Tom's autobiography:

There's one particular thing that I have noticed about the old buildings in Shenzhen, that is a strange, annoying smell that gets so fouling in those rainy days that my mom had to ask us to spend most of the day outside the house. A few months ago, it came to me that this smell is caused by no other, but Cladosporium spp. - a type of mold, which is what I saw behind the closet in my room: a dim, dark patch of small spots. At then we didn't really know the danger of it apart from feeling a bit dizzy around the head in that room. After a quick search on the pathogenicity of molds, it truly gave us a real scare that molds can be so much a health issue. We tried using mold removers, but the irritating smell just made the problem worse. Within a year, as we could no longer take it, we moved away. However, during the "Hui Nan" days, molds still thrive on the walls besides my bed, in the corners, below the tap, everywhere. Therefore, I brought this up as a central aim for GreatBay-SCIE 2025. With the power of synthetic biology, we are determined to tackle this problem for good.

General introduction of mold

Molds is a general term for a class of fungi that look like small-plants microscopically (Fig. 1a, 1b). They are composed of filaments that expand on or in every animal, plant or human-made structure to form a colony. They reproduce by spores that travel by the air and land on everywhere, starting growing rapidly if it finds warm and moist surface - and this is how our homes become infested (Fig. 1c, 1d) [6].

Fig. 1 | (a,b): microscopical view of mold; (c,d): Mold in household environment

Relative humidity (RH) and temperature [7] are the most critical factors for mold growth, studies proved that the optimal growth temperature for many mold species ranges from 25~30°C, and they begin proliferating once RH exceeds 75% [8]. Therefore, mold is mainly distributed in tropical and subtropical climate, (Fig. 2) such as eastern and southern China (the Yangtze River Delta and the Pearl River Delta) For these Chinese families, the most horrifying time of the year is the "Hui Nan" days - that is when the entire household become occupied by mold. This is a climatic phenomenon: every spring, when the weather has not yet fully warmed up, the warm air sweeping in from the sea meets the cooler surfaces of buildings, condensing into water droplets. The moisture provides humidity, the dust and organic materials on the walls offer nutrients, and the warm sea air contributes to the ideal temperature, allowing mold to thrive and proliferate. Except for China, mold occurrence is prevalent across the world - in 14 European countries plus Australia, India and New Zealand, the average relative indoor mold prevalence is 22.1%, while in Japan it is 9.7%, the United States reaches an astonishing 33% [9].

Fig. 2 | Global habitat suitability for Black Mold [10]

Mold also spreads like a wildfire in households. All corners of your house are at risk of mold colonization. In humid places like bathroom, kitchen, fridges and washing machine, mold population is the largest (Fig. 3) [11]. Building materials with high critical moisture levels, such as pine sapwood and plywood, chipboard and thin hardboard, are corroded easily [12].

Fig. 3 | Rooms most susceptible to mold colonization.

Harm of Mold

Damage to Health
The mold, their spores, their mycotoxins and the microbial volatile organic compounds (MVOCs) they produce all pose threats on our physical well-being. Mold itself, spores and its mycotoxins are significant allergens, inflicting the respiratory system. Sensitization of mold is greatly associated with increased risk of asthma and allergic rhinitis (Fig. 4) [13] [14]. In China, among the 8 million cases of children asthma, more than half is a result of the inhalation of mold [15]. If exposed to mold for an extended period, there is a significant risk that mold spores can penetrate deep into the respiratory tract, leading to more severe illnesses. An example is Allergic Bronchopulmonary Aspergillosis (ABPA), a hypersensitive case to Aspergillum. Patients with ABPA suffer from wheezing and dyspnea, if exacerbate, pulmonary fibrosis and respiratory failure may occur (Fig. 5) [16]. Another case is Hypersensitive Pneumonitis (HP), patients face the risk of irreversible lung damage if continuously exposed to the antigen [17]. In the diagnosis of HP, mold has a sensitivity of up to 84%, with Aspergillum and Penicillium being the most common cause [18].

Fig. 4 | Adjusted odd ratios (95%Cl) of children diagnosed with asthma and allergy associated with household mold in a northwest and southern city in China [13] [14]

Fig. 5 | the CT scan of the lungs of a patient with ABPA and Chronic Obstructive Pulmonary Disease (COPD) [16]

To the immunocompromised, mold infection has a much more detrimental effect. For example, invasive aspergillosis [19] is an aggressive fungal disease that occurs profoundly in patients with defective immune systems, such as those undergoing leukemia chemotherapy or suffering from HIV/AIDS. Its annual global case reports exceed 300,000, having an associated mortality rate ranging from 30% to 80%. Moreover, exposure to mold or mycotoxin could cause exacerbation of underlying pathophysiology including allergic and non-allergic chronic inflammatory diseases and autoimmune disorders, even HIV progression [20].

Damage to Buildings
The presence of mold not only compromises the aesthetic appeal of a building but also damages its structural integrity, posing a serious threat to its service life. Not long ago, on 7th of August, 2025, in Brighton, UK, an apartment with a mold-ridden ceiling collapsed, forcing the residents to lose their homes [21]. This case was not an exception: studies have reported that in coastal countries, up to 57% of buildings have mold invaded structures, while each building has an average of 3 structures affected by mold [22]. These constructions are all endangered by the mold proliferation within. Every year an astronomical sum of money is spent on repairing mold-damaged buildings. In some neighborhood maintanence project in Guangzhou, Guangdong, the quoted price can reach as high as 3.71 million Yuanes (0.52 million USD) [23].

Since mold is such a huge threat to buildings and our health, it is urgent to solve this problem. Although various domestic methods and commercial products that target mold exist already, they are either ineffective or have severe side effects that dramatically reduces the experience of the consumer.

Harmful to Human Health

Quaternary ammonium compounds (QACs) are effective antifungal agents and thus the components of some commerical products. However, they could cause dermatitis and other skin irritation [24]. Users are advised to wear gloves and special protective clothing when using these products, bringing unnecessary inconvenience and risks. Bleach and hydrogen peroxide are also commonly used [24]. Similar to QACs, however, they pose a health risk to the end user.

Weak Antifungal Effect

White vinegar often appears in household guides for mold removal [25], yet the antifungal effect of white vinegar is extremely limited. Baking soda solution is also frequently recommended as a readily available fungicide at home. It has the same downside to white vinegar, being relatively ineffective to mold.

Damage to Surfaces

The corrosiveness of bleach could cause degradation of painted surfaces and fabrics. This is why products with bleach require the user to test in small areas first before extensive use and ensure good ventilation. Hydrogen peroxide, as a potent oxidizing agent, incurs risk of wall surface damage and discoloring of fabric and leather.

Pungent Odor

Both bleach and ammonia produce a strong odor, which makes the consumer experience extremely unpleasant. Furthermore, the existing commerical products rarely deal with the pungent smell that mold brings.

Due to the obvious drawbacks that widely exist in the common mold-removal products, GreatBay-SCIE 2025 has conceived an innovative mold-remover - ArMOLDgeddon: (Fig. 6)

Fig. 6 | brief overview of ArMOLDgeddon

Enzymes

During initial conceptualization of our project, mold cell wall has become our main focus of attack. After brief analysis, we identified glucan, glycoproteins and chitin to be the main constituents of fungal cell wall and cell wall of its spores (Fig. 7) [26] [27]. Following this, we carried out extensive research on substances that damage fungal cell wall while remaining safe for humans and animals, and soon locked our view on specific glycoside hydrolases, (Fig. 8) namely chitinase, glucanase and lysozyme.

Fig. 7 | schematic representation of mold cell wall (a). composition of fungal cell wall; (b). composition of spore cell wall

Fig. 8 | schematic illustration of hydrolysis of cell wall carbohydrate by enzyme hydrolases.

It is noticed that different mold strains have cell walls that differ in composition and relative abundance of different polymers. Thus, we synthesized multiple enzymes with different characteristics, aiming to address different molds with a single, powerful anti-mold mixture. The chitinases we've chosen target different regions of the mold mycelia, ranging from the extending tip to the mature mold hyphal wall. For glucanases, we have chosen the enzymes that target different glycosidic bonds, aiming to address molds with a range of types of glucan in the cell wall.

Chitinase

Targeting β-1,4-N-acetylglucosamine polymers, or chitin, chitinases (EC 3.2.1.14) are glycoside hydrolases widely common in nature, acting as both a modulatory enzyme for fungal growth and as potent antifungal agents in innate defense systems in plant and animal kingdoms (Fig. 9) [28].

Figure 9 | illustration of the catalytic domain of an exempler chitinase PrChiA docked with chitin oligomer.

Chitinases hydrolyze chitin and disrupts fungal cell wall, leading to cell lysis due to osmotic imbalance. Considering the ubiquitous presence of chitin in common fungi species and highly specific nature of enzymes, chitinases fulfill the expectations of a safe, efficient, and environmentally friendly biofungicide. Continuing our research, we arrived at four chitinase candidates for further wetlab effort, namely rMvEChi, GlxChiB, PrChiA, and BcChiA1 [29] [30] [31] [32] [Fig.10], selected based on desirable properties such as high expression yield, strong antifungal effects, and soluble expression in E. coli host.

Fig.10 | protein structures of (a): rMvEChi; (b): GlxChiB; (c): PrChiA; (d): BcChiA1, generated with Alphafold 3

Among the enzymes, rMvEChi specifically targets the germinating mold hyphal tip, inhibiting further growth [29], while GlxChiB and PrChiA are capable of damaging both the hyphal tip and the mature hyphal wall [30] [33], not only inhibiting growth but also effectively killing the live molds. Due to such advantages, we decided to further improve GlxChiB and PrChiA from a modelling approach, aiming to increase expression, stability and activity. BcChiA1 is among the most active chitinases and possesses high chitinolytic activity, which will be, when used in combination with the other highly cell-wall-damaging chitinases, conducive for further fragmentation of the fungal cell wall [32]

Glucanase

Fungal cell walls are predominantly composed of glucans, which form an outer protective barrier safeguarding molds and their spores. Disrupting this glucan layer is therefore a key strategy for compromising fungal cell integrity. To maximise degradation efficiency, we incorporated a group of glucanases, each targeting distinct glycosidic linkages, including β-1,3-glucan, β-1,3-1,4-glucan, β-1,6-glucan and α-1,3-glucan, enabling multi-attack on the structural complexity of the cell wall glucan layer. (Fig. 11)

Fig. 11 | protein ribbon illustration of (a): BglS27; (b): Bglu1; (c): FlGlu30; (d): aglEK14 generated with alphafold

β-1,3-glucans constitute the predominant structural backbone of the fungal cell wall, accounting for 30% to over 80% of its composition [26]. Among β-1,3-glucanases, a variant BglS27 from Streptomyces sp. was selected due to its strong substrate-binding affinity and catalytic activity, leading to cell wall lysis. Moreover, the enzyme exhibits the ability to disrupt fungal hyphal membranes, showing enhanced antifungal effect [34] [35]. Bglu1, a β-1,3-1,4-glucanase from Bacillus velezensis ZJ20 [36], expands the spectrum of degradation by cleaving both β-1,3 and β-1,4 bonds. Similar to BglS27, Bglu1 has been shown to induce abnormal hyphal morphology. Furthermore, β-1,6-glucans act as cross-linkers that stabilize the glucan matrix by connecting β-1,3-glucan chains. To address this, FlGlu30 from Flavobacterium sp. was employed [37], not only due to its ability to degrade these cross-linkages, but also its ability to generate intracellular accumulation of reactive oxygen species (ROS), further damaging fungal cells from within. In a discussion with a professor, α-1,3-glucans was highlighted as a key structural component of the fungal cell wall. We therefore selected an α-1,3-glucanase from Flavobacterium sp. EK-14 [38], dubbed aglEK14, a highly specific enzyme that evolved to degrade this structural polymer in fungi. By acting on distinct yet interconnected components of the glucan matrix, these glucanases act in synergy. They weaken the structural backbone and break the cross-links, thereby destroying the outer protective layer of the cell wall and exposing the underlying chitin to our chitinase.

The combination of glucanases and chitinases is expected to act synergistically, enhancing individual enzymatic activities and achieving broad-spectrum antifungal activity against diverse mold species [39] [40].

Enzyme improvement

Wild type enzymes may possess features that are not suitable for realistic commercial application. Thus, to better promote the commercialization of our fungicide, we have identified three key downsides of our enzymes: low yield, low stability and low activity. We address them accordingly using protein modelling tools, namely ProteinMPNN and LigandMPNN, generating de novo protein sequences that may possess better qualities (Fig. 12) [41]. We then screened the sequences generated based on a matrix containing docking score, SASA, rosetta score, etc., selecting the best candidates for further wetlab effort. After screening, validation procedures are carried out to characterize the expression yield and activity of the de novo proteins. For more detail, please visit our Modelling page.

Fig. 12 | enzyme optimization using protein mpnn [41]

Lysozyme

Lysozymes, a class of small, disulfide-rich antimicrobial proteins from animals, has also been shown to display strong fungicidal effects according to multiple sources. (Fig. 13) For the modes of action for its antifungal effects, it is commonly believed that the highly cationic protein disrupts cell wall integrity and eventually leads to death of the fungus.

Fig. 13 | antifungal activity of human lysozyme [42]

We thus chose the well-studied human lysozyme, hLYZ, to be included in our project. Since literature suggests expression of hLYZ in E. coli host is toxic to the bacteria - even with co-expression of inhibitory chaperones - we decided to use the common eukaryotic chassis, P. Pastoris, for the protein's expression, both for the eukaryote's higher tolerance of toxicity as well as full-fledged ER for soluble expression of this disulfide-rich protein [43] [44] [45]. According to Xu, Kewei, et al., mutation V110S confers the recombinantly expressed lysozyme with higher activity and better soluble yield [46]. Thus we decide to incorporate such design into our project.

CBM addition

The main scenario of application for our product (sprayer, fogging, etc.) is the building wall surfaces, and among the chemicals in wall surface paints, cellulose and PET-like polymers has been identified as a widely used ingredient [47]. Thus, to better suit our product for mold deactivation on building wall surfaces, we decided to fuse our enzyme with a noval carbohydrate binding domain dubbed BaCBM2 [48] . This additional CBM domain confers binding ability to the most hydrocarbon chain polymer materials in wall paint, (Fig. 14) extending the period of enzyme activity on the targeted area of the wall, and thus lengthening the time period of fungal growth inhibition. Furthermore, the extra CBM domain allows us to integrate our enzymes onto AC filter materials without influencing enzyme activity, allowing more diverse product form and application scenarios for ArMOLDgeddon.

Fig. 14 | PET binding assay showing BaCBM2 with strongest affinity [48]

Terpene

Apart from enzymes, monoterpenes have also attracted our attention. Geraniol is a monoterpenoid alcohol with a pleasant rose-like aroma. As a common ingredient in many essential oils, geraniol is used commercially as a fragrance compound in cosmetic and household products [49], which, in our case, would hopefully cover up the musty smells of mold. Previous studies have shown that geraniol phytoconstituent (extracted from plant) has antifungal activity, with a minimal inhibitory concentration of 16-130 µg/mL. Furthermore, synthesized geraniol has antifungal activity and antibiofilm activity, with an inhibitory effect of around 30% [50]. The antifungal activity of geraniol is believed to be attributed to the disruption and permeabilization of fungal membranes, interference with ergosterol synthesis and production of reactive oxygen species that would eventually induce apoptosis and the downregulation of growth and secondary metabolites of fungal species [51] [52].

Therefore, we have decided to include geraniol as part of our product to give it a sweet scent and, at the same time, kill and inhibit mold. To produce geraniol, we use DH5α strain of E. coli to express two plasmids, combining the MVA pathway, which yields an increased amount of IPP/DMAPP, with the geraniol production pathway. (Fig. 15) The geraniol production plasmid contains geranyl diphosphate synthase from Abies grandis (AgGPPS2) and geraniol synthase from Ocimum basilicum (ObGES). A variant of ObGES (t65ObGES) truncated of its transit peptide is also included for investigation, as literature suggests such truncation could lead to higher expression efficiency [53].

Fig. 15 | metabolic pathway for the production of geraniol

It has been established in previous reports that the solubility of geraniol is very poor. Thus, we sought methods to improve the solubility of geraniol to better suit the implementation of our desired product. In our research, we came across a method that embeds geraniol in γ-cyclodextrin (γ-CD), which would provide geraniol better solubility in water. (Fig. 16) Both γ-CD-Ger, where geraniol is chemically grafted onto γ-CD, and γ-CD/Ger, where geraniol is physically encapsulated within the γ-CD have demonstrated good solubility compared to the mere solubility of geraniol [54]. To explore the feasibility of such design, we attempted physical encapsulation.

Fig. 16 | Degree of substitution (DS) is defined as the ratio of the number of grafted Ger to the number of sugar units, respectively. Note the solubility of geraniol(Ger) is significantly increased when it's directly grafted onto γ-cyclodextrin(γ-CD), instead of directly mixing the two to form inclusion complexes [54].

Furthermore, combining gerniol with γ-CD also reduces the volatiliity of geraniol, making the aroma more stable and long-lasting (Fig. 17) well suiting our aims [55].

Fig. 17 | CD/geraniol-IC-NFs are free-standing nanofibrous webs of cyclodextrin/geraniol-inclusion complex. HPβCD, MβCD, and HPγCD are different types of CDs. Note that the geraniol is not directly grafted onto CDs [55].

1. Advancing Enzyme Engineering:

2. Scaling and Application:

3. Spreading the Values of Synthetic Biology:

4. Expanding Inclusivity and Global Reach:

5. Advancing Sustainability Globally

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