Results

The goal of our iGEM project was to design and implement a novel microbial system that used Lactobacillus reuteri, a Generally Recognized as Safe (GRAS) probiotic strain, as a chassis for fruit peel degradation. A composite genetic circuit was constructed incorporating the ermB promoter for strong constitutive expression, the Usp45 engineered secretion signal to facilitate extracellular release, and the pgxC gene encoding polygalacturonase, an enzyme capable of cleaving pectin polymers abundant in fruit peels. The plasmid backbone pTRKH3 was used for stable replication and expression in L. reuteri. Through this strategy, we aimed to achieve efficient secretion of active polygalacturonase into the extracellular environment, enabling direct breakdown of fruit peel pectin into galacturonic acid and simpler sugars. By coupling probiotic safety with enzymatic efficiency, this system addressed the global issue of fruit waste accumulation while contributing to sustainable bioconversion. Furthermore, the project envisioned downstream applications, such as using the resulting degraded biomass as a substrate for bio-based products (biofuels, animal feed, or prebiotic supplements), thereby establishing a circular and eco-friendly waste-to-resource platform.

The wild-type pgxC protein sequence (highlighted in red) was compared with the secretion signal sequence derived from USP45 (highlighted in blue). To facilitate secretion of the engineered enzyme, the N-terminal region of pgxC was fused in-frame with the USP45 signal peptide, generating the proposed engineered pgxC construct (bottom panel). This design ensures that the protein maintains its catalytic core while acquiring an additional leader sequence that directs its secretion outside of the bacterial host cell. After obtaining the pTRKH3 plasmid backbone vector, the engineered pgxC with USP45 secretion signal was synthesized and cloned using standard restriction enzyme–based cloning methods (Figure 1).

The synthesis process involved codon optimization for efficient bacterial expression and seamless junction design between the USP45 signal peptide and pgxC coding sequence. Restriction sites were strategically introduced to allow precise ligation of the engineered insert into the pTRKH3 multiple cloning site without disrupting the reading frame. Following successful assembly, the construct was verified by sequencing, ensuring correct orientation and fidelity of the engineered sequence. The final recombinant plasmid, pTRKH3-USP45-pgxC, was designed to enable secretion of polygalacturonase enzymes into the extracellular medium, thus improving substrate accessibility and overall enzyme efficiency (Figure 2). Detailed steps of plasmid construction and cloning strategy are described in the engineering section.

The recombinant plasmid is 9,585 bp in size and was designed to enable secretion and expression of the engineered pgxC enzyme in a bacterial host system. The modified construct incorporates the USP45 signal peptide fused to the N-terminus of the pgxC coding sequence, driven by the strong ermB promoter to ensure robust transcription. The plasmid backbone, derived from pTRKH3, provides stability and compatibility for Gram-positive bacterial expression systems. Multiple cloning sites (MCS) are available and flanked by numerous restriction enzyme recognition sequences, which allow flexible cloning and potential future modifications.

In addition to the engineered pgxC region, the plasmid carries an antibiotic resistance marker for selective growth, a replication origin for stable maintenance, and regulatory elements such as the tet promoters and cat promoter, which provide tunable control over gene expression. The detailed arrangement of restriction sites around the insert ensures that the engineered signal peptide and coding sequence were seamlessly integrated without disrupting the reading frame.

The agar plate images illustrate the outcomes of transformation with the engineered pTRKH3-pgxC plasmid (Figure 3). The negative control plate (left, without plasmid) showed no colony growth, confirming the absence of background resistance. In contrast, the experimental plate (right, with plasmid) displayed abundant colony formation, indicating successful uptake and expression of the recombinant construct by L. reuteri competent cells. The observed colonies arose from bacteria harboring the pTRKH3-pgxC plasmid, which carries an antibiotic resistance marker, thereby allowing selective growth.

This result confirms the efficient introduction of the engineered plasmid into both E. coli and L. reuteri. Transformation of E. coli, used primarily as a cloning host, was achieved via the heat shock method, whereas L. reuteri, the intended expression host, was successfully transformed using electroporation. The ability to establish the construct in both bacterial systems demonstrates its broad host compatibility. The high density of colonies observed on the plasmid-containing plates highlights robust transformation efficiency, providing a solid foundation for downstream applications such as plasmid propagation, protein secretion analysis, and functional assays of the engineered pgxC enzyme.

To confirm expression and secretion of pgxC, proteins from both the culture supernatant (S) and cell lysate (L) were analyzed by SDS-PAGE (Figure 4). A distinct band corresponding to approximately 48.5 kDa, which matched the predicted molecular weight of pgxC, was observed in both the supernatant and cell lysate of L. reuteri containing the pgxC plasmid. In contrast, no such band was detected in the negative control samples lacking the plasmid. The presence of the pgxC band in the supernatant indicated that the engineered Usp45 secretion signal successfully directed protein export outside the cell. These results demonstrated that L. reuteri carrying the recombinant plasmid not only expressed pgxC but also secreted it into the extracellular medium, validating the effectiveness of the designed secretion system.

The successful detection of pgxC in the extracellular fraction demonstrated that our engineered secretion system was functional, enabling L. reuteri to release the enzyme directly into the surrounding medium. This is a critical outcome for the project, as extracellular secretion eliminates the need for costly cell disruption or purification steps and allows the enzyme to act directly on fruit peel biomass. Since pectin is a major structural component of fruit peels, the secretion of active pgxC provides a practical foundation for efficient biodegradation of agricultural waste. Furthermore, the use of a probiotic chassis such as L. reuteri enhances the safety and scalability of this system, opening applications in sustainable waste management.

A standard curve was generated using BSA concentrations ranging from 0 to 0.5 µg/µL, and absorbance at 595 nm was measured after Bradford reagent addition (Figure 5). The resulting linear regression equation was y = 0.401x + 0.091 with an R² value of 0.94, indicating a strong correlation between absorbance and protein concentration. Using this equation, protein concentrations were quantified in the culture supernatants of Lactobacillus reuteri. Samples without plasmid showed a protein concentration of approximately 0.04 µg/µL, while samples containing the pgxC plasmid exhibited a higher concentration of 0.10 µg/µL, suggesting successful expression and secretion of recombinant proteins.

The increase in protein concentration in the supernatant of plasmid-containing L. reuteri compared with the control confirmed that recombinant pgxC expression contributed to extracellular protein content. Although the total protein concentration measured included both secreted pgxC and native proteins, the relative increase in the plasmid-bearing condition is consistent with the SDS-PAGE results showing a clear 48.5 kDa pgxC band in the supernatant.

The strong linearity of the BSA standard curve (R² = 0.94) validated the accuracy of the Bradford assay in this experiment, though minor deviations from an R² of 1 likely reflect pipetting variability or uneven dye–protein binding efficiency. Together, these findings demonstrated that the engineered secretion system enabled detectable increases in extracellular protein concentration, supporting the functionality of pgxC expression and secretion in L. reuteri.

D-galacturonic acid was chosen as the calibration standard because it represents the major monosaccharide building block of pectin, a complex polysaccharide abundant in fruit peels. Pectin is composed mainly of α-(1→4)-linked D-galacturonic acid residues, often methyl-esterified or acetylated, forming homogalacturonan regions and rhamnogalacturonan domains. During enzymatic degradation, polygalacturonases such as pgxC hydrolyze the glycosidic linkages between these galacturonic acid units. This cleavage releases free D-galacturonic acid or shorter oligogalacturonides, which can then be detected as reducing sugars using the DNS assay. Thus, using D-galacturonic acid as the standard ensures that the assay directly reflects the extent of pectin depolymerization catalyzed by pgxC, providing an accurate measure of enzymatic activity during fruit peel degradation.

To evaluate the enzymatic activity of secreted pgxC, the DNS assay was performed to quantify the release of reducing sugars, specifically D-galacturonic acid, after incubating fruit peel pectin with culture supernatants. A standard curve generated using pure D-galacturonic acid (0–1.0 mg/mL) showed a strong linear correlation between concentration and absorbance at 540 nm (A = 1.207·C + 0.016, R² = 1.000) (Figure 6).

To further assess the kinetics of pectin degradation, supernatants from control (no pgxC) and engineered (pgxC-expressing) Lactobacillus reuteri were incubated with pectin for 0, 12, 24, and 48 hours, and the release of reducing sugars was quantified by DNS assay (Figure 7). In the engineered strain, absorbance values at 540 nm increased steadily over time, reaching 0.35 at 12 h, 0.61 at 24 h, and 0.67 at 48 h, reflecting continuous production of D-galacturonic acid. By contrast, the control strain showed only minimal changes in absorbance throughout the incubation period, remaining below 0.1 even at 48 h. These results demonstrated that the pgxC-expressing strain actively degraded pectin in a time-dependent manner, while the control strain exhibited negligible enzymatic activity. Together, these findings confirm that secreted pgxC enabled effective extracellular breakdown of fruit peel pectin, validating the engineered system’s functional role in waste bioconversion.

To evaluate real-world applicability, we tested fruit peels from banana, kiwi, pear, tomato, and watermelon as substrates (Figure 8). After 24 h incubation, engineered L. reuteri supernatants produced significantly higher levels of D-galacturonic acid compared to controls, reflecting efficient peel degradation. Pear and tomato peels exhibited the greatest release (~0.42–0.51 mg/mL), followed by kiwi (~0.30 mg/mL) and banana (~0.22 mg/mL). Watermelon peels produced minimal sugars (<0.1 mg/mL), consistent with their lower pectin content.

These results demonstrate both the substrate range and specificity of the engineered strain, highlighting its suitability for valorizing citrus- and pear-rich agricultural waste streams.

Through iterative design, verification, and testing, we successfully engineered L. reuteri to:

• Express codon-optimized pgxC fused with a secretion peptide.
• Stably maintain the recombinant construct and efficiently transform into L. reuteri.
• Secrete enzymatically functional pgxC extracellularly, confirmed by band detection and protein quantification.
• Hydrolyze fruit peel pectin into detectable levels of galacturonic acid, validated across multiple fruit substrates.

Together, these results establish our system as a practical proof-of-concept for fruit peel bioconversion. By combining probiotic safety with efficient extracellular enzyme action, this platform lays the groundwork for waste valorization routes such as feedstock generation, biofuel precursors, and prebiotic supplement production—contributing to a circular and sustainable bioeconomy.

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