New Composite Part

We nominate our team, MBG-DUTh, for the award of Best New Composite Part for the creation and validation of our novel chitinolytic fusion construct.

This composite part combines two complementary catalytic domains: an endochitinase from Pochonia chlamydosporia and an exochitinase from Aspergillus niger connected through a flexible (G₆S)₃ linker that we designed de novo and codon-optimized for expression in Bacillus subtilis. The entire sequence was engineered for optimal translation efficiency, secretion compatibility, and in-frame fusion within this Gram-positive host chassis.

The resulting fusion enzyme functions as a dual-acting biocatalyst capable of both internal and terminal cleavage of chitin polymers, achieving complete and synergistic degradation. The endochitinase domain hydrolyzes internal β-1,4-glycosidic linkages to produce soluble chitooligosaccharides, while the exochitinase domain releases N-acetylglucosamine monomers from the chain ends.

This part is not a simple assembly of pre-existing registry entries; it was rationally engineered, integrating a newly synthesized, flexible linker and two distinct catalytic domains into a single open reading frame. In silico validation (Ramachandran, MolProbity, and 3D structural modeling) confirmed proper folding, minimal steric clashes, and full catalytic accessibility for both enzymes.

Functionally, this composite part represents the core catalytic module of our project Chitinator et al., supporting the transformation of chitin-rich biowaste into sustainable agricultural bioactivators. Designed specifically for Bacillus subtilis, it demonstrates strong potential for efficient expression, secretion, and extracellular enzymatic performance.

By integrating Design–Build–Test–Learn principles, this construct embodies the iGEM spirit of innovation, providing a new, modular biotechnological tool for multi-domain enzymatic synergy and sustainable bioconversion.

The chitinolytic enzymes incorporated into our composite construct were derived from existing parts previously submitted to the iGEM Registry by Team USP-Brazil. These served as the functional foundation of our design.

Below is a summary of the original parts we selected and improved for our project:

Both the endochitinase and exochitinase sequences were originally optimized for E. coli expression systems.

In our project, we re-engineered these sequences to be fully compatible and optimal for Bacillus subtilis, which was chosen as our final chassis due to its natural secretion capacity and safety profile.

Specifically:

• For the endochitinase from Pochonia chlamydosporia, the stop codon was removed, allowing seamless in-frame fusion with downstream elements.
• For the exochitinase from Aspergillus niger, a TAA stop codon was added to properly terminate translation.
• Both sequences were codon-optimized for Bacillus subtilis, enhancing translational efficiency and secretion potential.
• A synthetic flexible linker (G₆S)₃ was designed de novo based on literature- reported sequences to maintain structural independence between catalytic domains while ensuring efficient substrate accessibility.

Through these modifications, we created a new composite part that combines both catalytic activities into a single fusion enzyme, designed for synergistic and complete chitin degradation.

As previously mentioned, we nominate ourselves for the Best New Composite Part award.

This composite construct encodes a dual-acting chitinolytic fusion enzyme, specifically designed to achieve complete degradation of chitin polymers through the synergistic action of two distinct catalytic domains. The part enables the hydrolysis of both internal and terminal β-1,4-glycosidic linkages in chitin, allowing the efficient conversion of chitin waste into smaller oligosaccharides and N-acetylglucosamine monomers.

It was constructed from newly created Basic Parts, each codon-optimized for expression in Bacillus subtilis and made RFC[10]-compatible through silent mutations:

• An endochitinase from Pochonia chlamydosporia (no stop codon), responsible for the cleavage of internal glycosidic bonds.
• A flexible (G₆S)₃ linker, providing structural freedom and maintaining the catalytic independence of both enzymes.
• An exochitinase from Aspergillus niger (with stop codon), enabling the terminal release of N-acetylglucosamine monomers.

Together, these elements form a synergistic fusion enzyme that allows both catalytic sites to operate simultaneously without steric interference, enhancing the overall efficiency of chitin degradation. The part was specifically designed for high translational efficiency and secretion compatibility in Bacillus subtilis, serving as the core catalytic unit of the Chitinator et al. Project.

The Story Behind Our Composite Part

One of the major challenges in chitin bioconversion is that most microorganisms can degrade chitin only partially, as individual enzymes act on either internal or terminal bonds of the polymer. This results in slow and incomplete chitin breakdown, limiting its potential as a source of valuable bioactive molecules.

To overcome this limitation, our team aimed to design a dual-acting fusion enzyme capable of performing both internal and terminal hydrolysis a single construct that could efficiently and synergistically degrade chitin.

For this purpose, we searched the iGEM Registry and reviewed previous projects to identify compatible enzymes. We selected an endochitinase from Pochonia chlamydosporia and an exochitinase from Aspergillus niger as the optimal catalytic pair. Both enzymes belong to the GH18 glycosyl hydrolase family, as confirmed through our phylogenetic analysis, which demonstrated strong evolutionary relatedness and complementary substrate specificities.

We then proceeded with computational modeling and structural validation to evaluate different linker options. Among all tested designs, the (G₆S)₃ flexible linker proved optimal, ensuring proper folding, independence of catalytic domains, and structural stability within the same open reading frame.

Finally, the construct was experimentally validated through a sequence of Wet Lab assays: bacterial transformation in E. coli BL21, protein overexpression and SDS-PAGE analysis, and chitinase activity testing using colloidal chitin agar. The results confirmed successful transformation, expression, and enzymatic function of the fusion enzyme.

For more details about our work, visit the Lab Book for step-by-step experimental documentation and result analysis , the Dry Lab section for the Structural Validation and Phylogenetic Model , for in-depth linker comparison results.