter is indicated by "+", with others following this pattern.
As shown in the results, fluorescence fold change measurements after 4 hours revealed that the grpE promoter performed most prominently when detecting the three target VOCs (1-octanol, 1-octen-3-ol, and phenylethyl alcohol). When exposed to 1-octanol, the "fluorescence value/OD₆₀₀" of BL21-grpE-mRFP strain was significantly higher than the control group (CK) and other promoter strains, with the highest response fold change, indicating that grpE had the strongest activation effect for this VOC. Under 1-octen-3-ol treatment, grpE similarly showed the strongest response, with both fluorescence output values and response fold changes significantly leading other promoters, demonstrating grpE's high sensitivity to this VOC. For phenylethyl alcohol, although there were some differences in responses among promoters, grpE still maintained high fluorescence levels, exhibiting good response stability. In contrast, the soxS promoter showed weaker responses. After most VOC treatments, its "fluorescence value/OD₆₀₀" showed limited improvement, indicating low correlation between soxS and these VOCs, making effective response difficult. lasI and recA promoters showed some response under certain VOC conditions, but their overall levels were significantly lower than grpE, indicating limited sensing capabilities. In summary, the grpE promoter demonstrated the highest sensitivity and stability in responses to all three target VOCs, making it the most suitable candidate promoter as the core element for subsequent biosensor construction.
Figure 12 Characterization of Response Properties of Four Promoters to VOCs at 4-hour Time Point
(A) Comparison of induction fold changes for each group relative to no-VOC control group (4h). Error bars represent ± standard deviation (n=3 biological replicates). Statistical significance was determined by one-way ANOVA and Tukey's post-hoc test；(B) Evaluation of promoter responses to VOCs. The number of "+" symbols is proportional to induction fold change
From the experimental results, it is evident that the four VOC-responsive promoters exhibited significant differences in their responses to different target volatile organic compounds. Among them, the
In contrast, the
Comprehensive analysis indicates that the
Based on previous screening that identified the PgrpE promoter as having the strongest response to individual VOCs, this experiment aimed to further evaluate the response characteristics of the PgrpE reporter strain to a VOC mixture simulating the strawberry spoilage environment (phenylethyl alcohol:1-octanol:1-octen-3-ol = 6:1:1). By measuring response fold changes, we validated the sensitivity and applicability of the PgrpE promoter in complex VOC environments, providing experimental evidence for constructing an early strawberry spoilage detection system.
The experiment used Escherichia coli DH5α carrying the PgrpE promoter-RFP reporter system (construction method as previously described). The strain was inoculated into 10 mL of LB liquid medium containing 100 μg/mL ampicillin (in 50 mL conical flasks) and cultured at 37°C with 250 rpm shaking. OD₆₀₀ was monitored, and subsequent experiments were conducted when the bacterial culture reached mid-logarithmic growth phase (OD₆₀₀ ≈ 0.6).
According to the typical composition ratio of VOCs during strawberry spoilage, a mixed VOC solution was prepared with phenylethyl alcohol:1-octanol:1-octen-3-ol = 6:1:1. VOC stock solutions (0.1% v/v aqueous solution) were mixed proportionally and diluted 10³-fold to simulate early spoilage VOC concentrations. For induction experiments, 90 μL of bacterial culture was aliquoted into each well of a 96-well black microplate (Corning, USA), and 10 μL of mixed VOC solution was added, making the total system volume 100 μL. Control wells received 10 μL of sterile water.
After mixing, samples were taken at time points 0 h (before treatment), 2 h, 4 h, 6 h, and 12 h to detect fluorescence intensity and OD600, calculating normalized fluorescence values. A multifunctional microplate reader (FlexStation 3, Molecular Devices, USA) was used to measure RFP fluorescence intensity (excitation wavelength 584 nm, emission wavelength 607 nm) and OD₆₀₀ to correct for cell density. Each group had three biological replicates, with two technical replicates each.
Calculation of normalized fluorescence value:
Using the NormFluo of the CK group at corresponding time points as baseline, calculate induction fold change (FC):
Each time point included three independent biological replicates, with results expressed as mean ± standard deviation (SD). One-way ANOVA was used to compare differences in FC across time points, followed by Tukey's post-hoc test, with significance threshold set at p<0.05.
The results showed that under mixed VOC treatment, the induction fold change of the PgrpE-mRFP reporter strain exhibited a continuous upward trend over time (Figure X). At 2 hours post-treatment, FC was approximately 1.3-fold, indicating the system could detect VOC signals within a short time. By 4 hours, FC increased to 2.6-fold; reaching 3.2-fold at 6 hours, and maintaining around 3.5-fold at high levels after 10 hours. This demonstrates that PgrpE has rapid response and high signal amplification effects to mixed VOC signals, further supporting its selection as the superior promoter.
Therefore, through testing with individual VOCs such as 1-octanol and their mixtures, the fluorescence signal mediated by grpE was significantly stronger than lasI, recA, and soxS, and could generate sustained responses to mixtures, confirming it as a superior VOC-responsive promoter.
Figure 13 Response fold changes of the PgrpE reporter strain to mixed VOCs at different time points. According to VOC composition distribution, the mixed VOCs had phenylethyl alcohol:1-octanol:1-octen-3-ol = 6:1:1.
The experimental results show that the induction fold change of the PgrpE-mRFP reporter system rapidly increased within a short time under VOC mixture treatment, peaking around 12 hours. This indicates that PgrpE can not only respond to individual VOCs but also maintain high comprehensive sensitivity in mixed gas environments.
The results demonstrate that the PgrpE reporter strain can generate detectable response signals within 1-2 hours and maintain detection effectiveness for a considerable duration, making it highly suitable for use as an early spoilage monitoring sensor. In practical fruit transportation or storage scenarios, the detection cycle could be set at 2-4 hours to achieve real-time early warning.
Before detecting violacein production levels, it is first necessary to determine its characteristic absorption wavelength and establish a standard curve for quantitative detection to ensure the accuracy and comparability of subsequent experimental data.
Chemically synthesized violacein standard was used to prepare a series of gradient concentration solutions (0–20 μg/mL) in 50% anhydrous ethanol. A spectrophotometer was used to scan the wavelength range of 300–700 nm to determine the maximum absorption peak wavelength of violacein in 50% anhydrous ethanol solution. The absorbance of each gradient concentration solution was measured at the maximum absorption wavelength, and a concentration-absorbance standard curve was plotted and linearly fitted.
Figure 10 Establishment of the Absorption Spectrum and Standard Curve for Violacein
The results indicate that violacein has a maximum absorption peak at 575 nm, and there is a good linear relationship between its absorbance and concentration. The linear regression equation is: Y = 1.733X + 0.08867 (R² = 0.99), where A represents the absorbance value and C represents the concentration (μg/mL).
Figure 11 Scanning Spectrum and Standard Curve of Violacein in 50% Anhydrous Ethanol Solution
The established standard curve can be used for quantitative determination of violacein concentration in subsequent experiments. The high correlation coefficient (R² = 0.99) of the linear fit indicates that this method has good accuracy and reproducibility, meeting the requirements for quantitative analysis.
This experiment aims to evaluate the violacein synthesis capability and dynamic changes in engineered bacteria during fermentation when using violacein as a reporter gene. Due to its distinct advantages in visible color output and quantitative detection, violacein holds potential as a signal output in VOC (volatile organic compound) response systems. Therefore, by analyzing the relationship between violacein production and bacterial growth, the stability and visualization effect of this reporter gene expression in engineered bacteria can be assessed, providing experimental evidence for the design and optimization of subsequent VOC sensing systems.
The violacein biosynthesis gene cluster (BCG) comprising vioAB and vioCDE was synthesized. A constitutively expressed promoter PJ23100 and a medium-strength RBS B0034 were added upstream to drive gene expression. Codon optimization was performed for E. coli, and EcoRI, XbaI, SpeI, and PstI restriction sites were eliminated to comply with the RFC#10 standard. The cluster was then cloned into the pSB1A3 vector via EcoRI and XbaI, yielding the recombinant plasmid pSB1A3-vioABCDE. The recombinant plasmid was transformed into E. coli BL21 as previously described.
A spectrophotometer was used to measure the absorbance of violacein at 575 nm, and its concentration (mg/L) was calculated using the standard curve. All experiments were performed in triplicate, and the average values were calculated.
The dry cell weight (DCW) increased slowly within the first 8 hours (from 0.05 g/L to 0.12 g/L), indicating an adaptation phase and early exponential growth phase. After 12 hours, the culture entered a rapid growth phase, reaching 0.85 g/L at 24 hours and peaking at 1.1–1.2 g/L during 36–48 hours. The biomass stabilized after 60 hours.
Violacein production was low in the initial phase (1–8 h), ranging from 0.02 to 0.05 mg/L. As the biomass increased rapidly, violacein production rose significantly between 12 and 24 hours, reaching 0.17 mg/L at 24 hours. It continued to increase to approximately 0.23–0.25 mg/L during 36–48 hours and remained at a plateau (around 0.24 mg/L) at 60 hours, closely aligning with the trend of biomass changes.
The calculation of violacein/DCW revealed that violacein synthesis increased synchronously with biomass growth throughout the fermentation process, with the highest specific productivity (around 0.20 mg/g) observed during 24–36 hours. This synchronization indicates that violacein expression is directly coupled with the growth of the engineered bacteria, with no significant lag, demonstrating efficient synthesis of the reporter gene and good compatibility with host metabolism.
Figure 16 Expression of vioABCDE driven by the J23100 promoter and B0034 RBS in BL21 using the pSB1A3 vector. DCW: dry cell weight. The medium used was M9 complete medium supplemented with glucose.
Violacein, as a reporter gene, demonstrates high efficiency, synchronization, and visualizability, making it suitable for real-time detection and result presentation in VOC response systems. This study provides a solid experimental foundation for subsequent system integration.
As the final visual output signal in the VOC response system, the yield of violacein directly affects signal visibility and detection sensitivity. Carbon source composition plays a critical role in metabolic flux distribution and the synthesis of secondary metabolites. To further improve violacein production, this experiment compared the synthesis of violacein in engineered bacteria cultured in M9 minimal medium supplemented with either 5% glucose or 5% glycerol as the primary carbon source. By analyzing the impact of different carbon sources on violacein yield, the most suitable carbon source supplementation strategy was identified to provide a more efficient and intuitive signal output for subsequent VOC response systems.
The engineered strain containing the violacein synthesis gene cluster (vioABCDE) was used as the experimental subject. Two culture systems were established:
① 5% glucose group;
② 5% glycerol group.
All cultures were incubated at 37°C with shaking at 220 rpm, with a culture volume of 50 mL.
Samples were collected after 24 hours of cultivation. Violacein was extracted from both cells and culture supernatant using the previously established ethanol extraction method:
The methodology for data processing was consistent with the violacein quantification described in Section 2.3. All experiments were performed in triplicate, with mean values taken as final results and subjected to statistical significance analysis.
The experimental results demonstrated that different carbon sources significantly influenced violacein synthesis (Table 2, Figure 3). Under 5% glucose cultivation, violacein production ranged from 0.132 to 0.162 mg/L, with an average of 0.146 mg/L. In contrast, under 5% glycerol cultivation, violacein production significantly increased, ranging from 0.245 to 0.323 mg/L, with an average of 0.281 mg/L—approximately 1.9 times higher than under glucose conditions.
Figure 17 Comparison of violacein content in BL21-Pj23100-RB0034-VioABCDE after 24 hours of cultivation with different carbon sources.
Supplementation with 5% glycerol significantly enhanced violacein production in the engineered bacteria, achieving nearly a 2-fold increase compared to the 5% glucose group, while also improving the stability and intuitiveness of the visual output. Therefore, glycerol is a more suitable carbon source choice for the subsequent construction of VOC-responsive sensing systems.
This experiment aimed to verify whether the PgrpE promoter can drive the expression of the vioABCDE violacein synthesis pathway upon detecting volatile organic compounds (VOCs) released during strawberry spoilage, thereby producing visible color changes. By integrating PgrpE with the vioABCDE pathway to construct a complete reporter system, we sought to transform invisible spoilage signals into a visual purple output. This approach aims to provide an instrument-free, low-cost, and real-time method for monitoring strawberry spoilage during cold chain transportation and storage.
The composite part
The recombinant plasmid was subsequently transformed into
To simulate volatile organic compounds (VOCs) released during early strawberry spoilage, a mixture of three major spoilage-related compounds -
In experimental setups, different volumes of the VOC mixture (
Samples were collected at
Absorbance was measured at
Control experiments were performed using bacterial cultures grown under identical conditions
Figure 18. Experimental workflow of the PgrpE-VioABCDE reporter strain
Figure 19. Response of PgrpE-VioABCDE reporter strain to VOCs.(A) Effect of different VOC concentrations on violacein production；(B) Visual observation of violacein accumulation under different VOC concentrations
Experimental results demonstrated that the
Post-incubation, cultures exposed to
This study systematically validates the application feasibility of the
In summary, the
Throughout the post-harvest supply chain—including transportation and storage—strawberries are highly susceptible to fungal infections. Fungal metabolic activities can lead to rapid spoilage, resulting in significant economic losses. The VOC detection module developed in the earlier phase of this study enables real-time monitoring of spoilage-related volatile organic compounds (VOCs), providing early warning and risk indication. While this module successfully detects spoilage, feedback from Human Practices (HP) interviews revealed that detection alone does not fundamentally reduce spoilage rates or economic losses. Without intervention measures, early warning alone may still lead to fruit decay and waste.
To address this limitation, this study further designed an independent antifungal strategy as a supplement to the detection module:
After harvesting and before transportation or cold-chain storage, strawberry surfaces are treated by spraying two hydrolytic enzymes that degrade fungal cell walls—
The main structural components of fungal cell walls are
Through synergistic action, these two enzymes target and break down the core structure of the fungal cell wall at the molecular level, causing destabilization and functional failure. This ultimately inhibits fungal growth and may lead to cell death.
By applying these enzymes before transportation, a protective barrier can be established on the strawberry surface, effectively suppressing fungal infection. This approach reduces the spoilage rate of strawberries during transit and storage, thereby mitigating economic losses caused by fungal decay.
This section aims to construct expression systems for chitinase and β-1,3-glucanase, and validate the molecular weights of both enzymes against theoretical values through SDS-PAGE analysis. This ensures the correctness and reliability of the expressed products, while providing high-purity enzyme sources for subsequent antifungal activity assays and strawberry spray validation, thereby establishing a foundation for strawberry spoilage prevention experiments.
To establish the antifungal enzyme expression system, this study selected two target genes capable of degrading major fungal cell wall components:
To enhance their expression levels in E. coli, both genes underwent
The optimized gene fragments were cloned into the
The pET28a(+) vector contains a
Following plasmid construction,
Figure 17. Construction of pET28a-Chitinase vector
Figure 18. Construction of pET28a-Glucanase vector
Recombinant plasmids were introduced into E. coli BL21(DE3) competent cells via
By comparing the positions of target bands with the molecular weight standard, the molecular weights of the expressed proteins were confirmed to match theoretical values:
Figure 19. Protein purification workflow
SDS-PAGE results are shown in Figure 20: After induction, a single clear band for
These results indicate that both target proteins were
Figure 20. SDS-PAGE of RmChi44 and MoGluB expressed in
This experiment successfully established
The band results demonstrate that both enzymes can be
This experiment aims to validate the in vitro broad-spectrum antimicrobial activity of purified chitinase and β-1,3-glucanase. Using the model fungus Saccharomyces cerevisiae as the target, the inhibitory effects of individual enzymes and their combination on fungal growth are evaluated. Since chitin and β-1,3-glucan are core structural components of the cell walls in most fungi (including strawberry spoilage pathogens such as Botrytis cinerea, Rhizopus spp., and Penicillium spp.), this study provides critical functional evidence for the subsequent application of these enzymes on strawberry surfaces to broadly prevent postharvest spoilage caused by various fungi.
Colonies grown on agar plates were counted to determine the number of colony-forming units per milliliter of the original suspension (CFU/mL).The CFU/mL value of the negative control group (heat-inactivated enzyme treatment) was designated as representing 100% survival, and the relative survival rate (%) of each treatment group was calculated accordingly. All experimental data were presented as the mean ± standard deviation (Mean ± SD) of three independent biological replicates. Statistical significance among groups was assessed using one-way analysis of variance (ANOVA), with p < 0.05 considered statistically significant.
Figure 21. Workflow for the expression and functional verification of chitinase and glucanase.
Quantitative results of the antifungal assays clearly demonstrated the inhibitory effects of chitinase, glucanase, and their combination against Saccharomyces cerevisiae (Figure 22, Table 1).
In the
Treatment with
The
The
This inhibition was significantly greater than that observed with either single-enzyme treatment (p < 0.001), revealing a strong
Figure 22. Synergistic inhibition of S. cerevisiae by the combined action of chitinase and glucanase.
Note: CFU = colony-forming unit. Relative survival rates were calculated based on the viable count of Group 2. Statistical significance was determined using one-way ANOVA; ### p < 0.001 vs. Group 2.
Through rigorous quantitative analysis, this study successfully verified the effectiveness of the enzyme formulation developed in this project.
The data clearly demonstrated that both purified enzymes possess strong in vitro antifungal activity, effectively degrading the cell wall of the model yeast and leading to a dramatic decrease in its survival rate. This provides direct evidence that functional proteins were successfully expressed and purified in Escherichia coli.
The performance of the dual-enzyme combination was particularly remarkable. Its antifungal activity was not merely additive but exhibited a clear synergistic effect (1 + 1 > 2). This finding indicates that our strategy—simultaneously targeting two key structural components of fungal cell walls (chitin and β-1,3-glucan)—achieves a more thorough and efficient disruption of cell wall integrity. Such a design represents a highly effective and rational engineering approach.
Given the similarity in cell wall composition between Saccharomyces cerevisiae and pathogenic fungi that infect strawberries, these results strongly support the development of this enzyme mixture as a broad-spectrum and efficient biological preservative. Its application in postharvest strawberry treatment is expected to markedly suppress fungal infection and reduce spoilage losses.
Moreover, this response module seamlessly integrates with the previously developed VOC-sensing and violacein-based reporting modules, together forming a complete synthetic biology system that encompasses
In the
In the
In the
Overall, the three modules form a functional synthetic biology loop that progresses from
This integrated system provides a feasible technological framework for real-time detection and prevention of postharvest spoilage in agricultural products. Beyond strawberries, it also lays the foundation for future applications in food safety monitoring and the development of intelligent packaging systems.
