Description

Fruit peel waste represents a major contributor to global agricultural biomass, yet its high pectin content offers untapped potential for sustainable bioconversion. In this project, we engineered Lactobacillus reuteri, a Generally Recognized as Safe (GRAS) probiotic, to secrete polygalacturonase (pgxC) for efficient fruit peel degradation. A recombinant construct was designed using the pTRKH3 plasmid backbone, driven by the ermB promoter, and fused with the Usp45 secretion signal to facilitate extracellular enzyme release. Transformation of E. coli and L. reuteri confirmed stable plasmid propagation, and SDS-PAGE analysis revealed a distinct 48.5 kDa protein band consistent with secreted pgxC.

Bradford assay quantification further demonstrated elevated protein levels in engineered strains compared to control strains without plasmid transformation, which showed only basal protein secretion and no detectable pgxC band (0.10 vs 0.04 µg/µL). Functional activity was validated using the DNS assay, with engineered L. reuteri producing significantly higher amounts of D-galacturonic acid from pectin than the plasmid-free control, which only generated background levels of reducing sugars (<0.1 mg/mL). Kinetic analysis showed time-dependent increases in sugar release, while substrate testing revealed fruit-specific degradation efficiencies (pear and tomato > kiwi > banana > watermelon). Collectively, these findings demonstrate that secretion of pgxC by a safe probiotic host enables direct extracellular breakdown of fruit peels, eliminating the need for costly downstream processing. This work establishes a proof-of-concept for a circular waste-to-resource platform, with potential applications in biofuels, animal feed, and prebiotic supplement production.

Food waste, particularly fruit and vegetable peels, is an increasingly urgent global issue that demands immediate attention. While food waste as a whole is widely recognized, peels often receive less focus despite representing a significant proportion of organic waste. Choosing to focus on fruit peels is important because they are (1) unavoidable in household and commercial food preparation, (2) generated in massive quantities globally, and (3) largely underutilized, despite containing valuable bioactive compounds. Addressing this issue not only reduces waste but also opens opportunities for innovation in sustainability and resource recovery. These various problems can be narrowed down into the following:

First, Environmental Impacts:

When fruit waste decomposes in landfills, it generates methane, a greenhouse gas 25 times more potent than carbon dioxide in trapping heat in the atmosphere. With over 1.3 billion tons of food waste generated annually worldwide, a significant portion of which consists of fruit and vegetable peels, the accumulation of this waste contributes substantially to climate change. Moreover, the physical presence of peel waste accelerates landfill overflow, straining already limited land resources and forcing municipalities to invest heavily in expanding or managing waste infrastructure. Decomposing fruit waste in landfills produces large amounts of methane, a greenhouse gas 25 times more potent than carbon dioxide.

Second, Landfill Overflow:

Over 1.3 billion tons of food waste are generated each year, a significant portion being fruit and vegetable peels. This contributes to landfill overflow, taking up valuable land and requiring costly waste management. This is illustrated in the figure below:

Third, Public Health Hazards:

Undegraded food waste attracts pests and spreads bacteria in urban areas. Studies show that poorly managed organic waste can lead to higher incidences of vector-borne diseases. Beyond environmental effects, unmanaged fruit peel waste poses serious public health risks. As organic waste sits in urban landfills or dumping grounds, it becomes a breeding ground for pests such as flies and rodents, which spread harmful bacteria and diseases. Research has shown that poorly managed organic waste correlates with higher incidences of vector-borne diseases, disproportionately affecting densely populated areas. In this way, fruit peel waste is not only an environmental concern but also a direct human health threat.

Our Problem Framework:

The left panel highlights the scale of food waste, with fruit and vegetable peels as major contributors to the 1.3 billion tons discarded annually. The middle panel illustrates conventional solutions—including landfilling, composting, biogas production, industrial pectin extraction, and animal feed conversion—each with significant limitations. The right panel presents our engineered solution: Lactobacillus reuteri secreting polygalacturonase (pgxC) to directly degrade fruit peels into D-galacturonic acid, enabling downstream applications such as biofuels, animal feed, and prebiotic supplements in a safe, scalable, probiotic-based system.

While many global sustainability challenges exist, food waste from fruit peels is especially urgent and solvable. Unlike plastic or e-waste, fruit peel waste is biodegradable and contains nutrients, antioxidants, and natural compounds that can be repurposed into value-added products such as biofuels, animal feed, or biodegradable packaging. Tackling this issue aligns with UN Sustainable Development Goal 12 (Responsible Consumption and Production) by addressing waste at its source. More importantly, it bridges multiple global challenges: reducing greenhouse gas emissions, easing landfill burdens, and lowering health risks. By focusing on fruit peels specifically, we target a problem that is widespread, visible, and solvable with innovative approaches, making it a strategic entry point for sustainable change.

Solution Overview:

Every year, millions of tons of fruit peels are discarded as food waste, contributing to landfill overflow and climate change. A major reason fruit peels persist is their high pectin content: a complex polysaccharide that is naturally resistant to degradation. Existing solutions, like composting and microbial fermentation, are slow, inefficient, and often require high temperatures, alkaline conditions, or long processing times. Our project focuses on building a synthetic biology-based solution that can efficiently break down fruit peels in a sustainable way.

Our Engineered System:

To address this, we are developing a synthetic biology-based system using Lactobacillus reuteri, a GRAS-grade probiotic that thrives in acidic environments like those found in fruit-waste sludge. Into this host, we introduce the pgxC gene from Aspergillus niger, which encodes polygalacturonase (PG) — an enzyme that specifically targets and breaks down pectin.

We fused the USP45 signal peptide to the PG enzyme, enabling direct secretion outside the cell. This design allows our engineered bacteria to act immediately on the surrounding fruit peels, bypassing the need for costly pre-treatments such as cell lysis or pH adjustment.

Transforming Waste into Value:

By efficiently breaking down fruit peels, we aim to upcycle food waste into valuable products:

• Compost and soil enhancers to support sustainable agriculture.
• Prebiotics such as galacturonic acid, a beneficial byproduct for gut health.
• Biofuels and other industrial intermediates.
• Biodegradable biomaterials for packaging and other uses

Through this approach, we not only reduce greenhouse gas emissions caused by untreated fruit waste but also contribute to a circular economy where waste is transformed into resources.

This image compares wild-type Lactobacillus reuteri and an engineered L. reuteri strain carrying the pgxC plasmid, highlighting a synthetic biology strategy for fruit peel degradation.

Left: Wild-Type L. reuteri (No Plasmid)

• Wild-type L. reuteri lacks the pgxC gene and cannot express polygalacturonase.
• As a result, it does not degrade pectin efficiently, so intact fruit peel persists with minimal degradation.

Right: Engineered L. reuteri with pTRKH3-pgxC (BBa_252WDOMF)

• Transformed L. reuteri harbors the recombinant pTRKH3-pgxC plasmid containing a pgxC gene fused to the Usp45 secretion signal.
• The engineered strain expresses and secretes pgxC, which is exported into the medium.
• Secreted pgxC hydrolyzes pectin, the main structural carbohydrate of fruit peels, releasing D-galacturonic acid and simple sugars.
• This process results in significant fruit peel breakdown compared to the control.

Synthetic Biology Significance

The project demonstrates a modular, food-grade bioconversion platform using a probiotic chassis. By overexpressing a targeted pectinase (pgxC), engineered L. reuteri efficiently upcycles fruit waste into valuable biomolecules.

Overall, this figure highlights the synthetic biology approach used in the project: introducing a plasmid-encoded pgxC construct into a probiotic chassis (L. reuteri) to enable efficient, food-grade bioconversion of fruit peel waste into valuable biomolecules.

Step 1: Selection and Cloning of pgxC

Aspergillus niger produces three major polygalacturonase enzymes—PgxA, pgxB, pgxC. We selected pgxC as it was the best fit and the most versatile, able to degrade both homogalacturonan (HG) and xylogalacturonan (XGA), the two main forms of pectin in fruit peels. This makes it more effective than enzymes that only target one type. DNA of pgxC was isolated and prepared for plasmid insertion.

Step 2: Insertion of the Gene into Bacteria

We used the pTRKH3 expression plasmid system that is known to function efficiently in Lactobacillus reuteri. The plasmid carries regulatory sequences like ermB promoter for a stronger expression of the pgxC gene. This enables the engineered bacteria to translate pgxC into a functional enzyme.

Step 3: Production of Recombinant Lactobacillus reuteri

When cultured under optimized conditions of pH ~6.0, the engineered L. reuteri secretes pgxC polygalacturonase into its surroundings. This secretion is essential since the enzyme must act directly on fruit peel pectin rather than remain inside the bacterial cells.

Step 4: Verification of Enzyme Activity

We used Bradford assay to ensure consistent protein production. We used enzyme activity assays to measure pectin degradation efficiency compared to controls. This ensured that pgxC was not only expressed but also functional.

Step 5: Test on Actual Fruit Peels

The engineered L. Reuteri was tested on actual fruit peels to simulate practical conditions. Degradation was measured by comparing against untreated control samples.

Promoter Innovation

• Successfully used the ermB constitutive promoter (BBa_K3183000) to drive continuous expression of pgxC in Lactobacillus reuteri.
• Avoided dependence on costly inducers like IPTG or nisin, ensuring stable enzyme production under variable, low-cost conditions.

Secretion Strategy

• Implemented the Usp45 secretion signal peptide (BBa_25KVZJA0) to export pgxC extracellularly, enabling direct access to fruit peel pectin.
• Designed and tested an engineered N-terminal secretion signal peptide (BBa_2596ET3M) to potentially enhance secretion efficiency beyond Usp45.

Functional Enzyme Expression

• Integrated the pgxC coding sequence (BBa_252AGPHV) into the pTRKH3 backbone, producing a ~48.5 kDa polygalacturonase band via SDS-PAGE.
• Demonstrated secretion of active enzyme, with Bradford assay showing higher protein levels in engineered strains (0.10 ± 0.01 µg/µL) compared to plasmid-free controls (0.04 ± 0.005 µg/µL).

Composite Part Construction

• Created the pTRKH3 composite plasmid (BBa_252WDOMF) combining ermB promoter, secretion signal(s), pgxC, and transcriptional terminator.
• Achieved stable cloning in E. coli and successful transformation of L. reuteri via electroporation.

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.

Functional Validation

• Used the DNS assay with D-galacturonic acid calibration to show activity of pgxC.
• Confirmed time-dependent pectin degradation: engineered strains reached ~0.61 absorbance at 24 h vs <0.1 for controls.
• Demonstrated fruit-specific degradation efficiencies: pear ≈ tomato > kiwi > banana >> watermelon.

Proof of Concept Established

• Engineered L. reuteri as a safe, food-grade chassis capable of directly breaking down fruit peel waste.
• Showed that this system can convert waste biomass into fermentable sugars, opening applications in biofuels, animal feed, and prebiotic production.

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