Chitinator et al.

Chitinator et al. is a project focused on the biodegradation of chitin, aiming o convert it into nutrient rich compounds that can be used as a bioactivator in crops such as garlic, using synthetic biology.

Chitinator et al. reflects a growing global interest in sustainable innovation, circular thinking, and environmentally conscious science. It belongs to a new generation of projects that move beyond theory, seeking to bring synthetic biology into real-world applications.

As the world faces challenges like waste accumulation, climate impact, and the need for greener agricultural practices, this project resonates with current scientific and societal priorities. It shows how biology can be used not only to understand the world, but to help improve it, by turning waste into value, and problems into solutions.

Our project, Chitinator et al., addresses two interlinked problems; one agricultural and one environmental, both of which are highly relevant today.

1. The agricultural problem:

Modern agriculture relies heavily on chemical fertilizers to sustain high crop yields. However, the overuse of these inputs often comes at a cost. Synthetic fertilizers can degrade soil quality, disrupt local ecosystems, and contaminate water sources. In the long term, they may even weaken plant health, making crops more vulnerable to pests and disease. There is an urgent need for sustainable alternatives that can nourish the soil without harming the environment.

2. The environmental waste problem:

Greece hosts a significant seafood processing industry, especially in shrimp packaging and cleaning facilities. While the country does not engage in large-scale shrimp farming, it imports and processes large volumes of crustaceans. This generates a considerable amount of chitin-rich waste, which often ends up in landfills or, worse, in marine ecosystems. These waste streams are rarely reused, despite being biologically valuable. The improper disposal of such biomass contributes to environmental pollution and represents a missed opportunity for resource recovery.

By addressing both the overreliance on harmful agricultural practices and the underutilization of chitinous seafood waste, our project aims to introduce a sustainable, bio-based solution that connects environmental responsibility with agricultural innovation.

We genetically modify Escherichia coli BL21 to express a fusion enzyme composed of an endochitinase and an exochitinase domain. These two enzymes work synergistically to break down chitin into its oligomeric units, primarily N-acetylglucosamine (GlcNAc).

This enzymatic activity is designed to occur directly in the substrate, within chitin-containing biomass such as shrimp shells, where the engineered bacteria act as biological tools for depolymerization. In the next stage, we plan to transfer this system into Bacillus subtilis 168, a chassis with GRAS status and an inherent ability for efficient protein secretion, making it highly suitable for large-scale and field applications.

The outcome of this process is nutrient-rich compounds derived from chitin degradation. To optimize and scale this production, we employ controlled bioreactor systems, ensuring consistent yields, process efficiency, and suitability for agricultural applications. These compounds can be formulated as a liquid bioactivator that is applied through foliar sprays in crops such as garlic cultivation, following chemical fertilizer treatments. In this way, they contribute to restoring soil health while simultaneously providing protective effects against fungal pathogens that threaten garlic fields.

In essence, we convert an otherwise inert and difficult to degrade waste material into a valuable agricultural input, using engineered microbes as active agents of transformation.

Our approach is scalable, modular, and aligned with the principles of the circular bioeconomy and environmental responsibility.

Chitin is a natural biopolymer, structurally similar to cellulose, and is considered the second most abundant polysaccharide on Earth. It is composed of repeating units of Nacetyl- D-glucosamine (GlcNAc) linked by β-(1→4) glycosidic bonds.

Chitin serves as a key structural component in several biological materials, including:

• The exoskeletons of arthropods, such as shrimp, crabs, and insects
• The cell walls of fungi
• The radula of mollusks and the beaks of cephalopods

Despite its abundance, chitin is not easily degradable in natural environments.

Certain microorganisms, such as chitinolytic bacteria and fungi, are capable of breaking it down. However, this process tends to be slow and inefficient, primarily due to the chemical and physical properties of chitin:

1. High crystallinity – Chitin molecules are tightly packed in crystalline microfibrils, making them resistant to enzymatic access.
2. Insolubility in water and most solvents – Chitin’s insolubility reduces its bioavailability.
3. Strong hydrogen bonding – The polymer chains form extensive hydrogen bonds, increasing structural rigidity.
4. Association with other biomolecules – In natural materials, chitin is often embedded in protein or mineral matrices (e.g., in shells), which further complicates its degradation.

Because of these characteristics, efficient biodegradation of chitin requires specialized enzymatic systems, often working in synergy, including endochitinases, exochitinases, and β-N-acetylglucosaminidases.

Our project aims to harness these enzymatic tools through synthetic biology, to turn this recalcitrant biomass into a useful agricultural input.

With Chitinator et al., our mission goes beyond solving a single problem, we aim to redefine how we view waste, sustainability, and the role of biology in the modern world.

We envision a future where biological tools unlock the hidden value of natural materials, turning underutilized resources like chitin into practical solutions for global challenges such as soil degradation, food insecurity, and environmental pollution.

Through this project, we want to demonstrate that synthetic biology can be accessible, scalable, and environmentally responsible. We believe that microbial engineering, supported by bioreactor-based production systems and ultimately transferred into robust chassis such as Bacillus subtilis 168, can pave the way for a circular and regenerative model of agriculture, where what was once discarded becomes essential.

Ultimately, our goal is to bridge the gap between innovation and impact, to create biological systems that not only work in the lab, but can be scaled in bioreactors, applied in the field, and serve communities, ecosystems, and the planet at large.

Greek white garlic is highly valued worldwide for its flavor, nutritional qualities, and cultural importance, but it is also particularly vulnerable to fungal diseases, most notably Fusarium proliferatum (causing Fusarium bulb rot) and Sclerotium cepivorum (causing white rot). These pathogens can devastate crops and remain in the soil for years, making them especially difficult to control.

Farmers often rely on chemical sprays and synthetic fertilizers to protect their fields, but these practices come with significant drawbacks: soil health declines, environmental burden increases, and long-term crop resilience is compromised.

This is where our bioactivators provide an ideal alternative. The monomers and oligomers of chitin (such as N-acetyl-glucosamine) not only enrich the soil with valuable nutrients but also act as elicitors of plant defense mechanisms, stimulating garlic plants to better resist fungal attacks.

By focusing on Greek garlic, we are addressing a real agricultural challenge while also protecting and promoting a national product of global reputation. In this way, Chitinator et al. combines biotechnology, circular bioeconomy principles, and environmental respect to deliver both sustainability and impact.

At the core of our approach lies the design of a synthetic fusion enzyme capable of efficiently breaking down chitin. This fusion protein combines two distinct enzymatic domains: an endochitinase, which cleaves internal bonds within the chitin polymer, and an exochitinase, which further digests the resulting fragments into oligomers and monomeric units such as N-acetylglucosamine (GlcNAc).

To express this dual-function enzyme, we genetically engineer:

• Escherichia coli BL21, a robust expression host used to produce and test the fusion construct.
• In the next stage, Bacillus subtilis 168, a GRAS-status chassis with an inherent ability for efficient protein secretion, making it highly suitable for large-scale applications.

The coding sequence of the fusion enzyme is inserted into compatible plasmids and optimized for high-level expression. A flexible peptide linker is included between the enzymatic domains to preserve proper folding and catalytic activity.

The engineered bacteria are then exposed to chitin-rich substrates, such as powdered shrimp shell waste. The fusion enzyme is secreted or released in situ, where it catalyzes the depolymerization of chitin into bioavailable nutrient-rich compounds. To ensure reproducibility and scalability, this process is conducted and optimized in controlled bioreactor systems, which allow precise regulation of growth conditions and product yield.

We monitor and evaluate the process using:

• In silico modeling and structural validation of the fusion enzyme prior to synthesis
• Cloning and transformation workflows using standard molecular biology techniques
• Qualitative and quantitative assays to assess enzymatic activity and degradation efficiency

Finally, the degradation products are collected and formulated as a liquid bioactivator. These can be applied through foliar sprays in crops such as garlic cultivation, following chemical fertilizer treatments, helping to restore soil health while simultaneously providing protective effects against fungal pathogens.

This modular and iterative methodology allows us to build, test, and optimize a living system capable of turning chitin waste into a valuable agricultural input.

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