The smell of rotten moist was the first thing to welcome Tom as he entered this ancient house in his hometown, Guangdong. There are tiny creatures lurking in the air, full of gloom and doom, forcing out from Tom a cough of discomfort. He stepped toward the bathroom, reaching to the sink that was crusted with something dark and dusky. It’s slick, coated in a film of black.
That wasn’t the single spot. In his house, black spots spread like veins — in the corners, along the baseboards, beneath the peeling wallpaper, everywhere.
Then he realized, it was the mold. But the mold was so stubborn that no detergent could peel them off the walls. Not after a while, Tom’s eyes were red and watery due to the anti-mold spray he used. He wasn’t the sole victim — every year, during the damp, rainy seasons before summer, mold sweeps across Southeast China like a hurricane, occupying every house, torturing every resident. Some rampant throughout the year, bringing numerous discomfort, even life-critical sufferings...
That's when he reached to us, reflecting these revolting creatures. Unfortunately, all the present mold repellant were either ineffective, or giving a devastating side effect. As a result, we decided to create our own Bio Anti-mold product.
As GreatBay SCIE 2025 blows the horn of Synthetic Biology, the enzyme archangels arrive at your home, and thus, begin the ArMOLDgeddon……

General Introduction of Mold

Molds are a general term for a class of fungi that look like small-plants microscopically. 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 occupied by them. [1]

Figure 1: (a): A college dormitory in southern China with walls covered in mold; (b): Mold growing on a petri dish; (c): Mold on a strawberry https://en.wikipedia.org/wiki/Mold ; (d): fruiting body of molds

In China, the estimated indoor mold prevalence is 12.1% with the most common type of molds as Cladosporium (16.5% ~ 85% among all indoor mold), Aspergillus (2% ~ 40.1%) and Penicillium (0.72% ~ 28.5%) [2] [3] . Mainly distributed in tropical and subtropical climate, the molds pervade the most in eastern and southern China, such as in Yangtze River Delta and the Pearl River Delta. The mold population expands across the world like wildfire – 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% [2] .

Figure 2: Global habitat suitability for Black Mold (Alkhalifah et al.)

Harm of Mold

Molds are harmful. They don't just look nasty; their growth is at the cost of our health and our home.

To our health

The mold, their spores, their mycotoxins and the microbial volatile organic compounds (MVOCs) all pose threats on our physical well-being.

Mold itself, spores and its mycotoxins are significant allergens. Sensitization of mold is greatly associated with increased risk of asthma and allergic rhinitis [4] [5] . In China, among 8 million cases of children asthma, more than half is a result of the inhalation of mold [6] .

Figure 3: Adjusted odd ratios (95%Cl) of children diagnosed with asthma and allergy associated with household mold in a northwest and southern city in China (Li et al.)

A more severe case 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. [7]

Figure 4: the CT scan of the lungs of a patient with ABPA and Chronic Obstructive Pulmonary Disease (COPD) (Sisodia and Bajaj)

Another case is Hypersensitive Pneumonitis (HP), causing symptoms varying from fever and chest tightness to irreversible lung damage if continuously exposed to the antigen [8] . Mold has a sensitivity of up to 84% in HP diagnosis, with Aspergillum and Penicillium being the most common cause [9] .

In addition, mold mycotoxins are known for their carcinogenicity: the International Agency for Research on Cancer has classified major aflatoxins (a class of mycotoxins) as human carcinogens [10] .

To the buildings

The more damp the house is, the more severe is the dwelling of mold. Building materials with high critical moisture levels, such as pine sapwood and plywood, chipboard and thin hardboard, are corroded easily [11] . Also, in places like bathroom, kitchen, fridges and washing machine, mold population is the largest [12]. You can surely say – all corners of your house are at risk of mold colonization.

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) [13]

Figure 5: Rooms most susceptible to mold colonization.

Since mold (Aspergillus spp., Penicillium spp., Cladosporium spp.) 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.

Weak Antifungal Effect

White vinegar often appears in household guides for mold removal [14] , 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.
Furthermore, both bleach and hydrogen peroxide are proved to be ineffective against colonies of Aspergillus niger, Cladosporium herbarum and Penicillium chrysogenum. [15]

Damage to Surfaces

Bleach is commonly found in commercial products. However, its corrosiveness could cause degredation of painted surfaces and fabrics. This is why products with bleach require the user to test on small area first before extensive use and ensure good ventilation.
Hydrogen peroxide is another popular ingredient in commerical mold remover. As a potent oxidizing agent, users also bear the risk of wall surface damage and discoloring of fabric and leather.

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. [16] Users are advised to wear gloves and special protective clothing when using these products, bringing unnecessary inconvenience and risks.
Ammonia is similar to bleach in its effectiveness, but it emits harmful vapours when mixed with other chemicals, especially bleach.

Pungent Odor

Both bleach and ammonia produce a strong odor, which makes the consumer experience extremely unpleasant. Furthermore, the existing commerical product 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 has conceived an innovative mold-remover -- Armoldgeddon:

Fig. 5

Fig. 6 : composition of fungal cell wall [17]

During initial conceptualization of our project, mold cell wall has become our main focus of attack. After brief analysis, we identified chitin and β-glucan to be the main constituents of fungal cell wall. Following this, we carried out extensive research on substances that damage fungal cell wall and are producible by microorganisms, and soon locked our target on specific bioenzymes, namely chitinase, glucanase and lysozyme.

Figure 6: composition of fungal cell wall [17]

During initial conceptualization of our project, mold cell wall has become our main focus of attack. After brief analysis, we identified chitin and β-glucan to be the main constituents of fungal cell wall. Following this, we carried out extensive research on substances that damage fungal cell wall and are producible by microorganisms, and soon locked our target on specific bioenzymes, namely chitinase, glucanase and lysozyme.

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. [18]

Figure 7: [19]

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 five chitinase candidates for further wetlab effort, namely rMvEChi, GlxChiB, PrChiA, CaChi19A, and BcChiA1, [20] [21] [22] [23] selected based on desirable properties such as high expression yield, strong antifungal effects, and soluble expression in E. coli host. Among these, BcChiA1 has been found to have no fungicidal activity though very high chitinolytic activity, [24] thus we have set it up for comparison.

Fearing that molds may in the long run develop resistance to our product, we decided to make adjustments to our enzymes to make them stronger ang more efficient at killing mold and suppressing fungal growth.

To do this, we have conducted extensive reasearch and decided the following methods:

• Optimizing enzyme sequence using software modelling tools such as protein and ligand mpnn. This may change enzyme structure in a way that favors faster degradation and higher stability, or higher yield due to higher solubility. [25]

Figure 8: enzyme optimization using protein mpnn [26]

• Adding or changing additional substrate binding domains to increase enzyme affinity, potentially increasing activity.

Figure 9: adding a ChBD domain in chitinase Chit42 [27]

• Fusing domains from different enzymes together, each domain possessing excelling properties in its own functions

Figure 10: [19]

• Linker enzyme: linking different enzymes together for potentially faster degradation of the fungal cell wall. [28]

Apart from chitinase, another major component of fungal cell wall is glucan

Glucanase

Beta 1 3 glucanase, known for its special function of endohydrolysis of (1->3)- or (1->4)-linkages in beta-D-glucans or hydrolysis of beta-D-glucose units from the non-reducing ends of (1->3)-beta-D-glucans. (EC 3.2.1.58)

Since stronger fungicidal effect is shown according to literature when glucanase is used with chitinase, and since beta-1,3-glucan is the second major component of targeted fungal cell wall, we decide to use beta - glucanase to break down β-glucan and thus increase the efficiency of our fungicide. Similar to chitinase, the damage to the fungal cell wall by glucanase causes osmotic imbalance and eventually cell lysis, inducing fungal death and suppressing further fungal growth. [29]

We chose F1Glu, Bglu1 and BglS27 for their studied antifungal activity and soluble expression yield. Also similar to chitinase, we may carry out the following improvement on our glucanases:

• Optimizing protein sequence using proteinMPNN or ligandMPNN
• Adding additional CBM(carbohhydrate binding module) to the enzyme
• Fusion enzyme to combine different domains

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. 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.

Figure 12: antifungal activity of human lysozyme [30]

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. [31] [32]

Terpene

Geraniol is a monoterpenic alcohol with a pleasant rose-like aroma. Known as an important ingredient in many essential oils, geraniol is used commercially as a fragrance compound in cosmetic and household products. [33] 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%. [34]

Therefore, based on our research, we have decided to include geraniol as part of our product to give it a sweet scent. We use DH5α strain of E. coli to express two plasmids. One plasmid contains truncated geranyl diphosphate synthase from Abies grandis (AgGPPS) and truncated geraniol synthase from Ocimum basilicum (ObGES). The other plasmid is named pMVA, which improves the production of DMAPP (IPP) in E. coli. [35]

Figure 13: metabolic pathway for the production of geraniol

We have also investigated the interaction between terpene and our three enzymes. (chitinase, glucanase and lysozyme) Geraniol is suspected to increase the activity of glucanase, since geraniol-treated S. cerevisiae were more susceptible to glucanase digestion compared to untreated control. [36]

γ-Cyclodextrin

γ-cyclodextrin (γ-CD) provides geraniol solubility in water. 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. γ-CD also slows down the release of geraniol, making the aroma more stable and long-lasting. A greater amount of HPγCD/geraniol-IC-NF preserves in comparison to PVA/geraniol-NF, the control. Furthermore, γ-CD increases the antioxidant activity of geraniol. [37] [38]

Figure 14: Degree of substitution (DS) are 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. [37]

Figure 15: 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. [38]

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