Engineering

Design Purpose

To achieve efficient fruit peel degradation, we designed a recombinant construct enabling Lactobacillus reuteri to secrete the polygalacturonase enzyme (pgxC). The construct was assembled in the shuttle plasmid pTRKH3, allowing stable propagation in E. coli and functional expression in L. reuteri. The design consists of the following elements:

• Promoter (ermB promoter) – a strong constitutive promoter ensuring robust gene expression in Gram-positive bacteria.
• Secretion Signal (Usp45) – a well-characterized leader peptide from Lactococcus lactis that directs the expressed protein into the extracellular medium.
• Coding Sequence (pgxC) – the polygalacturonase enzyme (48.5 kDa), responsible for cleaving the α-1,4-glycosidic linkages in pectin to release D-galacturonic acid.
• Terminator – native transcriptional terminator included for stability.

We registered this construct as a composite part in the iGEM Registry:

• Part ID: BBa_K5000001
• Name: [ermB promoter + Usp45 signal peptide + pgxC]
• Function: Extracellular secretion of polygalacturonase for fruit peel degradation
• Plasmid Backbone: pTRKH3 (erythromycin resistance marker)

The construct consists of the ermB constitutive promoter, followed by an EcoRI restriction site introduced at the 5′ end for cloning, the Usp45 secretion signal peptide, and the codon-optimized pgxC coding sequence. A downstream transcriptional terminator ensures stable expression. This design allows Lactobacillus reuteri to secrete active polygalacturonase for efficient fruit peel degradation.

The terminal region of the pgxC coding sequence was engineered to include a BamHI restriction site at the 3′ end, allowing for flexible downstream cloning. Sequence alignment highlights the codon-optimized pgxC region, with annotated restriction sites (PpuMI, PciI, SexAI) for verification and modular assembly. This design ensures that pgxC can be efficiently integrated and expressed in the final plasmid construct for extracellular polygalacturonase secretion.

The structure was modeled using the ESMFold program. The USP45 secretion signal peptide (left, red box) forms an extended flexible region, while the pgxC protein main body (right, blue box) adopts a stable β-sheet-rich fold characteristic of polygalacturonase enzymes. This fusion design facilitates extracellular secretion of the active enzyme by Lactobacillus reuteri.

Additional Notes

Besides the five new parts we added to the Registry, we added two additional parts to the Registry for engineering purposes. The additions are as follows:

• Engineered N-terminal Secretion Signal Peptide pgxC-silent PstI Scar Removal (https://registry.igem.org/parts/bba-25cmenhz)
• ermB Promoter with Engineered N-terminal Secretion Signal Peptide pgxC-silent PstI Scar Removal (https://registry.igem.org/parts/bba-25txyoa2)

*The pgxC signal peptide was modified by a silent PstI site removal to restore RFC 10 compatibility while maintaining the original amino acid sequence and secretion functionality.

Overview: Building Process

To engineer L. reuteri for extracellular polygalacturonase (pgxC) secretion, we constructed a recombinant plasmid based on the shuttle vector pTRKH3. The construct was designed with restriction enzyme sites to enable efficient insertion of the codon-optimized pgxC gene fused to the Usp45 secretion signal peptide under control of the ermB constitutive promoter.

Part 1: Cloning Strategy Using Restriction Enzymes

The codon-optimized pgxC sequence was synthesized with EcoRI (5′) and BamHI (3′) restriction sites flanking the gene to facilitate cloning into the pTRKH3 plasmid vector:

• EcoRI site: Positioned upstream of the promoter region, ensuring correct orientation and seamless ligation.
• BamHI site: Placed downstream, allowing the pgxC insert to be stably joined with the vector backbone.

The pTRKH3 plasmid was linearized with these enzymes, and the pgxC fragment was ligated in-frame with the Usp45 signal sequence. This design ensures secretion of the expressed protein into the extracellular medium.

Schematic representation of the recombinant pTRKH3 plasmid (9585 bp) containing the ermB promoter, Usp45 secretion signal peptide, and the codon-optimized pgxC gene. Restriction enzyme sites EcoRI and BamHI (red boxes) were introduced to enable insertion of pgxC. The construct provides erythromycin resistance for selection in both E. coli and L. reuteri.

Part 2: Verification of Cloning

The BamHI-only digest produced a single band at ~9585 bp, corresponding to the intact recombinant plasmid. The BamHI + EcoRI double digest generated two fragments: one corresponding to the vector backbone (8238 bp) and the other to the pgxC insert (1347 bp), consistent with the expected sizes of the engineered construct.

After ligation, the recombinant plasmid was first transformed into E. coli DH5α for propagation. Plasmid DNA was extracted and subjected to:

1. Restriction digestion analysis – BamHI-only digestion produced a single band (9585 bp), while BamHI + EcoRI double digestion produced two bands (8238 bp and 1347 bp), confirming correct pgxC insertion.
2. Agarose gel electrophoresis – Confirmed expected fragment sizes (Figure 6).
3. Sanger sequencing – Verified correct orientation and codon integrity of pgxC insert.

Once verified, the recombinant plasmid pTRKH3-pgxC was introduced into L. reuteri by electroporation, generating the engineered strain for subsequent protein expression and functional assays.

Rationale & Objectives

Following successful construction of the recombinant plasmid pTRKH3-pgxC, it was essential to evaluate whether the engineered Lactobacillus reuteri could express, secrete, and functionally utilize the pgxC enzyme. The rationale behind testing was to confirm that the designed genetic circuit not only worked at the DNA level but also translated into measurable protein production and functional pectin degradation. The specific objectives of the testing phase were:

1. To confirm expression of pgxC at the protein level by detecting the expected 48.5 kDa band using SDS-PAGE.
2. To quantify the amount of secreted protein in the engineered strain compared to controls using the Bradford assay.
3. To validate the enzymatic activity of pgxC through a DNS assay that measures release of reducing sugars (D-galacturonic acid) from pectin substrates.
4. To demonstrate functional application of the engineered strain in degrading real fruit peel biomass (orange and apple), thereby linking molecular results to practical bioconversion outcomes.

Together, these tests provide a comprehensive evaluation of the system — from protein secretion to enzymatic functionality and applied biomass degradation — ensuring that the design is effective beyond genetic assembly.

Part 1: Protein Expression and Secretion

To verify that pgxC was expressed and secreted:

• SDS-PAGE analysis revealed a distinct protein band at ~48.5 kDa in the culture supernatant of engineered L. reuteri, corresponding to the expected molecular weight of pgxC. This band was absent in the plasmid-free control strain.
• Bradford assay quantified total secreted protein, showing significantly higher levels in engineered strains (0.10 µg/µL) compared to controls (0.04 µg/µL).

These results confirmed successful extracellular secretion of the engineered enzyme.

Part 2: Enzyme Activity Validation

The functional activity of pgxC was tested using the DNS (3,5-dinitrosalicylic acid) assay:

• Engineered L. reuteri supernatants released significantly higher amounts of D-galacturonic acid from pectin substrate than control strains.
• Absorbance at 540 nm was used to quantify reducing sugars, with engineered strains showing a ~2.5-fold increase in activity.

This demonstrated that the secreted pgxC retained enzymatic functionality.

Part 3: Application to Fruit Peel Degradation
To evaluate real-world utility, tests were performed using fruit peels (orange&apple):

• Homogenized fruit peel incubations – Engineered strains showed visible degradation, including liquefaction and reduction in peel particle size, compared to controls.

These results confirmed that engineered L. reuteri can degrade fruit peel components, validating the practical application of the construct.

Through our Build and Test phases, we reflected on the outcomes and identified strategies to optimize our construct and experimental design. The Learn phase allowed us to evaluate the strengths and limitations of our system, and to plan improvements for future iterations.

Learn #1: Solving Project Problems Using Synthetic Biology Tools & Experimental Techniques

We aimed to confirm whether Lactobacillus reuteri engineered with pTRKH3-pgxC could effectively secrete active (pgxC) and degrade fruit peel waste. Our tests showed:

• Protein secretion was achieved, confirmed by SDS-PAGE with a visible 48.5 kDa band.
• Protein yield was modest (0.10 µg/µL vs. 0.04 µg/µL in controls), indicating successful expression but suggesting room for higher secretion efficiency.
• Enzymatic activity was validated through DNS assay, which showed ~2.5-fold more reducing sugar release in engineered strains compared to controls.
• Functional fruit peel degradation occurred, but the process was slower than expected, suggesting that enzyme activity or secretion levels may not yet be optimal at large-scale.

This confirmed that our synthetic biology approach successfully addressed the core challenge — enabling pectin degradation in a GRAS probiotic — with bottlenecks in yield and efficiency.

Learn #2: Next Steps and Improvements for Future Design Iterations
Based on these findings, we identified several improvements for the next engineering cycle:

1. Promoter Optimization
   Replace ermB with stronger or inducible promoters to increase protein expression while balancing host growth.
2. Secretion Signal Variants
   Test alternative signal peptides (SPuspA or SlpA) to enhance extracellular protein secretion compared to Usp45.
3. Codon Optimization Refinement
   Re-analyze codon usage in L. reuteri and design optimized pgxC variants to improve translation efficiency.
4. Enzyme Engineering
   Generate pgxC truncation variants or directed evolution mutants to improve activity and stability under acidic fruit waste conditions.
5. Adaptive Laboratory Evolution (ALE)
   Expose L. reuteri to fruit peel-rich environments to select strains with improved fitness and degradation capacity.
6. System-level Integration
   Explore co-expression of accessory enzymes (pectin methylesterase and or cellulases) to enhance complete biomass breakdown.

Summary of Learnings:

Our engineered construct proved functional in secreting pgxC and initiating fruit peel degradation, validating our design. However, efficiency limitations highlighted opportunities for promoter engineering, secretion optimization, and enzyme enhancement. These insights will guide the next iteration of the Design–Build–Test–Learn cycle to achieve higher degradation efficiency and practical scalability.

• Part 1: Cloning Strategy Using Restriction Enzymes
• Part 1: Protein Expression and Secretion
• Part 2: Enzyme Activity Validation
• Part 3: Application to Fruit Peel Degradation
• Learn #2: Next Steps and Improvements for Future Design Iterations