ArMOLDgeddon eliminates active mold growth mainly through targeting the mold cell wall. For such aim, we incorporated chitinase, glucanase and lysozyme as well as monoterpenoid geraniol into our anti-mold mixture. The two glycoside hydrolases, chitinase and glucanase, degrade chitin and glucan polymers respectively, while lysozyme disrupts cell membrane and attacks fungal walls through its cationic nature. Geraniol prohibits fungal growth through cell membrane permeabilization and interference of ergosterol synthesis, while providing a pleasant fragrance. Combining these active ingredients, our powerful fungicide can both deactivate mature mold and prevent mold regrowth through killing the spores. To better suit its realistic application, we engineered the enzymes thorugh computational modelling and fusion protein design, generating de novo protein sequences with improved characteristics and creating new protein complexes with enhanced affinity towards carbohydrates. To achieve all these, we have characterized numerous parts shown below.
| Part Numbers | Name | Type | Part Description |
|---|---|---|---|
| BBa_257ZGE31 | rMvEChi | Coding | Encodes a chitinase which hydrolyzes chitin in fungal cell wall, and damages hyphal tip. |
| BBa_250HUSWR | GlxChiB | Coding | Encodes a chitinase which hydrolyzes chitin in fungal cell wall, and damages both hyphal wall and hyphal tip. |
| BBa_K2380005 | BcChiA1 | Coding | Encodes a chitinase which has a high hydrolytic efficiency toward chitin in fungal cell wall. |
| BBa_258O0ON7 | PrChiA | Coding | Encodes a chitinase which hydrolyzes chitin in fungal cell wall, and damages both hyphal wall and hyphal tip. |
| BBa_259TEXAS | PrChiA-1 | Coding | An optimized version of PrChiA with enhanced folding fidelity, soluble yield and hydrolytic activity. |
| BBa_25CV1Y89 | PrChiA-2 | Coding | An optimized version of PrChiA with enhanced folding fidelity, soluble yield and hydrolytic activity. |
| BBa_2576A64D | PrChiA-3 | Coding | An optimized version of PrChiA with enhanced folding fidelity and soluble yield. |
| BBa_25RNFEFX | PrChiA-4 | Coding | An optimized version of PrChiA with enhanced folding fidelity and soluble yield. |
| BBa_25FMG3WH | PrChiA-5 | Coding | An optimized version of PrChiA with enhanced folding fidelity, soluble yield and hydrolytic activity. |
| BBa_25BH6KWF | PrChiA-6 | Coding | An optimized version of PrChiA with enhanced folding fidelity and soluble yield. |
| BBa_2558NS8D | GlxChiB-1 | Coding | An optimized version of GlxChiB with enhanced folding fidelity, soluble yield and hydrolytic activity. |
| BBa_25WWY3TT | GlxChiB-2 | Coding | An optimized version of GlxChiB with enhanced folding fidelity, soluble yield and hydrolytic activity. |
| BBa_251TQW39 | GlxChiB-3 | Coding | An optimized version of GlxChiB with enhanced folding fidelity and soluble yield. |
| BBa_250FFI5X | GlxChiB-4 | Coding | An optimized version of GlxChiB with enhanced folding fidelity and soluble yield. |
| BBa_25SQX00O | GlxChiB-5 | Coding | An optimized version of GlxChiB with enhanced folding fidelity, soluble yield and hydrolytic activity. |
| BBa_25MJGF3A | GlxChiB-6 | Coding | An optimized version of GlxChiB with enhanced folding fidelity and soluble yield. |
| Part Numbers | Name | Type | Part Description |
|---|---|---|---|
| BBa_25EGUS34 | Bglu1 | Coding | Encodes a β-1,3-1,4-glucanase that hydrolyzes mixed-linkage β-glucans and deforms mycelia. |
| BBa_25VOFUOT | BglS27 | Coding | Encodes a β-1,3-glucanase that targets β-1,3-glycosidic linkages. |
| BBa_25P7FVKR | FlGlu30 | Coding | Encodes a noval endo-β-1,6-glucanase that hydrolyzes β-1,6-glycosidic bonds and induce oxygen species accumulation. |
| BBa_25CNDPOK | aglEK14 | Coding | Encodes a noval ɑ-1,3-glucanase, able to hydrolyze ɑ-1,3 glycosidic bonds in fungal cell wall. |
| BBa_250FU2MS | BglS27-1 | Coding | An optimized version of BglS27, expected to exhibit enhanced folding fidelity, soluble yield and thermal stability. |
| BBa_25HW7929 | BglS27-2 | Coding | An optimized version of BglS27, expected to exhibit enhanced folding fidelity, soluble yield and thermal stability. |
| BBa_25MCI5ST | BglS27-3 | Coding | An optimized version of BglS27, expected to exhibit enhanced folding fidelity, soluble yield and thermal stability. |
| BBa_25OIC79W | BglS27-4 | Coding | An optimized version of BglS27, expected to exhibit enhanced folding fidelity, soluble yield and |
| BBa_25LQ623A | BglS27-5 | Coding | An optimized version of BglS27, expected to exhibit enhanced folding fidelity, soluble yield, thermal stability and hydrolytic activity. |
| BBa_25RRYZ4H | BglS27-6 | Coding | An optimized version of BglS27, expected to exhibit enhanced folding fidelity, soluble yield, thermal stability and hydrolytic activity. |
| Part Numbers | Name | Type | Part Description |
|---|---|---|---|
| BBa_K1228000 | hLYZ | Coding | Encodes human lysozyme, which possesses cationic peptide-like properties that can disrupt fungal cell wall and membrane integrity. |
| BBa_256U2VRO | mhLYZ | Coding | Encodes an optimized version of human lysozyme, which possesses cationic peptide-like properties |
| Part Numbers | Name | Type | Part Description |
|---|---|---|---|
| BBa_25H6DAJA | 𝛾-CGTase | Coding | Encodes 𝛾-cyclodextrin glycosyltransferase, which produces γ-cyclodextrin from starch. |
| BBa_K1653007 | ObGES | Coding | Encodes a geraniol synthase (GES), which catalyzes the conversion of geranyl diphosphate (GPP) into geraniol |
| BBa_259S50W0 | t65ObGES | Coding | Encodes a truncated version of geraniol synthase (GES), which catalyzes the conversion of geranyl diphosphate (GPP) into geraniol |
| BBa_25Q4A33W | t86AgGPPS2 | Coding | Encodes a truncated version of geranyl diphosphate synthase (GPPS), which produces geranyl diphosphate (GPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). |
| BBa_25G5W5N0 | t86AgGPPS2-ObGES | Composite | Coordinated enzyme system for high-yield production of geraniol. |
| BBa_25623N3I | t86AgGPPS2-t65ObGES | Composite | Coordinated enzyme system for high-yield production of geraniol. The two key geraniol synthases are truncated to increase conversion efficiency. |
Our collection consists of a set of functional genes designed for eliminating and inhibiting active mold and spores, including chitinase, glucanase, and human lysozyme genes, with geraniol and γ-cyclodextrin biosynthesis genes. Each part contributes to the overall antifungal goal with distinct biological functions. Chitinase and glucanase degrade the major polysaccharide components of fungal cell walls, while destabilizing hyphae. Lysozyme functions as a cationic peptide that disrupts negatively charged fungal cell wall and membranes. Geraniol serves as a natural antifungal compound that suppresses fungal growth, enhancing the overall fungicidal effect. Furthermore, when fused with carbohydrate-binding modules (CBMs), the enzymes can be immobilized onto specific materials for enhanced functionality. Together, these elements form a synergistic and comprehensive antifungal system. For each part, we provide detailed documentation, with clear sequences, expression vectors, experimental protocols, and results.
For each enzyme, we established a standardized
framework to ensure both functional validity and reproducibility. Expression
was first confirmed by cloning into suitable vectors and host strains,
followed by SDS-PAGE analysis to assess soluble expression. Enzyme activity
was then tested through concentration analysis and substrate degradation
assays.
As an example, wild-type PrChiA was only expressed as
inclusion bodies, whereas our redesigned variants achieved soluble
expression with detectable enzymatic activity. These results not only
validate the effectiveness of our optimization methodology but also provide
a reliable foundation for further functional testing. All experimental data
and protocols have been carefully documented to enable replication and
improvement by other teams. Moving forward, we aim to expand our assays to
additional substrates and fungal cell wall components to further evaluate
their antifungal potential.
This collection is characterized by its generality, modularity, and innovation. Because the design is based on the fundamental composition of fungal and spore cell walls, it can be broadly applied to any scenario involving fungal cell wall degradation. Geraniol extends potential applications to food preservation and agricultural mold prevention [1] [2]. γ-cyclodextrin not only stabilizes small molecules but can also be used for enzyme immobilization, improving real-world applicability. CBMs can be fused with a wider range of enzymes for enhanced enzyme stability. Moreover, our work demonstrates the feasibility of AI-assisted enzyme engineering to improve expression, stability, and activity. Each part can function independently or be combined into a multi-component system. Future iGEM teams can reuse or adapt individual parts for their own research. Thus, our collection provides both the synbio community with a novel strategy for antifungal part design and the society with a sustainable, bio-based alternative to chemical antifungal agents.
We present a comprehensive antifungal gene collection that targets multiple structural and physiological vulnerabilities in fungi through synergistic action. Our system has been experimentally validated and well-documented, offering the iGEM community a practical toolkit of chitinase, glucanase, lysozyme, and geraniol biosynthetic genes. Beyond the competition, this contribution delivers a biologically derived antifungal strategy that provides a safer, more sustainable alternative to chemical agents, while simultaneously inspiring future innovation in synthetic biology antifungal research.
